as an antimicrobial agent - eprints.qut.edu.au development of lactococcus lactis as an antimicrobial...
TRANSCRIPT
THE DEVELOPMENT OF
LACTOCOCCUS LACTIS
AS AN ANTIMICROBIAL AGENT
Yu Pei Tan
Bachelor of Laws (Honours)
Bachelor of Applied Science (Biotechnology)
Bachelor of Applied Science (Honours)
Institute of Health and Biomedical Innovation
School of Life Sciences
Queensland University of Technology
Brisbane, Queensland, Australia
A thesis submitted for the degree of Master of Applied Science
(Research) at Queensland University of Technology
2010
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ABSTRACT
Non-pathogenic lactic acid bacteria are economically important Gram-positive
bacteria used extensively in the food industry. Due to their “generally regarded
as safe” status, certain species from the genera Lactobacillus and Lactococcus
are also considered desirable as candidates for the production and secretion of
recombinant proteins, particular those with therapeutic applications.
The hypothesis examined by this thesis is that Lactococcus lactis can be
modified to be an effective antimicrobial agent. Therefore, the aims of this
thesis were to investigate the optimisation of the expression, secretion and/or
activities of potential heterologous antimicrobial proteins by the model lactic
acid bacterium, Lactococcus lactis subsp. cremoris MG1363.
L. lactis strains were engineered to express and secrete the recombinant CyuC
surface protein from Lactobacillus reuteri BR11, and a fusion protein consisting
of CyuC and lysostaphin using the Sep promoter and secretion signal. CyuC
has been characterised as a cystine-binding protein, but has also been
demonstrated to have fibronectin binding activity. Lysostaphin is a bacteriolytic
enzyme with specific activity against the Gram-positive pathogen,
Staphylococcus aureus. These modified L. lactis strains were then investigated
to see if they had the ability to inhibit the adhesion of S. aureus to host
extracellular matrix (ECM) proteins. It was observed that the cell extracts of the
L. lactis strain with the vector only (pGhost9:ISS1) was able to inhibit the
adhesion of S. aureus to fibronectin, whilst the cell extracts of the L. lactis strain
expressing lysostaphin was able to inhibit adhesion to keratin.
Finally, this thesis has identified specific lactococcal genes that affect the
secretion of lysostaphin through the use of random transposon mutagenesis.
Ten mutants with higher lysostaphin activity contained insertions in four
different genes encoding: (i) an uncharacterised putative transmembrane protein
(llmg_0609), (ii) an enzyme catalysing the first step in peptidoglycan
biosynthesis (murA2), (iii) a homolog of the oxidative defence regulator (trmA),
and (iv) an uncharacterised putative enzyme involved in ubiquinone
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biosynthesis (llmg_2148). The higher lysostaphin activity observed in these
mutants was found to be due to higher amounts of lysostaphin being secreted.
The findings of this thesis contribute to the development of this organism as an
antimicrobial agent and also to our understanding of L. lactis genetics.
Keywords: Lactococcus, recombinant protein secretion, lysostaphin, ECM
proteins
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TABLE OF CONTENTS
ABSTRACT iii TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xii LIST OF MANUSCRIPTS xv STATEMENT OF ORIGINAL AUTHORSHIP xvi ACKNOWLEDGEMENTS xvii LITERATURE REVIEW 1 CHAPTER 1 - LITERATURE REVIEW 1.1 AN OVERVIEW OF LACTIC ACID BACTERIA 2
1.1.1 Genomics of lactic acid bacteria 5 1.1.2 Applications 6 1.1.3 Extracellular proteins of Gram-positive bacteria 9
1.1.3.1 Covalent attachment of surface proteins 14 1.1.3.2 Non-covalent attachment of surface proteins 14
1.1.3.2.1 LysM domains 14 1.1.3.2.2 YG repeats or choline-binding domains 15 1.1.3.2.3 GW modules 15 1.1.3.2.4 S-layer homology domains 16 1.1.3.2.5 Unique domain – Sep 16 1.1.3.2.6 Non-specific anchored proteins 17
1.1.4 Transposon mutagenesis – tool for genetic analyses 18 1.2 AN OVERVIEW OF ANTIMICROBIAL PROTEINS 20
1.2.1 Attachment blocking proteins 20 1.2.1.1 Anti-adhesin antibodies 20 1.2.1.2 Adhesin analogues 21 1.2.1.3 Host-receptor analogues 21
1.2.2 Bacteriophage endolysins 22 1.2.2.1 Structure of endolysins 24
1.2.3 Bacteriocins 25 1.2.3.1 Modified bacteriocins (class I) 28 1.2.3.2 Unmodified bacteriocins (class II) 29 1.2.3.3 Large heat-labile bacteriocins (class III) 29 1.2.3.4 Therapeutics and other applications of bacteriocins 34
1.3 AIMS OF THIS STUDY 35 CHAPTER 2 - GENERAL MATERIALS AND METHODS 37 2.1 GROWTH MEDIA 38
2.1.1 Agar plates 38 2.1.2 Antibiotics 38 2.1.3 Brain Heart Infusion (BHI) medium 39 2.1.4 GM17 medium 39 2.1.5 GM17+LmB agar plates 39 2.1.6 GM17+SaB agar plates 39 2.1.7 GM17+SaU agar plates 39 2.1.8 Isopropylthio--D-galactoside (IPTG) plates 39 2.1.9 Lysogeny Broth (LB) 40
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2.1.10 de Man, Rogosa and Sharpe (MRS) medium 40 2.1.11 Psi medium 40 2.1.12 SGM17MC medium 40 2.1.13 SOC medium 40 2.1.14 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) plates 41 2.1.15 Escherichia coli JM109 41 2.1.16 Lactic acid bacterial strains 41 2.1.17 Pathogenic strains 42
2.2 BACTERIAL CULTURE METHODOLOGIES 42 2.2.1 Chemically competent E. coli JM109 cell preparation 42 2.2.2 Electrocompetent L. lactis cell preparation 43 2.2.3 Isolation of chromosomal DNA from L. lactis 43 2.2.4 Purification of plasmids from E. coli 44 2.2.5 Transformation of chemically competent E. coli 45 2.2.6 Transformation of electrocompetent L. lactis 45
2.3 SOLUTIONS FOR DNA ANALYSES 46 2.3.1 Agarose gel loading buffer 46 2.3.2 Tris-borate EDTA (TBE) buffer 46
2.4 METHODS FOR DNA ANALYSES 46 2.4.1 Agarose gel electrophoresis 46 2.4.2 DNA precipitation 47 2.4.3 Gel purification of DNA 47 2.4.4 Ligation reactions 47 2.4.5 Polymerase chain reaction (PCR) 47 2.4.6 Purification of PCR products 48 2.4.7 Quantitation of DNA 48 2.4.8 Restriction enzymes 48 2.4.9 Sequencing 48
2.5 SOLUTIONS FOR PROTEIN ANALYSES 49 2.5.1 CAPS transfer buffer 49 2.5.2 Coomassie stain 49 2.5.3 Electrode buffer 49 2.5.4 Phosphate buffered saline (PBS) (pH 7.0) 49 2.5.5 2x SDS loading buffer (non-reducing) 49
2.6 METHODS FOR PROTEIN ANALYSES 50 2.6.1 SDS polyacrylamide gel electrophoresis (SDS-PAGE) 50 2.6.2 Trichloroacetic acid (TCA) precipitation of supernatant proteins 50 2.6.3 Western blots 51 2.6.4 L. lactis cell associated protein extraction 52
CHAPTER 3 - APPLICATION OF CYUC-LYSOSTAPHIN FUSION PROTEIN SECRETED BY LACTOCOCCUS LACTIS TO PREVENT STAPHYLOCOCCUS AUREUS ADHERENCE TO EXTRACELLULAR MATRIX PROTEINS IN VITRO 53 3.1 INTRODUCTION 54 3.2 MATERIALS AND METHODS 56
3.2.1 Construction of L. lactis strains that secreted CyuC or CyuC-lysostaphin fusion protein 56 3.2.2 Cell fractionation, protein extraction and western blot analysis 61 3.2.3 Prediction of protein molecular weight based on sequence 61 3.2.4 Stock solutions of fibronectin, collagen, and keratin 62
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3.2.5 L. lactis crude cell extracts for adherence assay 62 3.2.6 Preparation of S. aureus for adherence assay 62 3.2.7 Adherence of S. aureus to immobilised ECM proteins and L. lactis cell extracts 63 3.2.8 Statistical data analyses of significance using Student’s t-test 64
3.3 RESULTS 64 3.3.1 Expression of CyuC and CyuC-Lss confirmed by western blot analysis 64 3.3.2 Optimisation of ECM proteins used in the S. aureus adherence assay 66 3.3.3 L. lactis cell extracts containing recombinant proteins have no effect on the adherence of S. aureus to immobilised collagen 67 3.3.4 Adherence of S. aureus to fibronectin is inhibited by the cell extracts of all L. lactis strains, including the L. lactis pGhost9:ISS1 68 3.3.5 Adherence of S. aureus to keratin is inhibited by cell extracts from L. lactis pGhost9-CyuC-Lss and L. lactis pGhost9-his1-lss-his2 69
3.4 DISCUSSION 70 CHAPTER 4 - LACTOCOCCUS LACTIS FACTORS INVOLVED IN THE EXPRESSION AND SECRETION OF ANTIMICROBIAL CELL WALL LYTIC ENZYMES 73 4.1 INTRODUCTION 74 4.2 MATERIALS AND METHODS 74
4.2.1 Construction of a lysostaphin expressing L. lactis strain suitable for random insertional mutagenesis 74 4.2.2 Construction of a L. lactis transposon library by random insertional mutagenesis 76 4.2.3 Screening the transposon library for mutants with altered lysostaphin activity 82 4.2.4 Characterisation of the pGhost9:ISS1 insertion site and isolation of stable ISS1-generated mutants 82 4.2.5 Prediction of operon structures 85 4.2.6 Prediction of subcellular locations of proteins 86 4.2.7 Cell fractionation, protein extraction, SDS-PAGE, and western blot 86 Endolysin 87 4.2.8 Ply511 expression and secretion in [lss] mutant strains 87 4.2.9 Lysozyme resistance test 87 4.2.10 Transmission electron microscopy (TEM) 88 4.2.11 Statistical data analysis of significance using Student’s t-test 88 4.2.12 Alignment and phylogenetic analysis 88
4.3 RESULTS 88 4.3.1 Isolation and identification of mutants with altered lysostaphin activity 88 4.3.2 Characterisation of the genes which affected lysostaphin secretion 90
4.3.2.1 The gene llmg_0609 is incorrectly annotated and is renamed lom 90 4.3.2.2 The murA2 gene encodes for the primary MurA in L. lactis 94 4.3.2.3 More lysostaphin is secreted by the trmA[lss] mutant strain under high temperature stress 96
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4.3.2.4 Basis for lysostaphin secretion in llmg_2148[lss] mutant is unclear 96
4.3.3 The lom, murA2, and trmA mutant strains secrete higher levels of the cell wall hydrolytic enzyme, Ply511, compared to wild-type 97 4.3.4 The murA2 and trmA mutants were more resistant to lysozyme hydrolysis 99
4.4 DISCUSSION 99 CHAPTER 5 - GENERAL DISCUSSION 105 CHAPTER 6 - REFERENCES 111
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LIST OF TABLES
Table 1.1 Non-exhaustive list of heterologous proteins produced in LAB.
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Table 1.2 Studies that used pGhost9:ISS1 to identify gene functions in
LAB 19
Table 1.3 Non-exhaustive list of functional bacteriophage endolysins. 26
Table 1.4 Non-exhaustive list of Gram-positive bacterial bacteriocins. 31
Table 3.1 Strains, plasmids, and oligonucleotides used in this study. 60
Table 4.1 Strains, plasmids, and oligonucleotides used in this study. 80
Table 4.2 Characteristics of mutants with lysostaphin activity greater than
that of the wild-type. 90
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LIST OF FIGURES
Figure 1.1 Phylogenetic tree of Gram-positive bacteria. 4
Figure 1.2 Phylogenetic tree of LAB and related bacteria. 4
Figure 1.3 Schematic representation of the B. subtilis protein translocation
pathway. 13
Figure 1.4 Modular structure of Sep. 17
Figure 1.5 Schematic representation of the structural motifs of the
lysostaphin protein. 34
Figure 3.1 Schematic representation of the PCR overlap strategy employed
to clone the CyuC (A), and CyuC-Lss (B) fusion protein to the
Sep promoter and secretion signal at the 5’ end, and the cyuC
operon transcription terminator at the 3’ end. 58
Figure 3.2 Expression of recombinant CyuC from L. lactis pGhost-CyuC,
CyuC-Lss from L. lactis pGhost-CyuC-Lss and Lss from L. lactis
pGhost-his1-lss-his2. 65
Figure 3.3 Adherence of S. aureus to wells coated with four different
concentrations of collagen, keratin, and fibronectin. 66
Figure 3.4 Adherence of S. aureus to wells coated with collagen and
exposed to cell extracts from, L. lactis pGhost9-CyuC (CyuC), L.
lactis pGhost9-CyuC-Lss (CLss), and L. lactis pGhost9-his1-lss-
his2 (Lss). 67
Figure 3.5 Adherence of S. aureus to wells coated with fibronectin and
exposed to cell extracts from, L. lactis pGhost9-CyuC (CyuC), L.
lactis pGhost9-CyuC-Lss (CLss) or L. lactis pGhost9-his1-lss-
his2 (Lss). 68
Figure 3.6 Adherence of S. aureus cells to wells coated with keratin and
exposed to cell extracts from L. lactis pGhost9-CyuC (CyuC), L.
lactis pGhost9-CyuC-Lss (CLss) or L. lactis pGhost9-his1-lss-
his2 (Lss). 69
Figure 4.1 The regions of the his operon cloned from the chromosome (A)
and the structure of pGhost-his1-lss-his2 (B). 77
Figure 4.2 Stable integration of the lss expression cassette into the L. lactis
chromosome. 78
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Figure 4.3 Representation of the pGhost9-transposed mutant between
duplicated ISS1 elements (A). 83
Figure 4.4 Schematic representation of the creation of a mutant by random
transposon mutagenesis using pGhost9:ISS1 and the excision of
the plasmid from the chromosome. 84
Figure 4.5 Example of a western blot used in semi-quantification. 86
Figure 4.6 Identification of the insertion sites for the nine over-secreting
mutants. 91
Figure 4.7 Phylogenetic tree showing L. lactis MurA2 is more closely
related to the primary MurA in other species. 93
Figure 4.8 Transmission electron micrographs of the control strain,
MG1363[lss] (A, B) and the murA2[lss] mutant (C, D). 94
Figure 4.9 Coomassie-stained SDS-PAGE of proteins from the supernatant
fractions. 95
Figure 4.10 Western blot detection of L. lactis strains secreting lysostaphin
and Ply511 in the cell associated and supernatant fractions. 97
Figure 4.11 Dilutions of cultures incubated for 18 h spotted onto GM17 agar
with various concentrations of lysozyme. 99
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LIST OF ABBREVIATIONS
2D 2 dimensional
6-histidine hexa-histidine
aa amino acids
ABC ATP-binding cassette
APF aggregating promoting factor
ATCC American Tissue Culture Collection
ATP adenosine triphosphate
BCV bovine corona virus
BHI brain heart infusion
BLAST basic local alignment search tool
bp (bps) base pair(s)
BSA bovine serum albumin
CAPS 3-cyclohexylamino-1-propanesulfonic acid
CFTR cystic fibrosis transmembrane conductase regulator
cfu colony forming units
ClfB clumping factor B
d day
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
dNTP deoxynucleotide triphosphate
ECM extracellular matrix
ECMBPs extracellular matrix binding proteins
EDTA ethylenediaminetetraacetate
Em erythromycin
FnBPA fibronectin binding protein A
FnBPB fibronectin binding protein B
g gravitational force
GAS group A streptococci
G+C guanine and cytosine
GRAS generally regarded as safe
HIV human immunodeficiency virus
IMAC immobilised metal affinity chromatography
kb kilo bases
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kDa kiloDaltons
LAB lactic acid bacteria
LB Lysogeny Broth
LysM lysin motif
MAb monoclonal antibody
Mbps mega base pairs
mol% percentage molarity
MRS de Man, Rogosa and Sharpe
MRSA methicillin-resistant Staphylococcus aureus
MSSA methicillin-susceptible Staphylococcus aureus
MSCRAMMs microbial surface components recognising adhesive
matrix molecules
NICE nisin-controlled gene expression system
NSP4 bovine non-structural protein 4
NucT staphylococcal nuclease
OD optical density
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PCR polymerase chain reaction
pI isoelectric point
PSep Sep promoter
QUT Queensland University of Technology
RE restriction enzyme
RNA ribonucleic acid
rRNA ribosomal RNA
scFv single-chain fragment variable
SD standard deviations
SDS sodium dodecyl-sulfate
SLH S-layer homology
SNPs single nucleotide polymorphisms
ssSep Sep secretion signal
subsp. subspecies
TBE Tris-borate EDTA
TBS Tris-buffered saline
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TBS-T Tris-buffered saline with Tween
TCA trichloroacetic acid
TE Tris-EDTA buffer
TEM transmission electron microscopy
TEMED N,N,N’,N’-tetramethylethylenediamine
UV ultraviolet
X-Gal 5-bromo-4-chloro-3-indolyl--D-galactosidase
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LIST OF MANUSCRIPTS
Contents related to thesis:
Random mutagenesis identified novel host factors involved in the secretion
of antimicrobial cell wall lytic enzymes by Lactococcus lactis.
Yu Pei Tan, Philip M. Giffard, Daniel G. Barry, Wilhelmina M. Huston and
Mark S. Turner
Applied and Environmental Microbiology (October, 2008), Vol 74, pp. 7490-
7496
Other work:
Inactivation of an iron transporter in Lactococcus lactis results in
resistance to tellurite and oxidative stress.
Mark S. Turner, Yu Pei Tan and Philip M. Giffard
Applied and Environmental Microbiology (October, 2007); Vol 73, pp. 6144-
6149
xvi
STATEMENT OF ORIGINAL AUTHORSHIP
The work contained in this thesis has not been previously submitted for a degree
or diploma at this or any other higher education institution. To the best of my
knowledge and belief, the thesis contains no material previously published or
written by any other person except where due references is made.
Signed:_______________________________________
Yu Pei Tan
LLB BAppSc(Hons)
Date:____________________
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ACKNOWLEDGEMENTS
Firstly, I would like to express my appreciation to my supervisory team, Dr
Mark S. Turner, Dr Wilhelmina M. Huston, and Associate Professor Philip M.
Giffard, for their support and encouragement. I would like to thank Mark for
his time, effort, and patience as a remarkable teacher, and his continued efforts
even after he has moved on from QUT. I have learnt a lot from Mark in these
past few years, from molecular microbiology and Gram-positive bacteria to golf
and beer drinking. I also wish to thank Willa for joining my supervisory team
half way through my research studies. Willa has been a wonderful and
enthusiastic mentor, always positive, and always believed in me. Finally, thank
you to Phil for challenging me to be a better scientist and for your support and
guidance even from as far away as Darwin.
I would also like to acknowledge the staff and students from the School of Life
Sciences and the Institute of Health and Biomedical Innovation, in particular
members of the Chlamydia and Reproductive Health research groups. I would
like to thank the following people for your invaluable friendship: Steven Bell,
Alison Carey, Shea Carter, Kelly Cunningham, Peter Cunningham, Tegan
Harris, Raquel Lo, Shreema Merchant, Candice Mitchell, and Alex Stephens. I
would also like to acknowledge Shea Carter and Callum Eastwood (University
of Melbourne) for mutual support during the thesis writing phase.
Next, I would like to thank my non-science friends: Malcolm and Janet Choi,
Melissa Gazsik, Angela Harris, Phil Kay, Eddie Leong, Eleanor Leung, and
Matthew Yates. These are people who have endured my eccentricities, and who
supported me, even though they had no idea what I was doing.
Finally, I would like to thank my parents for their constant support throughout
the decade of my QUT studies. I would not have been able to achieve all that I
have without their moral and financial support. I would like to dedicate this
thesis to my late mother: Thank you for everything, Mama.
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1
CHAPTER 1
LITERATURE REVIEW
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1.1 AN OVERVIEW OF LACTIC ACID BACTERIA
Lactic acid bacteria (LAB) are comprised of a diverse group of Gram-positive
bacteria. They are all catalase negative and they can range from being
aerophilic, aerotolerant to strict anaerobes. They occur as rods or cocci, and are
generally non-motile and non-spore-forming (with the exception of the genus
Sporolactobacillus) (Salk, 1973; Wood, 1992). Most importantly, all LAB
produce lactic acid as the sole or major product from fermentation of sugars,
therefore based upon this, they fall into two large groups: homofermentators and
heterofermentators. Homofermentors metabolise sugar via glycolysis (Embden-
Meyerhof-Parnas pathway). This results almost exclusively in lactic acid as the
end product under standard conditions, and the metabolism is referred to as
homolactic fermentation. Heterofermentors metabolise sugar via the 6-
phosphogluconate/phosphoketolase pathway, resulting in significant amounts of
other end products such as ethanol, acetic acid and carbon dioxide in addition to
lactic acid, and the metabolism is referred to as heterolactic fermentation
(Madigan et al., 2000; Salminen et al., 2004).
The classification of LAB into different genera is traditionally based on
morphology, mode of glucose fermentation, growth at different temperatures,
pH requirement, configuration of the lactic acid produced, ability to grow at
high salt concentrations, and acid or alkaline tolerance. Chemotaxonomic
markers, such as fatty acid composition and constituents of the cell wall are also
used in classification. With the advent of nucleotide sequencing, classification
based upon the sequence data of 16S and 23S ribosomal RNA (rRNA) and the
G+C content (i.e. the percentage moles of guanine plus cytosine content in the
genomic DNA) is the currently the most suitable approach.
Based on 16S and 23S rRNA sequence data, the Gram-positive bacteria form
two lines of descent (Figure 1.1). One phylum consists of Gram-positive
bacteria with a DNA base composition of less than 50 mol% G+C (the
Clostridium branch), whereas the other branch (Actinomyces) comprises
organisms with a G+C content that is higher than 50 mol%. The typical LAB
are of the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus,
Leuconostoc, Pediococcus, and Streptococcus (Figures 1.1 and 1.2). However,
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organisms such as those belonging to the genera Listeria and Staphylococcus,
ferment sugars with the production of lactic acid and are closely related to LAB
by 16S rRNA sequences, except they are catalase positive. Originally, the
genus Bifodobacterium was considered to be a member of the LAB, but based
on the high DNA G+C content and from 16S rRNA data it is now quite clear
that bifidobacteria belong to the actinomyces branch.
Lactobacillus, Leuconostoc and Pediococcus are traditionally treated separately
because of their different morphology and/or fermentation patterns. However,
phylogenetically they are intermixed (Figure 1.1). Based on 16S rRNA studies,
the genus Lactobacillus and other related genera were subdivided into three
groups: Leuconostoc, Lactobacillus delbrueckii and Lactobacillus casei-
Pediococcus (Collins et al., 1991). The Leuconostoc group is composed of all
members of the genus Leuconostoc and obligately heterofermentative
lactobacilli. The L. delbrueckii group comprises mostly of obligately
homofermentative lactobacilli. The L. casei-Pediococcus group is the largest of
the three subgroups and most of the members are facultatively
heterofermentative.
Lactococcus has its own phylogenetic cluster within the Clostridium branch
(Figure 1.1; Stackebrandt and Teuber, 1988). It was originally included in the
Streptococcus genus, but genetic evidence based on DNA-DNA and DNA-RNA
relatedness, clearly indicated that the lactic acid streptococci are a separate
species (Jarvis and Jarvis, 1981; Kilpper-Balz et al., 1982). Lactococcus was
conferred genus status and now accommodates non-motile, mesophilic
streptococci carrying a group N antigen (Schleifer and Kilpper-Balz, 1987).
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ClostridiumActinomyces ClostridiumActinomyces
Figure 1.1. Phylogenetic tree of Gram-positive bacteria.
The bar indicates 100% expected sequence divergence (Schleifer and Ludwig,
1995).
Figure 1.2. Phylogenetic tree of LAB and related bacteria.
The bar indicates 100% expected sequence divergence (Schleifer and Ludwig,
1995).
5
1.1.1 Genomics of lactic acid bacteria
The first lactic acid bacterium genome to be completely sequenced was
Lactococcus lactis subsp. lactis IL1403, a laboratory strain (Bolotin et al.,
2001). Since then, the complete genome sequences of several LAB species
have been published: Lactobacillus plantarum (Kleerebezem et al., 2003),
Lactobacillus johnsonii (Pridmore et al., 2004), Lactobacillus acidophilus
(Altermann et al., 2005), Lactobacillus sakei (Chaillou et al., 2005),
Lactobacillus bulgaricus (van de Guchte et al., 2006), Lactobacillus salivarius
(Claesson et al., 2006), Streptococcus thermophilus (Bolotin et al., 2004),
Lactobacillus gasseri, Lactobacillus brevis, L. casei, L. delbrueckii subsp.
bulgaricus, Leuconostoc mesenteroides, Oenococcus oenii, Pediococcus
pentosaceus (Makarova et al., 2006). In addition, the genome of two L. lactis
subsp. cremoris strains have also been published: L. lactis subsp. cremoris
MG1363, the model lactic acid bacterium (Wegmann et al., 2007), and L. lactis
subsp. cremoris SK11, the phage-resistant strain used commercially in cheese
fermentation (Makarova et al., 2006). In general, LAB genomes are small (1.8
to 3.3-Mbps), compared to Escherichia coli (approximately 5.5-Mbps; Hayashi
et al., 2001) and Bacillus subtilis (4.2-Mbps; Kunst et al., 1997).
Analysis of the genome sequences of all LAB revealed a central trend of loss of
ancestral genes and metabolic simplification (Makarova et al., 2006; Wegmann
et al., 2007). The number of predicted protein-coding genes range between
approximately 1,700 (O. oenii) to over 2,700 (L. casei), and all LAB genomes
include pseudogenes, and varying in numbers from 17 (L. mesenteroides) to
over 200 pseudogenes (S. thermophilus) (Makarova et al., 2006). Differences
can be found even within the same LAB species, as observed by comparison of
the three L. lactis strains. The genome of L. lactis subsp. cremoris MG1363 is
160-kbps and 90-kbps larger than L. lactis subsp. lactis IL1403 and L. lactis
subsp. cremoris SK11, respectively (Wegmann et al., 2007). The larger genome
means L. lactis subsp. cremoris MG1363 has 465 and 346 genes that are not
present in L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris SK11,
respectively (Wegmann et al., 2007). Forty-seven of these genes present in L.
lactis subsp. cremoris MG1363 and not in L. lactis subsp. lactis IL1403 were
characterised as involved in carbohydrate metabolism and transport. This is
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reflected in the greater ability of L. lactis subsp. cremoris MG1363 to
metabolise plant-derived sugars (Wegmann et al., 2007)
1.1.2 Applications
LAB are generally used in food production. Food-grade LAB are mostly from
the genera Lactobacillus and Lactococcus and are considered non-pathogenic.
They are often used as starter cultures in the fermentation of food, such as
cheese and yoghurt production, fermented meat products, fermented vegetables
(such as kimchi and sauerkraut), the Japanese rice wine sake, and sourdough.
Some strains of LAB may be selected for use in food preservation due to
naturally producing bacteriocins and other antimicrobial properties. LAB
produce organic acid (lactic, acetic and propionic acid) that causes significant
changes in the pH of the growth environment (sufficient to antagonise many
microorganisms), hydrogen peroxide and fatty acids (Earnshaw, 1992). These
metabolites are used to extend the shelf-life of food and to suppress spoilage
and food-borne pathogens in dairy products (Vandenbergh, 1993; Elmer et al.,
1996; Naidu et al., 1999).
LAB are also used as ‘probiotics.’ The definition of ‘probiotic’ is a “mono- or
mixed culture of live microorganisms which, applied to animal or [hu]man,
affect beneficially the host by improving the properties of the indigenous
microflora” (Havenaar and Huis In’t Veld, 1992). Probiotic LAB predominantly
belong to the genus Lactobacillus. The main mechanisms whereby probiotics
exert protective or therapeutic effects are believed to be caused by bacterial
interference, in which the presence of a microorganism limits the pathogenic
potential of another (Mcfarlane and Cummings, 1999; Sanders, 1999). The
most frequently used bacteria with well documented clinical effects are
Lactobacillus rhamnosus GG (used exclusively in probiotic yoghurts
manufactured by Parmalat®), L. acidophilus, L. casei Shirota strain (used
exclusively in the fermented milk drink, Yakult®), Lactobacillus reuteri
(Biogaia®) and L. johnsonii (used in probiotic yoghurts manufactured by
Nestlé®) (Alvarez-Olmos and Oberhelman, 2001). Lactobacillus-based
probiotics have long been used for antibiotic-associated diarrhoea. L. rhamnosus
GG is by far the most extensively studied probiotic organism in adults and
7
children. It has been reported to delay the first onset of pouchitis (complication
from proctocolectomy surgery) (Gosselink et al., 2004), alleviate the length and
intensity of antibiotic-associated diarrhoea in adults (Siitonen et al., 1999) and
children (Arvola et al., 1999; Vanderhoof et al., 1999), promote recovery from
rotaviral diarrhoea in children (Majamaa et al., 1995), and has the potential to
treat Clostridium difficile-associated diarrhoea (Gorbach et al., 1978; Biller et
al., 1995).
Some LAB species can be isolated from the mucosal surfaces of humans, such
as the oral cavity, gastrointestinal and urogenital tracts, as part of the natural
microflora. Lactobacilli are generally commensal and largely found in the
gastrointestinal and urogenital tracts, though in small numbers in the oral cavity
due to its poor ability to adhere to oral tissues (de Vrese and Schrezenmeir,
2008). L. casei and L. acidophilus are the most common lactobacilli isolated
from the gastrointestinal and urogenital tracts, and Lactobacillus fermentum is
most commonly found in the mouth and faeces. Lactococcus are non-
colonising, whilst Streptococcus colonises the mucous membranes of the mouth,
throat and respiratory and urogenital tracts, and to a lesser extent the
gastrointestinal tract. The genus Streptococcus also includes several species
that are important pathogens in humans. In addition to the highly virulent
species, such as Streptococcus pyogenes, Streptococcus pneumoniae and
Streptococcus agalactiae, many of the oral streptococci are capable of acting as
opportunistic pathogens under appropriate conditions (Hardie and Whiley,
1995). Streptococcus mutans is one of the most frequently isolated oral
streptococci and historically occupies a central position in the pathogenesis of
dental caries (Clarke, 1924; Loesche, 1986). Streptococcus milleri is a
commensal of the mouth, the gastrointestinal tract and the vagina (Bannantyne
and Randal, 1977), but has been associated with flexor sheath infection of the
hand (Lunn et al., 2001) and abscesses of the liver, brain and joints (Gossling,
1988; Rouff, 1988).
Due to their generally regarded as safe (GRAS) status, certain LAB are
considered ideal candidates either to be utilised as heterologous protein factories
(Nouaille et al., 2003), as potential live recombinant mucosal vaccines, delivery
8
vehicle for vaccines (Wells and Mercenier, 2008), and co-cultivation in food
products to suppress/prevent growth of food-borne pathogens (Cavadini et al.,
1998; Turner et al., 2007ba). As such, there have been a number of studies
conducted on protein expression and secretion systems in LAB, particularly in
lactobacilli and lactococci. Table 1.1 represents a non-exhaustive list of
heterologous proteins produced in LAB. Some of the heterologous proteins are
vaccine candidates, such as bovine corona virus epitope and the human
immunodefiency virus (HIV) envelope protein, whilst others are bacteriocins,
anti-bacterial peptides or proteins which inhibit the growth of or kill different
bacteria (more details provided in section 1.2.3).
Of the numerous LAB species with GRAS status, L. lactis has emerged as the
model bacterium for use in the production and secretion of therapeutic or
vaccine proteins (Le Loir et al., 2005). For this reason, L. lactis has been
extensively studied for the past two decades: its metabolism is relatively simple
and the genome of the international prototype for LAB genetics, L. lactis subsp.
cremoris MG1363, has been sequenced (Wegmann et al., 2007). L. lactis
provides several advantages in protein production and secretion. It only secretes
one major protein, Usp45, thus simplifying downstream purification processes
(van Asseldonk et al., 1993). The fermentation process of L. lactis can be easily
scaled up without the use of sophisticated equipment (Mierau et al., 2005a;
Mierau et al., 2005b). Numerous tools have also been developed for the
expression and secretion of heterologous proteins in the L. lactis MG1363
strain, such as inducible promoters, modified secretion signal sequences (Dieye
et al., 2001; Ravn et al., 2003), inactivation of proteases or the supplementation
with heterologous secretion machinery (Nouaille et al., 2006). Many of the
heterologous proteins produced in LAB listed in Table 1.1 were produced in L.
lactis or using tools originally developed in L. lactis. A few of these tools are
briefly described here.
The most extensively studied inducible expression system for LAB is the nisin-
controlled expression system (NICE; de Ruyter et al., 1996), where the nisA
promoter is induced by the antibacterial peptide nisin in a L. lactis host strain
that has been modified to abolish nisin production. A more recently described
9
inducible expression system utilised the pstF promoter of L. lactis subsp.
cremoris MG1363 to produce heterologous proteins comparable to the NICE
system (Siren et al., 2008). The cells are cultivated until they have consumed
phosphate in the growth medium to a concentration that induces the pstF
promoter. This new inducible promoter has the advantage that no inducing
agents need to be added and is functional without the need for modification of
the host strain. Another of these tools involves the inactivation of proteases. A
L. lactis strain was constructed deficient in the intracellular ClpP and
extracellular HtrA proteases, thus allowing heterologous proteins to be secreted
without degradation by host proteases (Cortes-Perez et al., 2006). In addition to
increase protein secretion level and stability, the L. lactis clpP-htrA mutant
strain also showed greater tolerance to high temperature stress and ethanol
resistance, and higher viability. The final tool described here involves the
supplementation of the L. lactis secretion machinery with components from
other bacteria. Supplementation of L. lactis with B. subtilis SecDF, a
component of the B. subtilis secretion machinery required for high-capacity
protein secretion, showed an increase in the levels of recombinant proteins
secreted (Nouaille et al., 2006).
1.1.3 Extracellular proteins of Gram-positive bacteria
Proteins that are expressed by LAB may be targeted to three different
subcellular locations including the cytoplasm, the extracellular environment or
the cell surface. As such, identification of signals which target secreted proteins
to the extracellular environment and signals which anchor proteins to cell
surface have been utilised to target heterologous proteins in LAB. To produce a
protein of interest, secretion is generally preferred to cytoplasmic production
because it allows continuous culture and simplifies purification. To use
lactobacilli or lactococci as a protein delivery vehicle, e.g. in the digestive tract
of humans or animals, secretion is also preferable because it facilitates
interaction between the protein and its environment, and does not rely on cell
lysis for the interaction. The following is a review on the mechanisms that
Gram-positive bacteria use to secrete and anchor proteins to the cell surfaces.
10
Table 1.1. Non-exhaustive list of heterologous proteins produced in LAB.
Proteins Expressing bacteria Reference Antigens Bovine corona virus (BCV) epitope L. lactis Langella and Le Loir, 1999 Bovine non-structural protein 4 (NSP4) L. lactis Enouf et al., 2001 Brucella abortus antigen L7/L12 L. lactis Ribeiro et al., 2002 Chlamydial antigen OmpA L. reuteri Turner and Giffard, 1998 HIV Env protein L. reuteri Turner and Giffard, 1998 Human cystic fibrosis transmembrane conductance regulator (CFTR) protein
L. reuteri Turner et al., 2003
Human E-cadherin L. lactis, L. reuteri, L. rhamnosus Turner et al., 2004a Human papillomavirus E7 L. lactis Bermudez-Humaran et al., 2002 Porcine parvovirus VP2 L. casei Xu and Li, 2008 scFv (against Human IgE) L. johnsonii Scheppler et al., 2005 TTFC L. lactis Wells et al., 1993 Bacteriocins Acidocin A L. casei Kanatani et al., 1995
Acidocin B L. plantarum Van der Vossen et al., 1994
Carnobacteriocin B2 L. sakei Axelsson et al., 1998
Colicin V L. lactis Divergicin A Leuconostoc gelidum
Van Belkum et al., 1997
Van Belkum et al., 1997
Enterocin A Enterococcus faecalis O’Keeffe et al., 1999
Helveticin J L. johnsonii Fremaux and Klaenhammer, 1994
Hiracin JM79 L. lactis, L. sakei, E. faecalis, Enterococcus faecium
Sanchez et al., 2008
Lactacin 3147 E. faecalis Ryan et al., 1999 Lactacin F L. gelidum Allison et al., 1995
Lactococcin A L. gelidum Leucocin A L. lactis
van Belkum and Stiles, 1995
11
Lysostaphin L. lactis, L. reuteri, L. rhamnosus, L. plantarum Turner et al., 2007b L. johnsonii Fremaux and Klaenhammer, 1993 Mesentericin Y105 Lactococcus cremoris Biet et al., 1998
L. lactis Chikindas et al., 1995
L. sakei Axelsson et al., 1998 Pediocin PA-1
E. faecalis, S. thermophilus Coderre and Somkuti, 1999
Listeria monocytogenes bacteriophage endolysin, Ply511
L. lactis, L. reuteri, L. rhamnosus, L. plantarum Turner et al., 2007b
Cell wall anchor S. aureus Protein A L. lactis Steidler et al., 1998a Enzymes L. delbrueckii cell surface proteinase PrtB L. lactis Germond et al., 2003
Staphylococcal nuclease (NucT) L. lactis, Streptococcus salivarus Le Loir et al., 1994 Immune modifiers/adjuvant Cholera toxin B subunit Lactobacillus paracasei, L. plantarum Slos et al., 1998 Human Interferon-beta 1b L. lactis Zhuang et al., 2008 Murine IL-2 L. lactis Murine IL-6 L. lactis
Steidler et al., 1998b Steidler et al., 1998b
Murine IL-10 L. lactis Schotte et al., 2000 Murine IL-12 L. lactis Bermudez-Humaran et al., 2003 Miscellaneous protein Brazzein (sweet-tasting) L. lactis Berlec et al., 2008
12
In LAB, like in other bacteria, proteins destined for translocation across the
cytoplasmic membrane contain a signal peptide (Blobel, 1980) that is generally
composed of a core of 15 to 20 hydrophobic residues flanked at the N-terminal
end by positively charged residues (Emr et al., 1980; Silhavy et al., 1983). For
secreted proteins, signal peptides are proteolytically removed by signal
peptidases upon translocation across the cytoplasmic membrane (Dev and Ray,
1990; Dalbey et al., 1997). Sometimes secreted proteins require subsequent
folding and maturation steps to acquire their active conformation (Pugsley and
Possot, 1993).
Signal peptides are necessary and sufficient for protein translocation across
membranes if the fused polypeptide substrate can be maintained in an export
competent state, a function that can be achieved in one of two separate pathways
(de Gier et al., 1997, Valent et al., 1998). In one pathway, a signal peptide
recognition protein can bind to the signal peptides of nascent chains and
temporally arrest their ribosomal translation (Walter and Blobel, 1980; Walter
and Blobel, 1981; Walter et al., 1981). Alternatively, signal peptide-bearing
precursors may be translocated after their synthesis has been completed, i.e. by
a post-translational translocation process.
Translocation of proteins involves different transport systems such as the
general secretory (Sec) pathway or the ATP-binding cassette (ABC) transporters
(Tjalsma et al., 2000). The highly conserved Sec pathway, which represents the
main pathway for protein transport in Gram-positive bacteria, has been studied
extensively in B. subtilis (Figure 1.3). This pathway mediates the translocation
of secretory and membrane proteins through a channel formed by the
membrane-embedded Sec-YEG protein complex, and driven by SecA, a
peripherally bound ATPase, which interacts with its substrate proteins and has
an affinity for Sec-YEG (Campo et al., 2004).
13
Signal peptide
Signal peptidecleavage site
Signal peptidase
Cell membrane Cell wall
Signal peptide
Signal peptidecleavage site
Signal peptidase
Cell membrane Cell wall Figure 1.3. Schematic representation of the B. subtilis protein translocation
pathway.
This pathway mediates the secretion of proteins across the cytoplasmic
membrane. To release the secreted proteins, the signal peptide is cleaved by
signal peptidases, and the mature protein is correctly folded by PrsA. Figure
has been adapted from Yamane et al., 2004.
The majority of extracellular proteins in Gram-positive bacteria are at some
stage anchored to the cell wall either via covalent or non-covalent cell wall
binding domains. Covalent attachment to the cell wall or cytoplasmic
membrane occurs via the carboxy-terminal LPXTG-type or amino-terminal
LXXC sorting signals, respectively (Navarre and Schneewind, 1999). Non-
covalent attachment to the cell wall or cell wall components occurs either via
specific repetitive LysM, choline-binding, or S-layer homology (SLH) domains
(Giffard and Jacques, 1994; Navarre and Schneewind, 1999; Buist et al., 2008)
or via non-specific cationic domains (Turner et al., 1997; Antikainen et al.,
2002). Varying amounts of both covalent and non-covalent surface anchored
proteins may be released into the environment due to cell wall turnover, cell
lysis, or proteolytic events (Buist et al., 1995; Piard et al., 1997; Rojas et al.,
2002; Roos and Jonsson, 2002).
14
1.1.3.1 Covalent attachment of surface proteins
Covalent attachment to the cell wall occurs via the C-terminal LPXTG sequence
motif, where X is any amino acid, followed by a C-terminal hydrophobic
domain and a tail of mostly positively charged residues (Fischetti et al., 1990).
The LPXTG motif is highly conserved within the sorting signals of all known
wall-anchored surface proteins of Gram-positive bacteria. LPXTG-carrying
proteins bind to the peptidoglycan peptide cross-bridge of the Gram-positive
bacteria cell wall. Cleavage has been demonstrated to occur between the
threonine and glycine residues at the LPXTG motif by a sortase (Navarre and
Schneewind, 1994). Examples of surface proteins with LPXTG include PrtP
(casein serine protease) from L. lactis (Vos et al., 1989), PrtP from L. paracasei
(Holck and Naes, 1992), M6 from S. pyogenes (Hollingshead et al., 1986), M
protein from streptococci (Talay et al., 1996), and Protein A (IgG binding
protein) from S. aureus (Uhlen et al., 1984; Shuttleworth et al., 1987).
Covalent attachment of proteins to the cytoplasmic membrane occurs via an N-
terminal LXXC secretion signal, where X is normally a small uncharged amino
acid. Following cleavage of this signal peptide by signal peptidase II, the
cysteine is then attached to a lipid (Hayanashi and Wu, 1990). Examples of
proteins with LXXC signal peptide include ScaA (manganese ion, Mn2+,
transporter) from Streptococcus gordonii PK488 (Kolenbrander et al., 1994;
Kolenbrander et al., 1998), PrtM (membrane-associated lipoprotein) from L.
lactis (Haandrikman et al., 1991) and OppA (oligopeptide transport protein)
from L. lactis (Tynkkynen et al., 1993).
1.1.3.2 Non-covalent attachment of surface proteins
1.1.3.2.1 LysM domains
The LysM domain is the most prominent way for proteins to attach to the cell
wall peptidoglycan in a non-covalent manner (Buist et al., 2008). This binding
has been recently demonstrated to be non-species-specific and the domain may
bind to many Gram-positive bacteria with different peptidoglycan structures
(Steen et al., 2003). The LysM domain occurs most often in cell wall degrading
enzymes where it anchors the catalytic domains to their peptidoglycan
substrates as repetitive sequences. LysM domains are typically found repeated a
15
number of times in the C-termini of non-covalently anchored surface proteins
such as in AcmA from L. lactis (contains three LysM domains) (Buist et al.,
1995), p60 and MurA from Listeria monocytogenes (contains two and four
LysM domains, respectively) (Goebel et al., 1991; Carroll et al., 2003).
1.1.3.2.2 YG repeats or choline-binding domains
Another non-covalent attachment mechanism to the cell wall is via choline-
binding domains, also known as YG repeats (Giffard and Jacques, 1994). The
most studied proteins which contain a choline-binding domain are that of
pneumococcal lytic enzymes (Lopez and Garcia, 2004). Pneumococcal
lipoteichoic or teichoic acids contain choline in their structure, an aminoalcohol
that plays a fundamental biological role in the physiology of pneumococcus
converting choline onto the cell surface of S. pneumoniae (Fischetti et al.,
2000). Pneumococcal bacteriophage endolysins, such as CPL-1 and -9 (Garcia
et al., 1990), and pneumococcal autolysins LytA, B and C, bind to choline
residues of the cell wall. This interaction is strictly required for activity. These
YG repeats are also found in several extracellular proteins of Gram-positive
bacteria such as glucan binding glucansucrases (Shah et al., 2004b) and
glucosyltransferases of oral streptococci (Wren, 1991; Von Eichel-Streiber et
al., 1992; Giffard and Jacques, 1994).
1.1.3.2.3 GW modules
Homologous domains of around 80-90 amino acids (aa) in length which are
present in a number of cell wall associated polypeptides from Gram-positive
bacteria have been shown to mediate cell wall anchoring of lysostaphin from
Staphylococcus simulans (Baba and Schneewind, 1996), autolysin of S. aureus
(Baba and Schneewind, 1998), Ami (amidase) and internalin B (invasion
protein) from L. monocytogenes (Braun et al., 1997) and SpaA (antigen) from
Erysipelothrix rhusiopathiae (Makino et al., 1998). These domains were
termed as GW modules due to the presence of GW dipeptide (Braun et al.,
1997). The cell wall components recognised by the GW modules are not
known, although it has been hypothesised that they may bind to a cell wall
component common to many Gram-positive bacteria, such as teichoic acid
moieties (Braun et al., 1997).
16
1.1.3.2.4 S-layer homology domains
Bacterial surface layers (S-layers) are two-dimensional crystalline arrays
covering the entire cell surface and are one of the most commonly observed
bacterial cell surface structures (Sleytr and Messner, 1988). The cell wall-
targeting mechanism of some S-layer proteins were found to be mediated by 10-
15 conserved amino acids, referred to as the S-layer homology (SLH) domain
(Fujino et al., 1993, Lupas et al., 1994). It has been found that S-layer proteins
lacking the SLH domain did not bind to cell walls in vitro (Olabarria et al.,
1996; Ries et al., 1997). The mechanism for SLH-mediated targeting of surface
proteins was not understood until recently, with the characterisation of the
csaAB operon of Bacillus anthracis (Mesnage et al., 2000). This conserved
operon encodes for the function of cell wall polysaccharide pyruvylation, a
modification that was necessary for the binding of the SLH domain to the cell
wall.
1.1.3.2.5 Unique domain – Sep
L. reuteri BR11 (formally classified as L. fermentum BR11) was isolated by
researchers at the Queensland University of Technology (QUT) from the female
guinea pig urogenital tract (Rush et al., 1994). Examination of supernatant
fractions from broth cultures revealed the presence of a 27 kDa small exported
protein (Sep; Figure 1.4). Sep is a 205 aa protein and contains a 30 aa secretion
signal and has overall homology (between 39 and 92%) with similar sized
proteins of Enterococcus faecium, S. pneumoniae, S. agalactiae and L.
plantarum. The C-terminal 81 aa of Sep showed strong homology to the
aggregating-promoting factor (APF) surface proteins of L. gasseri and L.
johnsonii (Turner et al., 2004a), which have been shown to determine cell shape
(Ventura et al., 2002). Sequence analysis of Sep for cell surface anchoring
domains revealed that it does not contain any typical covalent anchoring signals
such as cell wall-anchoring LXPTG or lipoprotein LXXC signal, but the N-
terminus of the mature protein unusually contain a single LysM domain, thus
making it distinct from APF proteins (Turner et al., 2004a). Within the C-
terminal domain, the presence of a YG motif was identified that was not related
to the LysM domain. LysM and YG domains are both functionally similar in
that they both recognise carbohydrates as ligands (Giffard and Jacques, 1994;
17
Bateman et al., 2000; Buist et al., 2008). Its biological function is unknown and
it is weakly anchored to the cell surface. Sep has also been shown to be useful
as a heterologous peptide fusion partner in L. reuteri BR11, L. rhamnosus GG
and L. lactis MG1363 (Turner et al., 2004a), and has the potential for
heterologous protein expression and export in LAB.
86% homology to C-terminal of APF1 from Lactobacillus johnsonii
Region rich in glutamine amino
acid
Lys M domain
Secretionsignal
YGQ-richLysMSS30 80 124 205
86% homology to C-terminal of APF1 from Lactobacillus johnsonii
Region rich in glutamine amino
acid
Lys M domain
Secretionsignal
YGQ-richLysMSS30 80 124 205
Figure 1.4. Modular structure of Sep.
Numbers above represents the number of amino acids. The YG motif is located
at amino acid position 150 (Y) and 155 (G) in the C-terminal region.
1.1.3.2.6 Non-specific anchored proteins
It is hypothesised that certain positively charged proteins may be anchored by
electrostatic interaction with acidic groups on the bacterial cell surface. One
such protein is the Lactobacillus S-layer protein. Unlike the S-layer proteins
described in section 1.1.3.2.4, S-layer proteins found in Lactobacillus lack the
SLH domain homologues, and the anchoring mechanism to the cell wall
remains uncharacterised (Engelhardt and Peters, 1998; Brechtel and Bahl,
1999). Based upon their genetic sequences, it was predicted that Lactobacillus
S-layer proteins range in size between 43 and 46 kiloDaltons (kDa) with basic
isoelectric points (pI > 9), and sequence variation in the N-terminal region
(Vidgren et al., 1992; Boot et al., 1993; Boot et al., 1995; Callegari et al., 1998;
Sillanpaa et al., 2000). The two well-characterised lactobacilli S-layer proteins
are CbsA of Lactobacillus crispatus (Sillanpaa et al., 2000), and SlpA of L.
brevis (Hynonen et al., 2002). Both these proteins exhibit binding activities to
extracellular matrix (ECM) proteins, with CbsA binding to collagen and SlpA
having affinity for epithelial cells and fibronectin. Furthermore, it was
demonstrated that the lysine-rich, C-terminal region of CbsA was responsible
18
for anchoring the S-layer protein to the cell wall peptidoglycan and this was
hypothesised to be based on electrostatic interactions involving the lysine
residues (Antikainen et al., 2002).
Another surface-located protein is CyuC (previously known as BspA), of L.
reuteri BR11 (Hung et al., 2005; Turner et al., 1997). CyuC is a high-affinity
L-cystine-binding protein, part of an ATP-binding cassette uptake transporter
system encoded by the cyuABC gene cluster (cyu for cystine uptake). CyuC was
found not to contain any lipoprotein cleavage and attachment motif (LXXC),
despite its origin in a Gram-positive bacterium. As the predicted isoelectric
point is 10.59, it was hypothesised that CyuC was anchored by electrostatic
interaction with the cell surface. This hypothesis was supported when CyuC
could be selectively removed from the surface by extraction with an acid buffer
(Turner et al., 1997).
1.1.4 Transposon mutagenesis – tool for genetic analyses
Much of the genetic potential of completely sequenced genomes is poorly
described or undefined. For example, 22% and 36% of the sequenced L. lactis
subsp. lactis IL1403 are of unknown function and poorly characterised,
respectively (Bolotin et al., 2001). The recently sequenced LAB prototype L.
lactis subsp. cremoris MG1363 is significantly larger (additional 160-kb) and is
predicted to encode 530 unique proteins (Wegmann et al., 2007). Therefore, to
take full advantage of functional genomics, it is essential to have efficient
genetic tools for mutagenesis.
In many bacteria, transposition has been a valuable genetic tool to study
chromosomal genes, their functions and regulators. Transposition is the random
non-sequence-specific insertion of a segment of DNA to a new position either
into the chromosome or plasmid. In L. lactis, transposition of the conjugative
elements Tn916 (Romero and Klaenhammer, 1990) and Tn919 (Hill et al.,
1987) have been reported. However, their use is limited by a requirement for
high-efficiency conjugal transfer and site-specific transposition in certain
strains. Maguin et al. (1996) developed an efficient insertional mutagenesis
method by associating the insertion sequence ISS1 (transposable bacterial
19
sequences) with the thermosensitive replicon pGhost. This mutagenic tool,
named pGhost9:ISS1, can be used even in poorly transformable strains, and has
been reported to be transformed in a number of species of on enterococci,
lactobacilli, lactococci, and streptococci (Table 1.2). High frequency
transposition using pGhost9:ISS1 allows efficient gene inactivation and direct
cloning of DNA surrounding the insertion. Efficient excision of the plasmid
replicon by a temperature shift gives rise to a stable food-grade mutant strain,
which doesn’t contain any antibiotic resistant markers. This is achieved when
pGhost9:ISS1 is first transformed in the LAB strain at a permissive temperature,
and transposition is selected for at a higher non-permissive temperature. This
temperature increase selects out plasmid replicating LAB. Replicative
transposition of the ISS1 sequences into the chromosome of LAB leads to the
integration of the plasmid vector. Transposition is thus revealed by selection for
antibiotic-resistant clones able to grow at a temperature restrictive for plasmid
replication.
Table 1.2. Examples of studies that used pGhost9:ISS1 to identify gene functions in LAB. Host species Function studied References
UV resistance Duwat et al., 1997 Secretion of NucT enzyme Nouaille et al., 2004 Biosynthesis of cell wall polysaccharides for bacteriophage adsorption
Dupont et al., 2004
L. lactis
Tellurite and oxidative stress resistance
Turner et al., 2007a
Branched-chain amino acid biosynthesis pathway for growth in milk
Garault et al., 2000
Phage resistance Lucchini et al., 2000
S. thermophilus
Defence against superoxide stress
Thibessard et al., 2004
S. agalactiae Signal transduction system regulating fibrinogen binding activity
Spellerberg et al., 2002
L. plantarum Regulation of phenolic acid metabolism
Gury et al., 2004
Streptoccus suis serotype 2 Exonuclease with cell-wall anchoring motif
Fontaine et al., 2004
20
1.2 AN OVERVIEW OF ANTIMICROBIAL PROTEINS
Antibiotic-resistant pathogens pose an enormous threat to the effective
treatment of a wide range of serious infections. Currently, some of greatest
causes for concern are infections by strains of S. aureus, enterococci and
pneumococci, displaying acquired resistance to six or more antibiotics. The
problem of antibiotic resistance has been compounded by the development of
many broad-spectrum antibiotics, whereas the patient population might be
served better by more selective medicines with activity restricted against small
groups of pathogens.
One option to combat infections caused by antibiotic-resistant bacteria is
vaccination. However, vaccines usually only elicit a systemic immune response
that may not, for example, efficiently reduce the mucosal carriage of the
pathogen (Mbelle et al., 1999; Veehoven et al., 2004). Other infectious disease
control methods for which resistance is rare and which are able to block the
initial entry into the host via mucosal sites are currently being investigated. One
such method is the use of antimicrobial proteins, such as attachment blocking
proteins, bacteriophage endolysins and bacteriocins.
1.2.1 Attachment blocking proteins
The identification of pathogen adhesins and host receptors has led to the
development of a new type of antimicrobial agent which blocks the initial stage
of infection, host attachment. Anti-adhesive strategies aimed at blocking this
interaction offer a means of preventing infection at an early stage. Three classes
of adhesion-blocking agent have been investigated: anti-adhesin antibodies,
adhesin analogues or receptor analogues (Kelly and Younson, 2000; Kelly et al.,
2001).
1.2.1.1 Anti-adhesin antibodies
This form of treatment involves passive immunisation of mucosal surfaces with
an anti-adhesin antibody. The uses of monoclonal antibodies (MAb) that
specifically target microbial adhesins are a means of enhancing the effectiveness
of this form of treatment. MAb were established to Helicobacter pylori and
inhibited adhesion to human cancer cell-line MKN45 (Osaki et al., 1998).
21
Adhesion-blocking MAb was also shown to be effective in protecting against
the re-colonisation of Porphyromonas gingivalis (possible cause of
periodontitis) in human trials (Booth et al., 1996).
However, the half-life of these molecules demands continuous administration
and raises the problems of bioavailability, safety and cost. This may be
overcome with the in situ delivery of passive immunity by non-pathogenic
bacteria producing anti-adhesin antibodies. This was demonstrated when a
single-chain fragment variable (scFv) antibody expressed by Lactobacillus zeae,
which was specific for the S. mutans surface antigen I/II, was able to reduce the
colonisation of the oral cavity by S. mutans and the development of dental caries
in rat models (Kruger et al., 2002). More recently, a L. casei strain was used to
secrete a scFv antibody specific for intercellular adhesion molecule 1, which
was able to block cell-associated HIV-1 transmission across an in vitro culture
model of the cervical epithelium (Chancey et al., 2006)
1.2.1.2 Adhesin analogues
Soluble forms of a microbial adhesin (or a fragment of it) may be used as
competitive inhibitors to block adhesion. A synthetic peptide corresponding to
residues 1025-1044 of SA I/II, which inhibited adhesion in vitro of S. mutans to
a salivary receptor (Kelly et al., 1995), was also shown to prevent infection in a
human clinical trial using a re-colonisation model (Kelly et al., 1999).
Other adhesin analogues may be found from another source, such as LAB.
Many commensal LAB have surface proteins that enable adhesion to host ECM
proteins just like their pathogenic relatives. It has been demonstrated that the
surface proteins of L. reuteri RC-14 was able to competitively inhibit adhesion
of E. faecalis to the surface of plastic (Heinemann et al., 2000) and of S. aureus
to surgical implants in mice (Gan et al., 2002).
1.2.1.3 Host-receptor analogues
Treatments using host-receptor analogues involve the use of analogues which
bind to microbial adhesins, thereby reducing the amount of microbial adhesins
available to bind to the real host-receptors. Many studies have investigated
22
soluble carbohydrate receptor analogues reflecting the frequency with which
these structures are recognised by microbial adhesins. Adhesion of S.
pneumoniae to human cell lines and to primary epithelial cells in vitro was
inhibited by sialylated oligosaccharides (Barthelson et al., 1998).
1.2.2 Bacteriophage endolysins
Near the end of the bacteriophage lytic cycle, the virus needs to coordinate
bacterial host lysis with the completion of viral assembly. For double-stranded
DNA bacteriophages, this is done by the production of a protein lytic system
consisting of a holin and lysin. The holin forms a pore in the bacterial
cytoplasmic membrane allowing the endolysin or lysin to gain access to the cell
wall. Degradation of the cell wall causes bacterial lysis by osmotic pressure and
therefore the release of progeny phage. Endolysins have particularly narrow
substrate specificities with generally only either intra-species or –genus
bacteriolytic activity. Table 1.3 lists examples of functional Gram-positive
bacteriophage endolysins with their specificity and the optimal pH for activity.
Endolysins from phages of Gram-positive hosts are able to quickly cause cell
lysis of the target bacteria when added exogenously (Loessner et al., 1995b).
This “lysis from without activity” is limited to Gram-positive bacteria, since
Gram-negative bacteria have an outer membrane. Although this activity has
been known for some time since the 1970’s (generally used to recover DNA,
RNA and proteins from cells) (Loessner et al., 1995b), it is surprising that
endolysins had not been investigated as bacterial control agents until the 21st
century. In the first study conducted this century, it was shown that 10ng of
purified endolysin from the streptococcal bacteriophage C1 was able to rapidly
kill 106 S. pyogenes in seconds (Nelson et al., 2001). The same killing effect
could also be observed in vivo with one oral endolysin treatment being sufficient
to eliminate S. pyogenes from mice with a heavily colonised oral pharynx. It
was also shown that the endolysin was only lethal to Group A, C and E
streptococci with no effects on other streptococci or other bacteria. This line of
research was extended to the treatment of S. pneumoniae infections using the
pneumococcal Dp-1 bacteriophage endolysin Pa1 (Loeffler et al., 2001). The
lethality of this enzyme was demonstrated on a range of pneumococci including
highly penicillin-resistant strains but was shown to be ineffective in killing oral
23
streptococcal strains, including S. mutans. Mice colonised intranasally with S.
pneumoniae revealed undetectable pneumococcal colony forming units five
hours after a single Pal treatment. Most recently the PlyG endolysin from the γ
bacteriophage from B. anthracis was shown to be specific in the killing of
members of the B. anthracis cluster of bacilli (Schuch et al., 2002). The PlyG
endolysin was able to rescue 77% of mice from what would be a lethal dose of
the test Bacillus cereus strain injected intraperitoneally.
As yet, no resistance has been observed for S. pyogenes, pneumococci or B.
cereus treated with varying amounts of endolysin. Even when B. cereus was
subjected to mutagenesis with methane sulphonic acid ethyl ester, which
increased the number of spontaneous antibiotic-resistant mutants, no endolysin-
resistant mutants were observed (Schuch et al., 2002). Spontaneous B. cereus
mutants resistant to γ bacteriophage are still sensitive to PlyG endolysin.
During the S. pyogenes and pneumococcal studies there was a rebound in
positive cultures for a few animals 1-2 days following endolysin treatment,
however none of these isolates were endolysin resistant. This suggests that
repeated administration of the endolysin treatment or a method whereby the
persistence of the endolysin at the mucosal surface can be increased may be
required for optimal effectiveness.
Endolysins may also have a potential as novel agents for the control of
foodborne pathogens such as L. monocytogenes (Loessner et al., 1995a) and
Clostridium perfringens (Zimmer et al., 2002) in human and animal foodstuffs.
The highly specific action of the endolysin, Ply3626, on C. perfringens forms
the basis for the potential applications of this enzyme, particularly as an
antimicrobial additive in poultry intestines and a biopreservative in raw chicken
or turkey (Zimmer et al., 2002). The use of lytic bacteriophages to reduce L.
monocytogenes on fruits has already been demonstrated (Leverentz et al., 2003).
In addition, the ability of the L. monocytogenes bacteriophage endolysin Ply511
to be cloned, expressed and secreted in L. lactis and other LAB (Gaeng et al.,
2000; Turner et al., 2007b) suggests the potential for this endolysin to be
utilised in fermented food products, such as dairy, meat and vegetables, the
24
contamination of which has been linked with human listeriosis (Farber and
Peterkin, 1991; Ryser and Marth, 1999).
1.2.2.1 Structure of endolysins
Most endolysins lack a secretory signal sequence and thus the holin, which
permeabilises the membrane, is required for the endolysin to gain access to the
peptidoglycan (Young et al., 2000). Depending on the peptidoglycan bond
which they hydrolyse, endolysins can be further grouped into N-
acetylmuramidases (lysozymes) which act on the carbohydrate components, N-
acetylmuramyl-L-alamidases which cleave the bond between the carbohydrate
and peptide components, endopeptidases which cleave the peptide interbridge,
or transglycosylases which cleave the glycosidic bond in a glycan strand of
bacterial cell wall (Young, 1992).
Little information has been published about the molecular structure and
mechanisms of functional endolysins. The focus has largely been on the holin
component of the bacteriophage lytic cassette as it controls the timing of cellular
lysis (Wang et al., 2000). It has been proposed that endolysins are modular
enzymes consisting of a catalytically active domain and a cell wall binding
domain. However, this hypothesis is mostly based upon sequence homologies,
and only recently experimental data has demonstrated which parts of the
endolysin contain the enzymatic activity and the cell wall binding capacity. In
most cases, the catalytic domains are located at the N-terminus and the cell-wall
binding domain is located at the C-terminus of endolysins.
Endolysins are typically between 30 and 60-kDa in size and sometimes function
as dimers (Grundling et al., 2000). The pH optimal for endolysin activity is
normally acidic (see Table 1.3), except for Listeria endolysins (pH 8.0-9.0)
(Loessner et al., 2002). Although most endolysins don’t contain secretion
signals, some like the L. plantarum phage g1e endolysin Lysg1e does. The N-
terminal region of Lysg1e, which is thought to be the catalytic domain of
endolysins in general, consists of a signal-peptide-like domain and a domain the
putative active sites of endolysin (Kakikawa et al., 2002).
25
1.2.3 Bacteriocins
Bacteriocins are anti-bacterial peptides or proteins ribosomally synthesised by
bacteria which either inhibit the growth or kill different bacteria. These toxins
have been found in all major lineages of bacteria, and it has been suggested that
99% of all bacteria may make at least one bacteriocin (Klaenhammer, 1988).
Bacteriocins can range from 19 to greater than 270 amino acids and vary in
action from forming holes in the cytoplasmic membrane to enzymatically
degrading the cell wall peptidoglycan.
Bacteriocins of Gram-positive bacteria differ from Gram-negative bacteria in
two fundamental ways: (i) bacteriocin production is not necessarily the lethal
event it is for Gram-negative bacteria; and (ii) Gram-positive bacteria have
evolved bacteriocin-specific regulation, whereas bacteriocins of Gram-negative
bacteria rely solely on host regulatory networks. The non-lethality of
bacteriocin production in Gram-positive bacteria is due to the transport
mechanisms Gram-positive bacteria encode to release the bacteriocin. Some
have evolved a bacteriocin-specific transport system, while others employ the
sec-dependent export pathway. From this point, only Gram-positive
bacteriocins will be discussed. Table 1.4 lists examples of Gram-positive
bacteriocins according to their class type.
26
Table 1.3. Non-exhaustive list of functional bacteriophage endolysins.
Lysin name Phage strain Host bacteria Optimum pH Specificity References C2(W) L. lactis 6.5-6.9 Group N and D lactic
streptococci Mullan and Crawford, 1985
Cp1-1 Cp-1 ND Garcia et al., 1987 Pa1 Dp-1
S. pneumoniae 8.0
Pneumococcal strains Sheehan et al., 1997
LysA L. reuteri ND L. fermentum, L. rhamnosus, L. casei, L. plantarum, Lactobacillus jensenii, L. delbrueckii subsp. lactis, L. lactis subsp. cremoris, S. pyogenes, S. agalactiae, S. aureus
Turner et al., 2004b
Lysg1e g1e L. plantarum L. gasseri, L. plantarum LysgaY gaY L. gasseri
ND B. subtilis, E.nterococcus hirae, L. casei, L. gasseri, L. plantarum, Lactococcus diacetylactis, L. lactis, L. mesenteroides, Micrococcus luteus, P. pentosaceus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hycus, S. simulans, Staphylococcus warneri, Staphylococcus xylosus
Yokoi et al., 2004
LytA US3 L. lactis ND 30 different lactococcal strains Platteeuw and de Vos, 1992
Mur LL-H L. delbrueckii 5.0 L. acidophilus, L. delbrueckii, Lactobacillus. helveticus, Pediococcus damnosus
Vasala et al., 1995
27
Mur-LH 0303 L. helveticus ND Thermophilic lactobacilli, lactococci, pediococci, B. subtilis, Brevibacterium linens, E. faecium
Deutsch et al., 2004
Ply118 A118 L. monocytogenes 8-9 Listeria species Loessner et al., 1995a Ply12 12826 B. cereus ND Bacillus species Loessner et al., 1997 Ply187 S. aureus phage
187 M. luteus ND Staphylococcus species Ashehov and Jevons, 1963
Ply21 TP21 B. cereus ND Bacillus species Loessner et al., 1997 Ply3626 3626 C. perfringens ND C. perfringens Zimmer et al., 2002 Ply500 A500 Ply511 A511
L. Monocytogenes 8-9 Listeria species Loessner et al., 1995a
PlyBa Bastille B. cereus ND Bacillus species Loessner et al., 1997 PlyGBS B30 S. agalactiae 5.5=6.0 Groups A, C, E and G
streptococci Pritchard et al., 2004
PlyV12 1 E. faecalis 6.0 E. faecalis, E. faecium Yoong et al., 2004 ND: Not determined
28
It is generally found that the killing range of bacteriocins is restricted to other
Gram-positive bacteria. The range of killing can vary significantly; from
relatively narrow (e.g. lactococcins A, B and M only kill Lactococcus) (Ross et
al., 1999) to extraordinary broad (e.g. nisin A have been shown to kill a wide
range of organisms including at least thirteen different genera) (Mota-Meira et
al., 2000). As an exception to the general rule, nisin A is also active against a
number of medically important Gram-negative bacteria, including
Campylobacter, Haemophilus, Helicobacter and Neisseria (Mota-Meira et al.,
2000). Bacteriocin genes are usually associated with a gene encoding the
‘immunity’ protein. This immunity protein protects bacteria from their own
bacteriocins, however the mechanism by which it does this remains unclear
(Hechard and Sahl, 2002).
Bacteriocins classification has been proposed on the basis of the primary
structures of bacteriocins produced by LAB (Klaenhammer, 1993; Nes et al.,
1996) (Table 1.4). Class I is composed of modified peptides, named
lantibiotics. Class II comprise of heat stable unmodified peptides. Class III
consists of larger heat labile proteins. Class I and II bacteriocins are the most
abundant and thoroughly studied. However, the state of bacteriocin
classification requires constant review as the knowledge concerning various
aspects of bacteriocin research rapidly accumulates and it appears that the term
bacteriocin has been used to cover a wide range of chemically diverse
substances which do not necessarily have much in common (Ennahar et al.,
2000).
1.2.3.1 Modified bacteriocins (class I)
A substantial proportion of the peptide bacteriocins of Gram-positive bacteria
undergo extensive post-translational modification before they are exported from
the cell (class I bacteriocins). These modified bacteriocins that contain non-
standard amino acids (e.g. hydroxyproline and selenomethionine), and are
therefore, given the designation ‘lantibiotics’ as an abbreviation for lanthionine-
containing peptide antibiotics.
29
Peptides classed as lantibiotics have a minimum of 19 and maximum of 38
amino acids. Lantibiotics are then further categorised into type-A and type-B
peptides (Jung, 1991). These classifications are based on structural and
functional aspects and take into account the fact that some lantibiotics are
elongated, flexible amphiphiles that form pores in bacterial membranes (i.e.
type-A), while others are globular, conformationally defined peptides that
inhibit enzyme functions (i.e. type-B).
1.2.3.2 Unmodified bacteriocins (class II)
Class II bacteriocins are unmodified, cationic and hydrophobic peptides of 20-
60 amino acids in length (Nes and Holo, 2000). They are divided into three
subclasses, IIa, IIb and IIc, on the basis of their primary structure. Their activity
mainly induces membrane permeabilisation and leakage of molecules from
sensitive bacteria. The inhibition spectrum is rather narrow, limited to species
or strains related to the producers. Accordingly, class II bacteriocins are mainly
active against low G+C gram-positive bacteria, such as lactic acid bacteria,
Listeria, Enterococcus and Clostridium.
The subclass IIa bacteriocins are those which share high similarities in their
primary structure as well as anti-listerial activity (Ennahar et al., 2000).
Subclass IIb includes bacteriocins whose activity depends on the
complementary action of two distinct peptides; therefore individual peptides
hardly display any activity. It is proposed that subclass IIc bacteriocins should
include miscellanous peptides with no structural similarity to subclass IIa or IIb
(Hechard and Sahl, 2002).
1.2.3.3 Large heat-labile bacteriocins (class III)
Large bacteriocins which cannot be identified as either class I or II are grouped
into this third class, including bacteriocins which function as cell wall degrading
enzymes.
The bacteriolytic activity and molecular structure of lysostaphin has been
thoroughly studied. Lysostaphin is an extracellular bacteriolytic enzyme
produced by a singly known staphylococcal strain, formerly designated as
30
Staphylococcus staphylolyticus (Schindler and Schuhardt, 1964) and now
designated as Staphylococcus simulans biovar staphylolyticus ATCC1362
(Sloan et al., 1982). The cell wall-degrading activity of lysostaphin is due to a
glycylglycine endopeptidase activity, which lyses practically all known
staphylococcal strains (Schindler and Schuhardt, 1964). The target of
lysostaphin is the interpeptide bridge of the peptidoglycan, which in S. aureus,
S. simulans, S. carnosus, and other staphylococcal strains is composed of five
glycine residues (Schleifer and Fischer, 1982). If one or more glycine residues
of the interpeptide bridge are replaced by serine residues, as in S. epidermidis
and S. simulans bv. staphylolyticus, the cell wall is less susceptible to
lysostaphin (Kloos and Schleifer, 1975; Robinson et al., 1979). Lysostaphin is
unable to hydrolyse glycylserine and serylglycine peptide bonds (Robinson et
al., 1979; de Hart et al., 1995). Lysostaphin seems to cleave specifically
between the third and fourth glycine residue of the pentaglycine cross-bridge, as
indicated by the release of LPXTG-containg staphylococcal cell wall bound
surface proteins following lysostaphin treatment. These proteins are covalently
anchored by their C-terminus to the pentaglycine cross-bridge of the
peptidoglycan (Schneewind et al., 1995; see 1.1.3.1).
Analysis of the lysostaphin gene (lss) sequence and the sequencing of the
amino-terminus of purified pro-lysostaphin and of mature lysostaphin revealed
that lysostaphin is organised as a preproprotein of 493 aa, with a signal peptide
of 36 aa, a propeptide of 211 aa from which 195 aa are organised in 15 tandem
repeats of 13 aa length, a mature protein of 246 aa (Thumm and Gotz, 1997;
Figure 1.5). Pro-lysostaphin is processed in the supernatant of S. simulans bv.
staphylolyticus by an extracellular cysteine protease. The mature lysostaphin
has 4.5-fold more activity than its preprotein. Therefore, it is hypothesised that
the tandem repeats of the propeptide are not necessary for protein export or
activation of lysostaphin, but rather keep the enzyme in a less active state
(Thumm and Gotz, 1997). Lysostaphin has optimal activity at pH 7.5 to 8.0
under low ionic conditions.
31
Table 1.4. Non-exhaustive list of Gram-positive bacterial bacteriocins. Class Bacteriocin Producing bacteria Sensitive bacteria References
Epidicin 280 S. epidermidis M. luteus, S. simulans, Staphylococcus carnosus Heidrich et al., 1998 Gallidermin Staphylococcus gallinarum Propionibacterium acne Kellner et al., 1988 Lacticin 481 L. lactis Clostridium tyrobutyricum Piard et al.,1993; Sahl and
Bierbaum, 1998 Lactocin S L. sakei Pediococcus acidilactici Mortvedt et al., 1991 Mutacin B-Ny266 S. mutans Actinomyces sp., Bacillus sp., Clostridium sp.,
Corynebacterium sp., Enterococcus sp., Gardnerella sp., Lactococcus sp., Listeria sp., Micrococcus sp., Mycobacterium sp., Propionibacterium sp., Streptococcus sp., Staphylococcus sp.
Mota-Meira et al., 2000
Nisin A Nisin Z
L. lactis B. cereus, C.. tyrobutyricum, L. lactis subsp. cermoris, L. monocytogenes, Micrococcus flavus, Sp. thermophilus
de Vos et al., 1993; Sahl and Bierbaum, 1998
Pep5 S. epidermidis S. carnosus, S. epidermidis, S. simulans Bierbaum et al., 1994 Salivaricin A Streptococcus salivarius M. luteus Ross et al., 1993 Subtilin B. subtilis Bacillus sp. Michener, 1953; Campbell and
Sniff, 1959
Class I Type-A lantibiotics
Variacin Micrococcus variants B. subtilis, B. cereus, Bacillus pumilis, Clostridium sp., E. faecalis, E. faecium, L. acidophilus, L. bulgaricus, Lactobacillus curvatus, L. delbrueckii, L. helveticus, L. plantarum, L. sakei, L. lactis, L. mesenteroides, Listeria innocua, L. monocytogenes, Listeria welhia, Staphylococcus sp., S. thermophilus
Pridmore et al., 1996
Type-B lantibiotics
Mersacidin Bacillus sp. C. difficile, Clostridium novyi, C. perfringens, Clostridium ramnosum, Clostridium septicum, Corynebacterium jeikeium, peptostreptococci, P. acnes, methicillin-susceptible (MS-) & methicillin-resistant (MR-) S. aureus, MS- & MR-S. epidermidis, S. pyogenes, S. agalactiae, Streptococcus bovis, S. pneumoniae
Niu and Neu, 1991
32
Bavaricin A Lactobacillus bavaricus L. monocytogenes Larsen et al., 1993 Carnobacteriocin B2 Carnobacterium piscicola Carnobacterium divergens, C. piscicola Quadri et al., 1995 Divergicin M35 C. divergens Carnobacteria sp., L. monocytogenes Tahiri et al., 2004 Enterocin A En. faecalis, L. plantarum, L. sakei, L. innocua, L.
monocytogenes, P. acidilactici, P. pentosaceus Aymerich et al., 1996
Enterocin P
E. faecium
S. aureus, L. monocytogenes, Clostridium botulinum, C. perfringens
Cintas et al., 1997
Leucocin A L. gelidium Hastings et al., 1991; Stiles, 1994
Mesentericin Y105 L. mesenteroides
Listeria sp.
Hechard et al., 1992 Mundticin Enterococcus mundtii C. botulinum, L. monocytogenes Bennick et al., 1998 Pediocin AcH C. perfringens, L. monocytogenes, S. aureus Bhunia et al., 1988 Pediocin PA-1
P. acidilactici L. monocytogenes Rodriguez et al., 2002
Piscicocin V1b C. piscicola C. divergens, E. faecalis, L. curvatus, L. plantarum, L. sakei, L. mesenteroides, L. innocua, L. monocytogenes, P. acidilactici
Bhugaloo-Vial et al., 1996
Class II Subclass IIa
Sakacin A Sakacin P
L. sakei C. piscicola, E. faecalis, E. faecium, Lactobacillus alimentarius, L. curvatus, L. sakei, Leuconostoc paramesenteroides, L. monocytogenes
Schillinger and Lucke, 1989; Tichaczek et al.,1994
Acidocin J1132 L. acidophilus L. acidophilus, L. brevis, L. casei, L. fermentum, L. plantarum Tahara et al., 1996 Enterocin 1071 E. faecalis C. tyrobutyricum, Enterococcus durans, E. faecalis, E.
faecalis subsp. liquifaciens, E. faecium, L. salivarius subsp. salivarius, L. innocua, Micrococcus sp., Peptostreptococcus aerogenes, Propionibacterium freudenreichii subsp. shermanii, S. agalactiae
Balla et al., 2000
Lactacin F L. acidophilis E. faecalis, L. delbrueckii subsp. bulgaricus and subsp. lactis, L. fermentum, L. helveticus
Muriana and Klaenhammer, 1991
Plantaricin EF L. casei, L. casei subsp. casei, L. plantarum, L. sakei, Lactobacillus viridescens, P. acidilactici, P. pentosaceus
Subclass IIb
Plantaricin JK
L. plantarum
L. plantarum, L. sakei, L. viridescens, P. Pentosaceus
Anderssen et al., 1998
33
Thermophilin 13 S. thermophilus B. cereus, Bifidobacterium bifidum, C. botulinum, E. faecium, L. acidophilus, L. helveticus, L. fermentum, L. cremoris, L. cremoris, L. mesenteroides, L. monocytogenes, S. thermophilus
Marciset et al., 1997
Lactococcin A L. lactis subsp. cremoris and subsp. lactis biovar diacetylactis
L. lactis subsp. cremoris, L. lactis subsp. lactis (bv. diacetylactis), Lactococcus raffinolactis, Lactococcus garvieae
Holo et al., 1991 Subclass IIc
Plantaricin A L. plantarum L. casei subsp. casei, L. plantarum, L. sakei, L. viridescens, P. pentosaceus
Anderssen et al., 1998
Helveticin J L. helveticus L. bulgaricus Joerger and Klaenhammer, 1986 Enterolysin A E. faecalis E. faecium, L. brevis, L. curvatus, L. sakei, L. cremoris, L.
lactis, P. acidilactici, P. pentosaceus Nilsen et al., 2003
Class III
Lysostaphin S. simulans bv. staphylolyticus
S. aureus, S. carnosus, Schindler and Schuhardt, 1964; Zygmunt and Tavormina, 1972; Schleifer and Fischer, 1982
34
The information for target cell specificity of lysostaphin is encoded in its 92aa
C-terminus (Baba and Schneewind, 1996). Experiments, whereby deletions of
the targeting signal did not interfere with endopeptidase activity but abolished
the bacteriolytic killing of S. aureus cells, indicated that this domain functions
to address specifically the bacteriocin molecule to its target cells (Baba and
Schneewind, 1996).
36 247 401 493
Cell walltargeting
15 tandem repeatsSignalpeptide
Propeptide Mature lysostaphin
36 247 401 493
Cell walltargeting
15 tandem repeatsSignalpeptide
Propeptide Mature lysostaphin
Figure 1.5. Schematic representation of the structural motifs of the lysostaphin
protein.
Numbers above represents the number of amino acids
1.2.3.4 Therapeutics and other applications of bacteriocins
The best characterised Gram-positive bacteriocins are from lactic acid bacteria.
Many of these LAB are food grade organisms that are already widely used in
the food industry in the production of fermented food, but now offer the further
prospect of application to improve food preservation. As LAB have been used
for centuries to ferment foods, they enjoy GRAS status worldwide. This
permits their use in fermented food with relatively little regulatory approval.
The best example of a commercially successful naturally produced inhibitory
agent is nisin. Known since 1928 to be produced by some L. lactis isolates
(Rogers, 1928) and structurally characterised in 1971 as a lanthionine-
containing peptide (Gross and Morell, 1971), nisin and nisin-producing strains
have had a long history of application in food preservation, especially in dairy
products.
A more recent example where bacteriocin is used as a therapeutic is the use of S.
salivarius K12 as an oral probiotic to treat halitosis and maintain throat health
35
(Blis K12 Throat Guard ®). S. salivarius K12 produces two unique types of
lantibiotics, salivaricin A2 and salivaricin B, which exhibit strong inhibitory
activity against S. pyogenes (Ross et al., 1993; Wescombe et al., 2006; Hyink et
al., 2007). S. salivarius is a primary coloniser of oral mucosal surfaces in
healthy human and is not known to initiate infections (Burton et al., 2006), in
contrast to the pathogenic S. pyogenes which has also adapted to exist on human
oral mucosal surfaces. Therefore, S. salivarius K12 can be used
prophylactically to colonise oral mucosal surfaces to reduce the incidences of S.
pyogenes infections (Tagg and Dierksen, 2003).
Although bacteriocins have been used as a food preservative since the 1950s,
the recent increase and spread of multi-drug resistant bacterial pathogens has led
to renewed interest in bacteriocins as a potential alternative anti-microbial
treatment to conventional antibiotics. S. aureus infection remains one of the
most common nosocomial and community-acquired infections. With the
emergence of methicillin-resistant S. aureus (MRSA) (Hiramatsu et al., 2001),
strains of S. aureus intermediately resistant to glycopeptides (Smith et al., 1999)
and the isolation of the first clinical strain of S. aureus fully resistant to
vancomycin (CDC, 2002), research has focused on lysostaphin as a potential
therapeutic agent rather than a research tool for DNA isolation, formation of
protoplasts and differentiation of staphylococcal strains for staphylococcal
genetic studies (Polack et al., 1993; Climo et al., 1998; Patron et al., 1999;
Dajcs et al., 2000; Climo et al., 2001). Lysostaphin has been reportedly
successful in treating systemic S. aureus infection in a mouse model (Kokai-
Kun et al., 2007), and also rapidly clearing S. aureus nasal colonisation in the
cotton rat model using a cream application (Kokai-Kun et al., 2003).
1.3 AIMS OF THIS STUDY
L. lactis is a Gram-positive bacterium that is considered a desirable candidate to
be utilised as a heterologous protein factory, and as a recombinant protein
delivery vehicle. Several tools have been designed for the purpose of
expressing and secreting heterologous proteins efficiently in L. lactis. One such
tool developed by the LAB research group at QUT utilises the promoter and
36
secretion signal from the highly abundant, non-covalently bound surface protein
Sep (patent number: PCT/AU2004/001461). Despite its unknown function, the
Sep system has been successfully utilised as a fusion partner for the
heterologous expression and secretion of several different proteins in L. lactis
and other LAB (Liu et al., 2006; Turner et al., 2004a; Turner et al., 2007b).
It is the overall hypothesis of this thesis that L. lactis can be modified to be an
effective antimicrobial. To this end, the expression and optimisation of
heterologous antimicrobial proteins in L. lactis were investigated. The first part
of the investigation (Chapter 3) was to produce several novel proteins in L.
lactis, including a fusion protein consisting of CyuC and lysostaphin to test their
potential in reducing S. aureus attachment to ECM proteins, whilst the second
part of the investigation (Chapter 4) sought to identify L. lactis factors which
affected the secretion of lysostaphin, the chosen heterologous protein of interest.
37
CHAPTER 2
GENERAL MATERIALS AND METHODS
38
2.1 GROWTH MEDIA
The following solutions were resuspended in Milli-Q H2O (water that has been
purified and deionised until the electrical resistance of the water measured 18
m) and sterilised by autoclaving at 121C for 15 min or by filter sterilisation
using a 0.22µm filter (Millipore).
2.1.1 Agar plates
Bacteriological agar (Oxoid Australia) was added to liquid media to achieve a
concentration of 1.4% w/v and the suspension autoclaved. This was distributed
in 15mL lots into 150mm petri dishes (Crown Scientific). For the addition of
supplements (e.g. antibiotics, lysozyme, autoclaved bacterial cells), the
sterilised solution was placed in a 50C water bath immediately following
autoclaving. Supplements were added to the desired concentration once the
solution has reached 50C.
2.1.2 Antibiotics
The addition of antibiotics to growth media was done following sterilisation.
The media were allowed to cool to approximately 50C before antibiotics were
added to the desired concentration.
Ampicillin
A stock solution of 100mg ampicillin mL-1 (Sigma Aldrich) was made up by
dissolving 100mg of ampicillin in 1mL of Milli-Q H2O. This solution was
filter-sterilised and stored at -20°C.
Erythromycin
A stock solution of 10mg erythromycin mL-1 (Sigma Aldrich) was made by
dissolving 10mg of erythromycin in 1mL of 70% v/v ethanol and stored at -
20C. This solution did not require filter sterilisation. This solution was only
used where a concentration of 2 to 5g erythromycin mL-1 was required. Where
a higher concentration was needed, the required amount of erythromycin was
dissolved in 70% v/v ethanol at a much higher concentration. This was
prepared fresh when required.
39
2.1.3 Brain Heart Infusion (BHI) medium
Brain Heart Infusion (BHI) medium was prepared by dissolving 3.7g of BHI
broth (Oxoid Australia) in 100mL of Milli-Q H2O and sterilised by autoclaving.
2.1.4 GM17 medium
GM17 medium was prepared by dissolving 3.72g of M17 broth (Oxoid
Australia) in 100mL of Milli-Q H2O and sterilised by autoclave. Once the
solution was cooled to room temperature (24C), 2.5mL of 20% w/v glucose
solution (filter-sterilised) was added.
2.1.5 GM17+LmB agar plates
These are GM17 agar plates that contain 300mL of autoclaved 100X
concentrate of stationary phase L. monocytogenes cells, and buffered with
potassium phosphate pH 7.0 to a final concentration of 200mM.
2.1.6 GM17+SaB agar plates
These are GM17 agar plates that contain 100mL of autoclaved 100X
concentrate of stationary phase S. aureus cells, buffered with potassium
phosphate buffer pH 7.0 to a final concentration of 200mM.
2.1.7 GM17+SaU agar plates
These are the similar to the GM17+SaB agar plates except without potassium
phosphate buffer.
2.1.8 Isopropylthio--D-galactoside (IPTG) plates
A stock solution was made by dissolving 2g of IPTG (Sigma Aldrich) in 2mL
Milli-Q H2O. The solution was filter-sterilised and stored at -20°C. IPTG (7L
of stock solution per agar plate) was added on the surface of pre-poured LB agar
plates containing 100g ampicillin mL-1. This solution was spread across the
surface of the agar with a sterile glass spreader until all the IPTG has been
absorbed by the agar plate.
40
2.1.9 Lysogeny Broth (LB)
Lysogeny Broth (LB) was prepared by dissolving 10g of tryptone (Oxoid
Australia), 5g of yeast extract (Oxoid Australia) and 10g of NaCl (Sigma
Aldrich) in 1L of Milli-Q H2O and sterilised by autoclaving.
2.1.10 de Man, Rogosa and Sharpe (MRS) medium
de Man, Rogosa and Sharpe (MRS) medium was prepared by dissolving 5.2g of
MRS broth (Oxoid Australia) in 100mL of Milli-Q H2O and sterilised by
autoclaving.
2.1.11 Psi medium
Psi medium was prepared by dissolving 20g of tryptone, 5g of yeast extract and
5g of MgSO4 (Sigma Aldrich) in 1L of Milli-Q H2O. The pH of the solution
was adjusted to 7.6 using 1M KOH (Sigma Aldrich) and sterilised by
autoclaving.
2.1.12 SGM17MC medium
SGM17MC medium was prepared by dissolving 7.45g M17 broth in 92mL
Milli-Q H2O and sterilised by autoclaving. Once this has cooled to room
temperature (24C), 5mL of sterile 20% w/v glucose solution, 1.6mL of sterile
2.5M MgCl2, 2mL of sterile 0.2M CaCl2 and 100mL of sterile 1M sucrose were
added. This medium was stored at 4°C.
2.1.13 SOC medium
SOC media was prepared by dissolving 20g of tryptone, 5g of yeast extract and
0.5g of NaCl in 1L of Milli-QH2O. In addition, 3.73mL of 20% w/v KCl
(Sigma Aldrich) were added to the media and sterilised by autoclaving. Once
the solution has cooled to room temperature (24C), 10mL of sterile 2M MgCl2
(Sigma Aldrich) and 18mL of sterile 20% w/v glucose solution was added to the
media.
41
2.1.14 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) plates
A stock solution was made consisting of 20mg of X-Gal (Sigma Aldrich)
dissolved in 2mL of N,N’-dimethyl-formamide. This was stored at -20°C in a
bottle wrapped in aluminium foil. X-Gal (40µL of the stock solution per agar
plate) was spread onto the pre-poured LB agar plates containing 100µg
ampicillin mL-1. This solution was spread across the surface of the agar with a
sterile glass spreader until all the X-Gal has been absorbed by the agar plate.
BACTERIAL STRAINS
2.1.15 Escherichia coli JM109
This strain was originall purchased from Promega Australia for routine cloning.
It was cultured in LB (with aeration), on LB agar plates or on BHI agar plates,
supplemented with antibiotics as required. They were incubated at 30C or
37C.
2.1.16 Lactic acid bacterial strains
Lactococcus lactis subsp. cremoris MG1363
This is a well recognised model strain used extensively in the research of the
genetics and molecular biology of lactic acid bacteria, and was originally
donated by Scott Chandry, CSIRO, Werribee, Victoria, Australia. It was
cultured in GM17 medium or on GM17 agar plates, supplemented with
antibiotics as required, and incubated at 30C.
Lactobacillus plantarum ATCC 14917
This strain was first isolated from pickled cabbages and was purchased from the
American Tissue Culture Collection (ATCC). It was cultured in MRS medium,
or on MRS agar plates (in anaerobic jars with gas generating sachets from
Oxoid Australia), at 37C.
Lactobacillus rhamnosus GG
This strain is a commercial probiotic strain. It was purchased from the ATCC
(ATCC 53103) and was first isolated from human faeces. It was routinely
42
cultured in either MRS medium, or on MRS agar plates (in anaerobic jars with
gas generating sachets), at 37C.
Lactobacillus reuteri BR11
This strain was previously isolated from a guinea pig vaginal tract by
researchers at the Queensland University of Technology (Rush et al., 1994). It
was cultured in either MRS medium, or on MRS agar plates (in anaerobic jars
with gas generating sachets), at 37C.
2.1.17 Pathogenic strains
Listeria monocytogenes ATCC 19112
This strain was purchased from the ATCC and was first isolated from the spinal
fluid of a patient in Scotland, UK. It was cultured in either BHI medium or on
BHI agar plates at 37C.
Staphylococcus aureus ATCC 49476
This strain was generously donated by Dr Graeme Nimmo (Queensland Health)
and was characterised to be a MRSA strain. It was cultured in either BHI
medium or on BHI agar plates at 37C.
2.2 BACTERIAL CULTURE METHODOLOGIES
2.2.1 Chemically competent E. coli JM109 cell preparation
JM109 cells were plated out onto LB agar plates and incubated at 37°C for 18 h.
The following day, a colony was picked and inoculated into 3mL of LB, and
incubated at 37°C for 18 h. The stationary phase culture was then diluted 1 in
100 into 100mL of Psi medium, and was incubated at 37°C till the OD550nm
reached 0.4-0.6. The Cells were then incubated on ice for 15 min and then
pelleted by centrifugation at 3-5000 x g for 5 min at 4°C. The supernatant was
discarded and 0.4 volume (i.e. of starting volume) of TbfI buffer (30mM
potassium acetate, 100mM RbCl, 10mM CaCl2, 50mM MgCl2, 15% v/v
glycerol, filter-sterilised; Sigma Aldrich) at a pH of 5.8 (pH was adjusted with
100mM acetic acid) was used to resuspend cells. Cells were then incubated on
43
ice for 15 min, and pelleted as described previously. The supernatant was
discarded and cells were resuspended in 0.04 volume (i.e. of starting volume) of
TbfII buffer (10mM MOPS, 75mM CaCl2, 10mM RbCl, 15% v/v glycerol,
filter-sterilised; Sigma Aldrich) at pH 6.5 (pH was adjusted with 100mM
NaOH). Cells were used fresh for transformation or frozen for later use. When
freezing, cells were distributed into 200L aliquots and snap frozen in liquid
nitrogen before storing at -80°C. Cells were thawed on ice prior to use in
subsequent transformation reactions.
2.2.2 Electrocompetent L. lactis cell preparation
MG1363 cells were plated out on GM17 agar plates and incubated at 30°C for
18 h. The following day, a colony was picked and inoculated into 10mL of
GM17 medium and incubated at 30°C for 18 h. The stationary phase culture
was then diluted 1 in 100, 1 in 50 or 1 in 20 into 10mL of GM17 medium and
incubated at 30°C. After 3 hours, the OD600nm of whichever dilution reached 0.5
was used to dilute 1 in 100 into GM17 supplemented with glycine (final
concentration 2.5% w/v). Cells were then incubated at 30°C until they reached
an OD600nm of 0.45-0.55. The cells were pelleted by centrifugation at 3000 x g
for 15 min at 4°C. The supernatant was discarded and the cells were
resuspended in 12.5mL ice-cold (-20°C) poration-storage buffer (0.5M sucrose,
10% v/v glycerol, filter-sterilised through Millipore 0.22m filter), and the cells
pelleted as previously described. This was done twice. After the final wash, the
cells were resuspended in 1mL poration-storage buffer and used fresh for
transformation or frozen for later use. Cells were distributed into 200L
aliquots and snap frozen in liquid nitrogen before storing at -80°C. Cells were
thawed on ice prior to use in subsequent transformation reactions.
2.2.3 Isolation of chromosomal DNA from L. lactis
L. lactis was grown for a minimum of 18 h in 2mL of media and was
centrifuged at maximum speed (18,000 x g) in a bench top centrifuge for 10
min. After discarding the supernatant, the cells were resuspended in 850L of
TEN buffer (10mM Tris at pH 8.0, 1mM EDTA, 0.1M NaCl) and 150L of
lysozyme (100mg lysozyme dissolved in 1.5mL Milli-Q H2O) was added. This
44
mixture was incubated at 37°C in a water bath for 30 min, after which 115L of
10% w/v sodium dodecyl sulphate (SDS) and 1L Proteinase K (100mg
Proteinase K mL-1; Roche Applied Science) was added. The mixture was
incubated at 37°C in a water bath for 60 min. Then, 130L of 5M NaCl and
750L of chloroform-isoamyl alcohol mix (24:1 choloroform:isoamyl alcohol;
Sigma Aldrich) were added and the mixture was shaken vigorously. The
mixture was centrifuged at maximum speed (18,000 x g) in a bench top
centrifuge for 5 min. The top layer was removed and transferred into a 2mL
microfuge tube. Aftewards, 700L propan-2-ol was added to the top layer and
shaken vigorously. The mixture was centrifuged at maximum speed (18,000 x
g) in a bench top centrifuge for 5 min. The supernatant was discarded and the
resultant pellet of genomic DNA was washed with 1mL 70% v/v ethanol, and
centrifuged at maximum speed on a bench top centrifuge for 5 min. The
supernatant was discarded and the pellet was air dried or vacuum desiccated
before being dissolved in 150L TE buffer (10mM Tris at pH 8.0, 1mM EDTA)
and 1L Rnase A (10mg RnaseA mL-1; Roche Applied Science).
2.2.4 Purification of plasmids from E. coli
LB (3-5mL; supplemented with antibiotics) was inoculated with a colony from
an agar plate or from glycerol stocks and incubated at either 30°C or 37°C for a
minimum of 18 h. The stationary phase culture was centrifuged at maximum
speed (18,000 x g) in a bench top centrifuge for 5 min to pellet cells and the
supernatants discarded into disinfectant. Plasmids were either purified from the
cells using the QIAprep Spin Miniprep kit (Qiagen) following the
manufacturer’s instructions or by the following method. The pellet was
resuspended in 100L of solution I (25mM Tris-Cl at pH 8.0, 10mM EDTA,
filter-sterilised, stored at 4°C). Then, 200L of solution II (0.2M NaOH, 1%
w/v SDS, made fresh before use) was then added and mix by inversion 5 times.
Finally, 150L of solution III (3M potassium acetate adjusted to pH 5.4 with
5M acetic acid, stored at 4°C) was added and mixed by inversion 5 times. This
mixture was then centrifuged at maximum speed (18,000 x g) in a bench top
centrifuge for 5 min to pellet the cell debris, and the supernatant decanted into a
new microfuge tube. An optional step is the addition of 150L of chloroform-
45
isoamyl alcohol (24:1 choloroform:isoamyl alcohol; Sigma Aldrich) to the
tubes, then mixed by inversion 5 times and centrifuged at maximum speed
(18,000 x g) in a bench top centrifuge for 5 min. The top layer was removed
into a new 1.5mL microfuge tube and 1mL of 100% v/v ethanol was added.
The tube was mixed by inversion 10 times and centrifuged as previously
described for 5 min. The supernatant was discarded and the pellet was washed
with 100L of 70% v/v ethanol and centrifuged as previously described for 10
s. The supernatant was discarded and the pellet air dried or vacuum desiccated.
The pellet was resuspended in 30L of Milli-Q H2O with RnaseA (350g Rnase
A mL-1) and stored at 4°C.
2.2.5 Transformation of chemically competent E. coli
Cells were thawed on ice and then dispensed into 50L aliquots in 1.5mL
microfuge tubes. The ligation reaction (5µL) or the plasmid (2µL) was added to
the E. coli competent cells. (Specific details of the ligation reaction or plasmid
can be found in the material and methods section of chapters 3 and 4.)
Competent cells were gently mixed with the plasmids by flicking the tube.
Cells and plasmids were left on ice for 30 min. Cells were then heat shocked at
42°C in a water bath for 50 s, and then quickly returned to ice for 2 min. Cold
(4°C) SOC medium (900µL) was added to each tube. All tubes were then
incubated at either 30°C or 37°C (depending on the plasmid) in a shaker for 1 h.
Cells were then plated out onto selective agar plates. Inoculated agar plates
were incubated at 30°C (for 2 to 4 days) or 37°C (for 18 h). The plasmid,
pUC19 (Promega), was used as a transformation control was used to ensure
competent cells were able to be transformed.
2.2.6 Transformation of electrocompetent L. lactis
Cells were thawed on ice and then dispensed into 50L aliquots in ice-cold
electroporation cuvettes (Bio-Rad Laboratories). The ligation reaction (2µL) or
the plasmid of interest (2µl) was added to the L. lactis competent cells.
Competent cells were gently mixed by flicking the cuvettes. The cells were
pulsed (200, 2.5kv, 25F charge duration 4.8 msec, 2mm electrode gap) and
immediately transferred into 1.5mL microfuge tubes with 960L of ice-cold
46
SGM17MC medium and placed on ice for 10 min. Cells were then incubated at
30°C for 2 h. Cells were plated out onto selective agar plates, and incubated at
30°C for 18 h. The plasmid, pGhost9:ISS1, was used as a transformation
control in order to ensure competent cells were able to be transformed.
2.3 SOLUTIONS FOR DNA ANALYSES
2.3.1 Agarose gel loading buffer
Gel loading buffer for visualisation of DNA on agarose gel was made with
0.25% w/v bromophenol blue, 30% v/v glycerol and in a solution of 100mM
Tris (pH 8.0). Gel loading buffer was diluted 6-fold into DNA solutions before
being loaded onto the agarose gels.
2.3.2 Tris-borate EDTA (TBE) buffer
Tris-borate EDTA (TBE) buffer was prepared by combining 10.8g of Tris-base,
5.5g of boric acid and 0.93g of Na4EDTA in 1L of Milli-Q H2O. The pH was
adjusted to 8.3.
2.4 METHODS FOR DNA ANALYSES
2.4.1 Agarose gel electrophoresis
Agarose gels (0.7-2% w/v) were prepared by combining low electroendosmosis
agarose (Roche Applied Science) in TBE buffer to the desired percentage
depending on the anticipated product size. Agarose was dissolved in the TBE
buffer by microwaving the mixture in 30 s bursts until completely dissolved.
The agarose was allowed to cool to 50°C, then ethidium bromide (Bio-Rad
Laboratories) was added to achieve a final concentration of 0.5µg ethidium
bromide mL-1, and the solution poured into 7cm x 10cm or 15cm x 10cm gel
moulds (Bio-Rad Laboratories) containing an agarose gel comb (Bio-Rad
Laboratories). DNA solution was combined with agarose gel loading buffer
prior to electrophoresis. Bands were visualised under ultra-violet light (UVP
Gel-documentation system, UVP Incorporated).
47
2.4.2 DNA precipitation
To precipitate DNA, 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volume
of 100% v/v ethanol were added to samples and placed on ice for 30 min. The
samples were then centrifuged at maximum speed (18,000 x g) in a bench top
centrifuge at 4C for 20 min. The supernatant was discarded and the pellet was
washed with 70% v/v ethanol and then centrifuged as previously described at
room temperature (24C) for 5 min. The supernatant was discarded and the
pellets air dried or vacuum desiccated. The pellets could then be resuspended in
any desired solution.
2.4.3 Gel purification of DNA
Digested plasmid DNA was resolved on a 0.7% w/v TBE agarose gel at 100V
for 60 min, excised from the gel, and purified using QIAgen Gel Purification kit
(Qiagen) following the manufacturer’s instructions.
2.4.4 Ligation reactions
Ligation reactionss were performed using T4 DNA ligase (Roche Applied
Science). The reactions were set up as directed by the manufacturer. Briefly,
DNA from digested vectors was resuspended in 17µL of Milli-Q H2O and 2µl
of 10x Ligation Buffer (660mM Tris-HCl, 50mM MgCl2, 10mM
dithioerythritol, 10mM ATP; Roche Applied Science), and 1µl of T4 DNA
ligase was added. The reactions were incubated at 4°C for a minimum of 18 h.
2.4.5 Polymerase chain reaction (PCR)
A standard Polymerase chain reaction (PCR) amplification mix contained
2.75mM MgCl2, 3.75U Expand Long Template Enzyme mix (Roche Applied
Science) or Pfu DNA polymerase (Promega), 400µM of deoxynucleoside
triphosphates (dNTPs) (Roche Applied Science), 500nM of primers (Sigma
Proligo), 1l of DNA template and sterile Milli-Q H2O added to a total volume
of 50l. Thermal cycling was done on an MJ Research PTC-200 Thermal
Cycler (Geneworks). Cycle conditions for gene amplification was as follows:
Step 1: 94°C for 2 min, then 30 cycles of 93°C for 10 s,55°C for 30 s, and 68°C
for 1 min, followed by final extension step of 68°C for 7 min. The cycle
48
conditions were varied at times to optimise specific reactions. Full details are
provided in the “Materials and Methods” sections of the relevant chapters.
2.4.6 Purification of PCR products
PCR products were purified using the Hi-Pure PCR Purification kit (Roche
Applied Science) following the manufacturer’s instructions. An additional
centrifugation step was introduced whereby the final eluted product was
centrifuged at maximum speed (18,000 x g) in a bench top centrifuge for 1 min,
and the supernatant removed to a 1.5mL microfuge tube. This step was
introduced to remove any contaminating resin from the filter of the purification
column.
2.4.7 Quantitation of DNA
DNA concentrations were determined by measuring the A260nm and its quality
determined by measuring the ratios of A260nm and A280nm. For absorbance
readings, DNA solutions were diluted 50-fold and analysed using a DU800
UV/visible spectrophotometer (Beckman Coulter).
2.4.8 Restriction enzymes
All restriction enzymes used in this study were purchased from Roche Applied
Science and digest reactions were performed following the manufacturer’s
directions. The average digest reactions were set up so that 20L contained
4L of plasmid DNA, 2L of 10x Incubation Buffer (SuRE/Cut Buffer B or H
depending upon the restriction enzyme in use; Roche Applied Science), and 10
units of enzyme. The reactions volumes varied with the amount of plasmid
DNA or PCR product to be digested. All reactions were incubated at 37C for a
minimum of 2 h.
2.4.9 Sequencing
The DNA template (plasmid) and primer (Sigma Proligos) was provided to the
Australia Genome Research Facility (http://www.agrf.org.au) for sequencing.
49
2.5 SOLUTIONS FOR PROTEIN ANALYSES
2.5.1 CAPS transfer buffer
CAPS transfer buffer for protein transfer in western blots were prepared by
dissolving 2.21g of 3-cyclohexylamino-1-propanesulfonic acid (CAPS; Sigma
Aldrich) in 800mL Milli-Q H2O, and pH adjusted to 11.0 using NaOH. Then
100mL of methanol was added and the total volume was adjusted to 1L with
Milli-Q H2O. The buffer was prepared at least 1 h prior to use and kept at 4°C
with stirring.
2.5.2 Coomassie stain
Coomassie stain consisted of 0.25g of Comassie Brilliant Blue R250 (Sigma
Aldrich) dissolved in 125mL of methanol. Then 25mL of acetic acid and
100mL Milli-Q H2O were added.
2.5.3 Electrode buffer
Electrode buffer (10x) was prepared by dissolved 30.3g of Tris-base, 144.2g of
glycine in 950mL Milli-Q H2O, and then adding 50mL of 10% w/v SDS
solution. This solution was stored at 4°C. The electrode buffer was diluted 10-
fold in Milli-Q H2O prior to use.
2.5.4 Phosphate buffered saline (PBS) (pH 7.0)
Phosphage buffered saline (PBS) was prepared either by using PBS tables
(Oxoid Australia) following the manufacturer’s instructions or by dissolving 8g
of NaCl, 0.2g of KCl, 1.44g of Na2HPO4 and 0.24g of KH2PO4 in 1L of Milli-Q
H2O. The pH was adjusted to 7.0 using NaOH. The solution was sterilised by
autoclaving.
2.5.5 2x SDS loading buffer (non-reducing)
The 2x SDS sample loading buffer consisted of 100mM Tris (pH 6.8), 4% w/v
SDS, 0.2% w/v bromophenol blue and 20% v/v glycerol. Protein samples that
were to be separated via electrophoresis were mixed 1:1 v/v with the 2x SDS
loading buffer.
50
2.6 METHODS FOR PROTEIN ANALYSES
2.6.1 SDS polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was carried out using the Bio-Rad Mini-PROTEAN 3 apparatus,
according to the manufacturer’s instructions. Briefly, a 12% v/v resolving gel
was prepared by combining 3mL of 30% w/v acrylamide, 4.35mL of Milli-Q
H2O, 2.5mL of resolving gel buffer (1.5M Tris, pH 8.8) and 0.1mL of 10% w/v
SDS. Finally, 5µL of N,N,N’,N’-tetramethylethylenediamine (TEMED) and
50µL of 30% w/v ammonium persulphate were added, and the solution was
carefully mixed and quickly poured into the glass plate cassette. Milli-Q H2O
(500µL) was layered over the top to prevent the gel from drying. The resolving
gel was left to polymerise for 20 min. Once the gel had set, the Milli-Q H2O
was carefully tipped off and a 4% v/v spacer gel solution was added to the top
of the resolving gel. A 4% v/v spacer gel was prepared by combining 1mL of
30% w/v acrylamide, 6.4mL of Milli-Q H2O, 2.5mL of spacer gel buffer (1M
Tris, pH 6.8) and 0.1mL 10% w/v SDS. Finally, 10µL of TEMED and 50µL of
30% w/v ammonium persulfate were added, and the solution was carefully
mixed and quickly poured on top of the resolving gel. A Teflon comb was
quickly inserted into the spacer gel solution and the gel allowed to polymerise
for 20 min. Following polymerisation, the Teflon comb was removed and the
gel cassette placed in an electrophoresis tank. The tank was filled with 1x
electrode buffer, and protein samples in non-reducing SDS loading buffer were
loaded into the wells using capillary pipette tips. The gel was run at 170V until
the dye front reached the bottom of the resolving gel. If the gel was for a
western blot, the gel was pre-treated with CAPS transfer buffer for a minimum
of 5 min at room temperature (24C). If the gel was for visualisation of protein
bands, it was place into Coomassie stain for a minimum of 2 h. Gels were de-
stained using multiple changes of de-stain solution (45% v/v methanol, 10% v/v
acetic acid).
2.6.2 Trichloroacetic acid (TCA) precipitation of supernatant proteins
Proteins in the culture supernatant were precipitated using the following
method. The cultures were centrifuged at 3000 to 5000 x g for 10 min at 4°C.
The supernatant was then filtered through 0.22m filter (Millipore). 1.8mL of
51
the filtered supernatant was transferred into a 2mL microfuge tube and chilled
on ice for a minimum of 5 min. Cold (4C) 80% w/v TCA (125µL) was added
and the solution mixed by inversion 10 times, and placed on ice for a minimum
of 30 min (average 60 min). The proteins were pelleted by centrifugation at
maximum speed (18,000 x g) in a bench top centrifuge for 5 min at 4°C. The
supernatant was discarded and the pellet was washed with 700L of cold (-
20°C) acetone and centrifuged as previously described. The supernatant was
discarded and the pellet air dried or vacuum desiccated. The pellet was
resuspended in equal volumes of NaOH (50mM) and 2x SDS loading buffer to
the desired volume.
2.6.3 Western blots
SDS-PAGE gels were carried out as described in section 2.6.1 and pre-treated
with CAPS transfer buffer. Protein was transferred from a SDS-PAGE gel to a
nitrocellulose membrane (GE Healthcare) using CAPS transfer buffer at 70V for
90 min on ice. Following transfer the nitrocellulose membrane was removed
from the tank and blocked in a solution of 1% w/v casein (Roche Applied
Science) in Tris-buffered saline (TBS; 50mM Tris, 150mM NaCl, pH 7.5) and
Tween-20 (TBS-T; TBS with 0.1% v/v Tween-20) for a minimum of 1 h. After
blocking, the membrane was washed with TBS for a minimum of 2 min, and
then incubated with the primary antibody for 1 h with gentle rocking. The
primary antibody used was a mouse anti-His6 monoclonal antibody (Sigma
Aldrich) diluted 3000-fold in a solution of 0.5% v/v casein in TBS. After
incubation, the membrane was washed twice with TBS-T for 10 min. Rabbit
anti-mouse-HRP-conjugate (Dako) was used as the secondary antibody, diluted
1000-fold in a solution of 0.5% v/v casein in TBS, and incubated for 1 h with
gentle rocking. The membrane was then washed three times with TBS-T for 5
min. The bound secondary antibodies were detected using the Lumi-Light
chemiluminescence kit (Roche Applied Science). The membrane was exposed
on Autorade X-ray films (AGFA), with exposures ranging from 10 s to 1 min
for films with strong signals, and from 10 min up to 18 h for weak signals.
52
2.6.4 L. lactis cell associated protein extraction
Two different cell associated protein extraction methods were used. This
involved either boiling the cells in 2x SDS loading buffer or homogenising the
cells using glass beads. For both methods, the L. lactis culture was centrifuged
at 3000-5000 x g for 10 min at 4°C, and the supernatant removed. For the
boiling method, the cells were resuspended in 50µL of 2x SDS loading buffer,
giving a total volume of approximately 200µL. The cells were incubated at
97°C for 5 min in a heating block prior to loading of SDS-PAGE. For the glass
bead method, the pelleted cells were washed in 3 to 5mL of PBS, and
centrifuged at 3000-5000 x g for 10 min at 4°C. The cells were resuspended to
10 or 100 times concentration in PBS and transferred to a 2mL cryovial with
0.75mL of 0.1mm diameter zirconia/silicone glass beads (Daintree Scientific).
The mixture was homogenised with a Mini-Beadbeater-8 cell disruptor
(Daintree Scientific) for 1 min, then placed on ice for 1 min. When thicker cell
suspensions were used, the homogenisation step was repeated. The
homogenised suspension was centrifuged at maximum speed (18,000 x g) in a
bench top centrifuge for 10 min at 4°C to remove the glass beads and cell
debris. The supernatant was transferred into a 1.5mL microfuge tube and mixed
with an equal volume of 2x SDS loading buffer. The samples were incubated at
97°C for 5 min in a heating block prior to loading of SDS-PAGE.
53
CHAPTER 3
APPLICATION OF CYUC-LYSOSTAPHIN FUSION
PROTEIN SECRETED BY LACTOCOCCUS LACTIS TO
PREVENT STAPHYLOCOCCUS AUREUS ADHERENCE TO
EXTRACELLULAR MATRIX PROTEINS IN VITRO
54
3.1 INTRODUCTION
L. lactis is a Gram-positive bacterium widely used in the dairy manufacturing
industry. Due to its GRAS status; it is often regarded as a promising host for
the production of recombinant proteins of therapeutic interest. One such protein
is lysostaphin, an endopeptidase that is naturally produced by S. simulans biovar
staphylolyticus ATCC1362 (Schindler and Schuhart, 1964). Lysostaphin
specifically cleaves the pentaglycine cross bridges of S. aureus peptidoglycan,
which results in cell wall weakening, and consequentially, cell death. Interest in
this antimicrobial enzyme has increased in recent years due to the worsening
problem of MRSA, and several studies have demonstrated its usefulness in the
treatment of infections (Patron et al., 1999; Dajcs et al., 2000; 2001; Kokai-Kun
et al., 2007; Oluola et al., 2007). Recombinant lysostaphin was first produced
in E. coli in 1987 (Recsei et al., 1987). More recently, it has been expressed as
an intracellular protein in L. lactis using the NICE system (Mierau et al., 2005a;
2005b). Recent work by Turner et al. (2007b) has demonstrated the expression
and secretion of active lysostaphin in several lactic acid bacteria, including L.
lactis.
S. aureus is an important human pathogen in nosocomial and community
acquired infections (Boucher and Corey, 2008; Chastre, 2008). It is the primary
causative agent of wound infections, bacteraemia, and sepsis, culminating in
high mortality rates. With the decrease in the efficacy of antibiotics to treat S.
aureus infections, this pathogen has become a considerable problem on a
worldwide basis. Of particular concern are MRSA, and strains with reduced or
complete resistance against vancomycin (Tenover et al., 1998; CDC, 2002) and
teicoplanin (Kaatz et al., 1990), antibiotics which are the last line of defence
against MRSA. Escalation in the spread of antibiotic-resistant S. aureus
(Grundman et al., 2006) highlights an urgent need for new measures to prevent
and treat S. aureus infections. An alternative to antibiotics which has attracted
significant interest in recent years is the antimicrobial protein, lysostaphin.
More importantly, lysostaphin can also kill antibiotic resistant strains such as
MRSA (Wu et al., 2003). S. aureus strains resistant to lysostaphin become
hypersusceptible to -lactam antibiotics, including methicillin (Stranden et al.,
55
1997; Climo et al., 2001). Therefore, a combination treatment of lysostaphin
and a -lactam antibiotic for MRSA would not lead to any lysostaphin resistant
survivors (Clino et al., 2001).
S. aureus is also present as a commensal organism in humans and colonises
multiple sites, such as the nasal passage, which is the most frequent site of
carriage (Fierobe et al., 1999; Kluytmans et al., 1997; Lee et al., 1999; White
and Smith, 1963). S. aureus adhere to host ECM proteins, such as fibronectin,
collagen, and keratin, via surface proteins called microbial surface components
recognising adhesive matrix molecules (MSCRAMMs), and these interactions
are important for initiating infection. Clumping factor B (ClfB), and fibronectin
binding proteins A and B (FnBPA and FnBPB), have been identified as S.
aureus MSCRAMMs which bind to keratin and fibronectin, respectively
(Peacock et al., 1999; O’Brien et al., 2002). Specifically, ClfB has been shown
to bind to the keratin of human nasal epithelial cells, and that mutants lacking
ClfB adhered poorly to keratin and showed overall reduction in adherence to
human nasal epithelial cells (O’Brien et al., 2002). In addition, intranasal
immunisation of mice with ClfB was able to reduce S. aureus nasal colonisation
(Schaffer et al., 2006).
Previous research has demonstrated that the CyuC protein (Turner et al., 1999),
found in abundance on the cell wall of L. reuteri BR11, can bind to fibronectin
in ligand blots (M. S. Turner, personal communication). It has also been
reported that the use of whole cells or cell surface extracts (which contain
CyuC) of the related strain L. reuteri RC-14 (Reid et al., 1992) were able to
significantly inhibit the adherence of S. aureus to silicone implants, and thus
reduced S. aureus surgical implant infections (Gan et al., 2002). The
hypothesised mechanism for this was that the biosurfactant of L. reuteri RC-14
contained cell surface extracellular matrix binding proteins (ECMBPs) which
may effectively compete with S. aureus’ own MSCRAMMs for binding to host
sites. This suggests that native proteins from L. reuteri, including CyuC, may
be able to reduce S. aureus colonisation and infection by competitive inhibition
of binding to ECM proteins.
56
Therefore, the aim of this study was to investigate the potential of lysostaphin
and CyuC when expressed by L. lactis singularly or as a fusion protein to act as
S. aureus inhibitory agents. This involved determining if: (i) L. lactis can be
used to express CyuC and a CyuC-lysostaphin fusion protein, and (ii) the CyuC-
lysostaphin fusion protein will have superior anti-staphylococcal activity by
being able to inhibit S. aureus adherence to immobilised ECM proteins.
3.2 MATERIALS AND METHODS
3.2.1 Construction of L. lactis strains that secreted CyuC or CyuC-
lysostaphin fusion protein
L. lactis strains were constructed which expressed and secreted CyuC or CyuC
fused to lysostaphin (CyuC-Lss) using the Sep promoter (PSep) and the Sep
secretion signal (ssSep) The strategy was to use overlap extension PCR to create
long DNA fragments from smaller fragments (Higuchi et al., 1988).
Oligonucleotides (Figure 3.1, Table 3.1) were designed to amplify the following
region of genes as discrete fragments with 20-bp complementary overlaps, as
described below:
- PSep, ssSep, and 6xhis,
- the mature CyuC peptide (corresponding to amino acids 26 to 264),
- the stem-loop transcription terminator that followed the CyuC sequence
(Turner et al., 1999), and
- the mature lysostaphin peptide.
The CyuC expression cassette was assembled from two DNA fragments. The
first fragment was the PSep-ssSep-6xhis fragment, which was amplified from
pGhost9-his1-lss-his2 (Table 3.1, Figure 4.1B) using the primers SepUSEco-5F
and 6HSepUS-3R (Figure 3.1A, Table 3.1). The second fragment encoded the
mature CyuC protein and contains the cyuC operon transcription terminator
(6xhis-CyuC-term), which was amplified from chromosomal DNA extracts of L.
reuteri BR11 (prepared by R. Galea) using the primers 6HCyuC-5F and
CyuCTermHind-3R (Table 3.1). DNA encoding 6xhis was also inserted at the
57
N-terminus of the CyuC encoding fragment (Figure 3.1A). The PCR products
of PSep-ssSep-6xhis and 6xhis-CyuC-term were purified as described in section
2.4.6, and dilutions of the purified products were combined and used as
templates for the amplification of PSep-ssSep-6xhis-CyuC-term using the primers
SepUSEco-5F and CyuCTermHind-3R (Figure 3.1A). The resulting 1.6-kb
PCR product was ligated into pGEM-3Zf to form pGEM3-CyuC following
digestion with EcoRI and HindIII. This cloned expression cassette was
sequenced and verified to contain the predicted DNA sequence.
In a similar fashion, the CyuC-Lss expression cassette was amplified from three
fragments. The first fragment was PSep-ssSep-CyuC, which encoded the Sep
promoter and Sep secretion signal with a 20-bp region complementary to the
cyuC sequence at the 3’ end. This was amplified from pGhost9-his1-lss-his2
(Table 3.1) using the primers SepUSEco-5F and SepUS-3R (Figure 3.1B; Table
3.1). The second fragment was ssSep-CyuC-Lpx, which encoded the sequence
for the mature CyuC protein with a 20-bp complementary region to the Sep
secretion signal at the 5’ end and to the LPXTG linker at the 3’ end. This
fragment was amplified from L. reuteri chromosomal DNA extracts using the
primers SepCyuClss-5F and LpxCyuC-3R (Figure 3.1B; Table 3.1). The third
fragment, Lpx-6xhis-Lss-term, was amplified from pGEM3-CyuC-Lss-term
using the primers Lpx6HLss-5F and CyuCTermHind-3R (Figure 3.1B; Table
3.1). This fragment encoded the mature lysostaphin protein and the cyuC
operon transcription terminator, with a 20-bp complementary region to the
LPXTG linker at the 5’ end. Amplification was undertaken in a two-step
process. Firstly, ssSep-CyuC-Lpx and Lpx-6xhis-Lss-term were gel purified (as
described in section 2.4.3), and used as templates and amplified using the
primers SepCyuClss-5F and CyuCTermHind-3R (Figure 3.1B). Then, the ssSep-
CyuC-Lpx-6xhis-Lss-term PCR product was gel purified, and used as template
along with the PSep-ssSep-CyuC fragment and amplified using SepUSEco-5F and
CyuCTermHind-3R (Figure 3.1B). The resultant PCR product was cloned into
pGEM-3Zf to generate pGEM3-CyuC-Lss. This cloned expression cassette was
sequenced and verified to contain the predicted DNA sequence.
58
PSepssSep
CyuC
PSepssSep
CyuC
Lss
SepUSEco-5F SepUSEco-5F
6HSepUS-3F
6HCyuC-5FCyuCTermHind-3R
CyuCTermHind-3R
SepUS-3F
LpxCyuC-3RSepCyuClss-5F
Lpx6HLss-5F
PSepssSep
CyuC
PSepssSep
CyuC Lss
PSepssSep
CyuC Lss
A BPSepssSep
CyuC
PSepssSep
CyuC
Lss
SepUSEco-5F SepUSEco-5F
6HSepUS-3F
6HCyuC-5FCyuCTermHind-3R
CyuCTermHind-3R
SepUS-3F
LpxCyuC-3RSepCyuClss-5F
Lpx6HLss-5F
PSepssSep
CyuC
PSepssSep
CyuC Lss
PSepssSep
CyuC Lss
A B
59
Figure 3.1. Schematic representation of the PCR overlap strategy employed to clone the CyuC (A), and CyuC-Lss (B) fusion protein to the Sep
promoter and secretion signal at the 5’ end, and the cyuC operon transcription terminator at the 3’ end.
Symbols: Sep promoter, PSep; Sep secretion signal, diagonal lines and ssSep; 6xhis, black box; LPXTG linker, horizontal lined box; CyuC, dotted
box; lysostaphin (Lss), grey box; lollipop symbol, cyuC operon transcription terminator; primers, broken arrows.
60
Table 3.1. Strains, plasmids, and oligonucleotides used in this chapter. Details Source Strains E. coli JM109 Cloning host Promega L. lactis MG1363 Plasmid free L. lactis subsp. cremoris Gasson, 1983 S. aureus ATCC 49476 Plasmids pGEM-3Zf Ampr ; 3.2-kb pGhost9:ISS1 Emr ori (Ts); 4.6-kb ; non-replicative in L. lactis at 37°C pGEM3-CyuC-Lss-term Ampr ; 5.5-kb ; pGEM-3Zf containing CyuC, LPXTG linker, 6xhis, lysostaphin, and CyuC transcription terminator M.S. Turner. pGEM3-CyuC Ampr ; 4.8-kb ; pGEM-3Zf containing PSep-ssSep-6xhis-CyuC-term This study pGEM3-CyuC-Lss Ampr ; 5.6-kb ; pGEM-3Zf containing PSep-ssSep-CyuC-LPXTG-6xhis-Lss-term This study pGhost9-CyuC Emr ; 5.3-kb ; pGhost9 containing PSep-ssSep-6xhis-CyuC-term This study pGhost9-CyuC-Lss Emr ; 6.1-kb ; pGhost9 containing PSep-ssSep-CyuC-LPXTG-6xhis-Lss-term This study pGhost9-his1-lss-his2 Emr ; pGhost9:ISS1 containing the lss cassette within the his operon (7.6-kb) Chapter 4 Oligonucleotides SepUSEco-5F ATAGAATTCAACCTTCCTGCTGACCT This study SepUS-3R ATCGGTGTAGATAGTGTCAGCAT This study 6HSepUS-3R GTGATGATGGTGATGATGATCGGTGTA This study 6HCyuC-5F CATCATCACCATCATCACGCATCTTCGGCAGTAAATTC This study SepCyuClss-5F CTGACACTATCTACACCGATGCATCTTCGGCAGTAAATTC This study LpxCyuC-3R TTCTCCTGTTGATGGTAATCCTCCTTCTGTAATATCCGCACCAA This study Lpx6HLss-5F GGAGGATTACCATCAACAGGAGAACATCATCACCATCATCAC This study CyuCTermHind-3R TAGCAAGCTTTCACCCACTCATTCGTCAGGC This study Restriction enzyme sites are underlined.
61
The inserts from both pGEM3-CyuC and pGEM3-CyuC-Lss were digested
using EcoRI and HindIII, and cloned into pGhost9:ISS1, replacing the ISS1
element. The resulting plasmids, pGhost9-CyuC and pGhost9-CyuC-Lss, were
transformed first into E. coli, and then into L. lactis. L. lactis pGhost9-CyuC-
Lss was grown on GM17+SaB agar plates (see section 2.1.6) containing 5µg
erythromycin mL-1 (GM17+SaB+5Em). These agar plates containing
autoclaved whole S. aureus cells are opaque. Colonies that produced active
lysostaphin, which lyses the S. aureus cells, will exhibit a visible zone of
clearing (or halo) around the colonies itself. This zone of clearing indicates that
the lysostaphin expressed was active. As a control, L. lactis pGhost9-CyuC did
not generate any zones of clearing on GM17+SaB+5Em, thus indicating that the
halos were specific to the production of lysostaphin.
3.2.2 Cell fractionation, protein extraction and western blot analysis
Cell associated and supernatant proteins were prepared from cultures from three
different incubation times: late-log phase of growth (cultures grown for 18 h
diluted 50-fold into fresh media and incubated until the OD600nm reached 1.0),
minimum 18 h incubation, and two days incubation. Proteins in the supernatant
were precipitated using TCA as described in section 2.6.2. For the cell
associated proteins, 10mL of cultures were centrifuged at 3000 to 5000 x g for
10 min. The cell pellet was then resuspended in 50µL of 2x SDS-PAGE
loading buffer giving a total volume of 200µL per 10mL of cells.
The protein samples were incubated at 97°C for 5 min, and 20µL were
separated by SDS-PAGE as described in section 2.6.1, and then transferred to
nitrocellulose membrane and the proteins were detected as described in section
2.6.3.
3.2.3 Prediction of protein molecular weight based on sequence
The predicted molecular weights of recombinant CyuC and CyuC-Lss were
calculated by the Protein Molecular Weight Calculator
(http://www.bioinformatics.org/sms/prot_mw.html).
62
3.2.4 Stock solutions of fibronectin, collagen, and keratin
In this study, fibronectin, collagen, and keratin are collectively referred to as
ECM proteins. Human fibronectin (BD Biosciences) was resuspended in sterile
Milli-Q H2O to obtain a stock concentration of 1mg fibronectin mL-1. Collagen
type I from calf skin (Sigma Aldrich) was resuspended in Milli-Q H2O to obtain
a stock concentration of 1.67mg collagen mL-1. Type I keratin from human
epidermis (Sigma Aldrich) was purchased in solution, and the concentration
varied depending on the batch purchased. Stocks of fibronectin and collagen
were stored at -20°C. Once thawed, they were stored at 4°C and used within 2
weeks. Keratin was stored at 4°C.
3.2.5 L. lactis crude cell extracts for adherence assay
L. lactis pGhost9:ISS1, L. lactis pGhost9-CyuC, L. lactis pGhost9-CyuC-Lss,
and L. lactis pGhost9-his1-lss-his2 (Table 3.1) were inoculated into 400mL of
GM17 medium containing 5µg erythromycin mL-1 (GM17+5Em), and
incubated at 30°C for two days. The cells were centrifuged at 5000 x g for 15
min. The cells were washed with 5mL of PBS, centrifuged at the same speed,
and the supernatant discarded. The cells were then resuspended in PBS to
achieve at 100 times concentration (i.e. 400mL of cell culture in 4mL of PBS),
and aliquots of 1.0mL were transferred into cryovials with approximately
0.75mL of 0.1mm zirconia/silicone beads (Daintree Scientific). The cells were
homogenised with a Mini-Beadbeater-8 cell disruptor (Biospec) for 1 min,
placed on ice for 1 min and homogenised again. Cell debris and beads were
removed by centrifugation in a benchtop centrifuge at maximum speed (18,000
x g) for 10 min at 4°C. Approximately 700µL of the supernatant was removed
from each aliquot and pooled.
3.2.6 Preparation of S. aureus for adherence assay
S. aureus was inoculated into 10mL of BHI media and incubated at 37°C for a
minimum of 18 h. The overnight incubated cultures were diluted 50-fold into
50mL of BHI and incubated at 37°C until the OD600nm reached 0.8. The cells
were centrifuged at 5000 x g for 10 min. The cells were washed once in 5mL of
PBS, centrifuged at the same speed, and the supernatant discarded. The cells
were then resuspended in PBS to obtain an OD600nm of 4.0.
63
3.2.7 Adherence of S. aureus to immobilised ECM proteins and L. lactis
cell extracts
Adherence experiments were performed in 96-well flat-bottomed polystyrene
tissue culture plates (Nunc) coated with ECM proteins and protein extracts from
L. lactis pGhost9:ISS1, L. lactis pGhost9-CyuC, L. lactis pGhost9-CyuC-Lss or
L. lactis pGhost9-his1-lss-his2 (Table 3.1). The protocol followed was that
published by Walsh et al. (2004) with some modifications. Each well was
coated first with 100µL of ECM proteins (at a concentration of 20µg ECM
protein mL-1) in coating buffer (20mM sodium carbonate buffer, pH 9.6).
Coating buffer without ECM proteins was used as a blank control. The plate
was sealed with parafilm, covered in foil, and incubated at 4°C for a minimum
of 18 h. Any unbound ECM proteins were discarded and the wells were
washed three times with 100µL PBS. The wells were then blocked with 100µL
of 5mg bovine serum albumin mL-1 (BSA) in PBS at 37°C for 2 h. The BSA
solution was discarded and the wells were washed three times with 100µL PBS.
Crude cell extracts from L. lactis pGhost9:ISS1, L. lactis pGhost9-CyuC, L.
lactis pGhost9-CyuC-Lss or L. lactis pGhost9-his1-lss-his2 (prepared according
to section 4.2.5) were dispensed into each well (100µL/well) and incubated at
4°C for a minimum of 18 h. Wells containing PBS only was used as a positive
control for S. aureus adherence to the ECM proteins. The crude extracts (or
PBS) were discarded and the wells were washed three times with 100µL PBS.
S. aureus cells (prepared according to section 4.2.6) were added to each well
(100µL/well) and then incubated at 37°C for 2 h. Unattached S. aureus were
discarded and the wells were washed three times with 100µL PBS. Cells that
remained attached were fixed with 100µL of 25% v/v formaldehyde (Sigma
Aldrich) for 30 min at room temperate (24C), after which the wells were
washed three times with 100µL PBS. The fixed cells were stained with 100µL
of 0.5% w/v crystal violet solution (Sigma Aldrich) for 1 min and discarded.
The wells were washed twice with 100µL PBS and once with 200µL PBS. The
stain was solubilised with 100µL of 5% v/v acetic acid for 10 min at room
temperature (24C). The A570nm was measured in a microplate
spectrophotometer (Benchmark Plus Microplate Spectrophotometer, Bio-Rad).
64
3.2.8 Statistical data analyses of significance using Student’s t-test
All data from the S. aureus adherence assays represent the mean SD of at least
two different experiments performed in triplicate for each condition tested. An
exception was made for the collagen adherence assays and in the fibronectin
adherence assays (as indicated) where the data represent the mean SD of a
single experiment performed in triplicate for each condition tested. The
determination of statistical significance, as indicated by a two-tailed p value,
was performed using unpaired Student’s t-test as calculated by the software
GraphPad QuickCalcs (http://www.graphpad.com/quickcalcs/ttest1.cfm).
3.3 RESULTS
3.3.1 Expression of CyuC and CyuC-Lss confirmed by western blot
analysis
The expression of the recombinant CyuC and CyuC-Lss proteins from L. lactis
was confirmed by western blot analysis. Cell associated and supernatant
proteins from three different incubation times were analysed by western blotting
using mouse anti-His6 monoclonal antibodies (Figure 3.2). Previously, it has
been demonstrated that wild-type L. lactis do not contain any anti-His6 cross-
reactive proteins from the cell associated or supernatant fractions (Turner et al.,
2007b). Wild-type CyuC from L. reuteri BR11 resolves on SDS-PAGE as a 32-
kDa protein (Turner et al., 1997), whilst the recombinant CyuC resolved at
approximately 36-kDa (Figure 3.2). The recombinant CyuC is larger due to the
addition of the 6xhis tag and 5 amino acids from the mature Sep protein that
remained after the secretion signal has been processed. The lysostaphin protein
is predicted to be 29-kDa (Turner et al., 2007b) and resolved at approximately
36-kDa (Figure 3.2). The CyuC-Lss fusion protein is predicted to have a
combined size of 61-kDa and resolved at approximately 66-kDa (Figure 3.2).
The most recombinant CyuC and CyuC-Lss proteins were found in the cell
associated fractions of cultures after two days of incubation (Figure 3.2),
whereas equal amounts of lysostaphin were found in the cell associated and
65
supernatant fractions of late-log phase cultures. Therefore, to maximise the
amount of extractable recombinant CyuC and CyuC-Lss, the cultures were
incubated for two days, and the cells were lysed by homogenisation with 0.1mm
zirconia/silicone beads (section 3.2.5).
37
29
50
97104
3729
C S C S C S2 days O/N late-log
CyuC
CyuC-Lss
Lss
37
29
50
97104
3729
C S C S C S2 days O/N late-log
CyuC
CyuC-Lss
Lss
Figure 3.2. Expression of recombinant CyuC from L. lactis pGhost-CyuC,
CyuC-Lss from L. lactis pGhost-CyuC-Lss and Lss from L. lactis pGhost-his1-
lss-his2.
Cell associated (C) and supernatant (S) fractions were taken from cultures
incubated for two days, minimum 18 h (O/N), and until it reached late-log
phase. The amount of protein loaded was equivalent to 1mL of culture for cell
associated, and 900µL for supernatant fractions. Molecular mass standards are
indicated in kDa. Proteins were detected with mouse anti-His6 monoclonal
antibody.
66
3.3.2 Optimisation of ECM proteins used in the S. aureus adherence assay
The wells of a 96-wells flat-bottomed tissue culture plate were coated with
100µL of ECM proteins at the following concentrations: 1µg, 5µg, 10µg, and
20µg per mL. S. aureus cells were prepared as described in section 3.2.6 and
the adherence was measured as described in section 3.2.7. The results indicate
that at the concentration of 20µg ECM protein mL-1 the wells have almost
reached saturation point, as indicated by the maximal adhesion of S. aureus
observed (Figure 3.3). Therefore, 20µg ECM protein mL-1 was determined to
be the optimal concentration to be used in the S. aureus adherence assays.
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
1 5 10 20
ECM proteins (µg/mL)
A57
0nm
Collagen Keratin Fibronectin
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
1 5 10 20
ECM proteins (µg/mL)
A57
0nm
Collagen Keratin Fibronectin
Figure 3.3. Adherence of S. aureus to wells coated with four different
concentrations of collagen, keratin, and fibronectin.
Each data point represents the mean of replicate wells (n = 3). Error bars are
represented by ± SD.
67
3.3.3 L. lactis cell extracts containing recombinant proteins have no effect
on the adherence of S. aureus to immobilised collagen
Cell extracts from L. lactis pGhost9:ISS1, L. lactis pGhost-CyuC, L. lactis
pGhost-CyuC-Lss, and L. lactis pGhost9-his1-lss-his2 were added to wells pre-
coated with collagen, and the adherence of S. aureus was measured as described
in section 3.2.7. The results indicate that S. aureus adhered to wells coated with
collagen only (Figure 3.4, column PBS), and that cell extracts from L. lactis
pGhost9:ISS1, L. lactis pGhost-CyuC, L. lactis pGhost-CyuC-Lss, and L. lactis
pGhost9-his1-lss-his2 had no effect on the adherence of S. aureus (Figure 3.4).
0.0
0.5
1.0
1.5
2.0
2.5
PBS pG9 CyuC CLss Lss Blank
A57
0nm
0.0
0.5
1.0
1.5
2.0
2.5
PBS pG9 CyuC CLss Lss Blank
A57
0nm
0.0
0.5
1.0
1.5
2.0
2.5
PBS pG9 CyuC CLss Lss Blank
A57
0nm
Figure 3.4. Adherence of S. aureus to wells coated with collagen and exposed
to cell extracts from, L. lactis pGhost9-CyuC (CyuC), L. lactis pGhost9-CyuC-
Lss (CLss), and L. lactis pGhost9-his1-lss-his2 (Lss).
Wells containing collagen only (PBS) were used as a positive control for S.
aureus adherence, whilst wells treated with cell extracts from L. lactis
pGhost9:ISS1 (pG9) were used as a negative control for the L. lactis strains
expressing recombinant proteins. Blank refers to where S. aureus cells were
added to wells blocked with BSA only. The columns represent the mean of
replicate wells (n = 3). Error bars represent ± SD.
68
3.3.4 Adherence of S. aureus to fibronectin is inhibited by the cell extracts
of all L. lactis strains, including the L. lactis pGhost9:ISS1
Cell extracts from L. lactis pGhost9:ISS1, L. lactis pGhost-CyuC, L. lactis
pGhost-CyuC-Lss, and L. lactis pGhost9-his1-lss-his2 were added to wells pre-
coated with fibronectin, and the adherence of S. aureus were measured as
described in section 3.2.7. The results demonstrate that extracts from all the L.
lactis strains, including the strain containing the vector only (pGhost9:ISS1),
significantly inhibited the adherence of S. aureus to fibronectin (Figure 3.5).
0.00.20.40.60.81.01.2
PBS pG9 CyuC CLss Lss Blank
A 570
nm
0.00.20.40.60.81.01.2
PBS pG9 CyuC CLss Lss Blank
A 570
nm
Figure 3.5. Adherence of S. aureus to wells coated with fibronectin and
exposed to cell extracts from, L. lactis pGhost9-CyuC (CyuC), L. lactis
pGhost9-CyuC-Lss (CLss) or L. lactis pGhost9-his1-lss-his2 (Lss).
Wells containing fibronectin only (PBS) were used as a positive control for S.
aureus adherence, whilst wells treated with cell extracts from L. lactis
pGhost9:ISS1 (pG9) were used as a negative control for the L. lactis strains
expressing recombinant proteins. Blank refers to where S. aureus cells were
added to wells blocked with BSA only. All the columns represent the mean of
replicate wells (n = 9, except CLss and Lss, where n = 3). Error bars represent
± SD.
69
3.3.5 Adherence of S. aureus to keratin is inhibited by cell extracts from
L. lactis pGhost9-CyuC-Lss and L. lactis pGhost9-his1-lss-his2
Cell extracts from L. lactis pGhost9:ISS1, L. lactis pGhost9-CyuC, L. lactis
pGhost9-CyuC-Lss, and L. lactis pGhost9-his1-lss-his2 were added to wells pre-
coated with keratin, and the adherence of S. aureus were measured as described
in section 3.2.7. The results indicate that keratin coated wells treated with cell
extracts from L. lactis pGhost9-CyuC-Lss had significantly less adherent S.
aureus cells as compared to keratin coated wells treated with cell extracts from
L. lactis pGhost9:ISS1 (Figure 3.6). Cell extracts from L. lactis pGhost9-his1-
lss-his2 were able to completely inhibit the adherence of S. aureus to keratin
(Figure 3.6).
0.00.20.40.60.81.01.2
PBS pG9 CyuC CLss Lss Blank
A57
0nm
*
**
0.00.20.40.60.81.01.2
PBS pG9 CyuC CLss Lss Blank
A57
0nm
0.00.20.40.60.81.01.2
PBS pG9 CyuC CLss Lss Blank
A57
0nm
*
**
Figure 3.6. Adherence of S. aureus cells to wells coated with keratin and
exposed to cell extracts from L. lactis pGhost9-CyuC (CyuC), L. lactis pGhost9-
CyuC-Lss (CLss) or L. lactis pGhost9-his1-lss-his2 (Lss).
Wells containing keratin only (PBS) were used as a positive control for S.
aureus adherence, whilst wells treated with cell extracts from L. lactis
pGhost9:ISS1 (pG9) were used as a negative control for the L. lactis strains
expressing recombinant proteins. Blank refers to where S. aureus cells were
added to wells blocked with BSA only. All the columns represent the mean of
replicate wells (n = 12, except CLss and Lss, where n = 6). Error bars represent
± SD. * indicates p < 0.02 between CLss and pG9. ** indicates p < 0.0001
between Lss and pG9.
70
3.4 DISCUSSION
The aim of this study was to investigate the potential of lysostaphin and CyuC
when expressed by L. lactis, singularly or as a fusion protein, to act as S. aureus
antimicrobial agents. The two proteins (CyuC and CyuC-Lss) were expressed
in L. lactis using the Sep promoter and the Sep secretion signal, and were then
tested for their abilities to reduce S. aureus adhesion to the extracellular matrix
proteins fibronectin, collagen, and keratin.
Recombinant L. lactis strains were successfully generated which expressed and
secreted recombinant CyuC and CyuC-Lss using the Sep promoter and secretion
signal. Examination of the expression and secretion of recombinant CyuC and
CyuC-Lss by L. lactis by western blot analysis showed that substantial
quantities of CyuC and CyuC-Lss were present in the cell associated fractions
after two days of growth, whereas recombinant lysostaphin was present in
abundance at late-log phase (Figure 3.2). This expression and secretion profile
may be attributed to the nature of CyuC. CyuC is a positively charged protein
(pI of 10.6) which is anchored to the negatively charged cell wall by
electrostatic interactions (Turner et al., 1997). The absence of CyuC and CyuC-
Lss in the supernatant fractions from late-log phase and overnight cultures is
supported by previous studies where the yield of CyuC from log phase cultures
of L. reuteri BR11 was significantly lower than at stationary phase (i.e.
incubated for a minimum of 18 h) (M.S. Turner, personal communication). The
absence of CyuC and CyuC-Lss in the supernatant fraction of overnight culture
(i.e. incubated for a minimum of 18 h) is supported by the fact that the native
CyuC is not a secreted protein, i.e. not detected in the supernatant of stationary
phase cultures of L. reuteri BR11 (Turner et al., 1997). The Sep secretion
signal exports the recombinant CyuC and CyuC-Lss, but the positive charge of
CyuC likely anchors it to the negatively charged lactococcal cell surface. The
presence of CyuC and CyuC-Lss in the supernatant fraction after two days of
incubation is most likely due to cell lysis. Therefore, crude extracts of CyuC
and CyuC-Lss were made from harvesting whole cells incubated for two days
and lysed by homogenisation.
71
Recombinant CyuC, CyuC-Lss, and lysostaphin were investigated for their
ability to affect the adhesion of S. aureus to immobilised ECM proteins. Three
ECM proteins, type I collagen from calf skin, fibronectin purified from human
plasma and keratin type I from human epidermis, were selected for testing on
the basis of their importance in the pathogenesis of S. aureus. S. aureus
colonises the host by adhesion to components of the host ECM proteins
mediated by bacterial cell wall associated proteins called MSCRAMMs (Patti
and Hook, 1994; Foster and Hook, 1998). S. aureus can express up to 20
different potential MSCRAMMs that are covalently anchored to the cell wall
peptidoglycan through the action of sortase (Navarre and Schneewind, 1994;
Mazmanian et al., 1999). Specific MSCRAMMs have been identified that bind
to fibronectin (Signas et al., 1989; Jonsson et al., 1991), collagen (Switalski et
al., 1989), and keratin (O’Brien et al., 2002).
Crude cell extracts prepared from lactococcal cultures incubated for two days
were added into wells pre-coated with ECM proteins and then discarded before
S. aureus cells were added. By doing so, it was hypothesised that only CyuC or
CyuC-Lss would bind to the ECM proteins, thereby blocking the adhesion of S.
aureus. The results from the adhesion assays were different for each of the
three ECM proteins.
Cell extracts from all the lactococcal strains tested did not have any effect on the
adhesion of S. aureus to collagen (Figure 3.4). This may be due to the fact that
recombinant CyuC or CyuC-Lss did not strongly bind with the immobilised
collagen, and thus unable to affect S. aureus adhesion.
The S. aureus adhesion assay on immobilised fibronectin showed that the cell
extracts of all the recombinant lactococcal strains tested were able to completely
inhibit adhesion (Figure 3.5). In particular, it was observed that the negative
control strain, L. lactis pGhost9:ISS1, was also able to inhibit adhesion (Figure
3.5, column pG9). This observation was confirmed by repeated
experimentation. As such, it was concluded that any possible role that the
recombinant CyuC, CyuC-Lss and lysostaphin may play would be masked by
native lactococcal proteins. The mechanism by which L. lactis pGhost9:ISS1
72
was able to block S. aureus adhesion to fibronectin is unclear. L. lactis itself
does not express any surface proteins that adhere to fibronectin, and it is this
trait that lends itself to studies involving the recombinant expression of
fibronectin binding proteins from other bacteria, such as L. brevis S-layer
protein (Avall-Jaaskelainen et al., 2003), S. pyogenes M1 protein (Cue et al.,
2001), and S. aureus fibronectin binding proteins (Sinha et al, 2000; Que et al.,
2001). While L. lactis may not have any fibronectin adherence properties on the
cell surface, the cell extracts used in the adhesion assays also contain
intracellular proteins which may play a role. However, there has been no
previous study reporting the ECM protein adhesion properties of intracellular
proteins.
Unexpectedly, the cell extract from L. lactis pGhost9-his1-lss-his2 was able to
significantly inhibit the adherence of S. aureus to keratin (Figure 3.6). There
has been no previous study reporting the ability of lysostaphin to bind to keratin,
although it has been shown to bind to plastic and still maintained its lytic
activity against S. aureus (Shah et al., 2004a). A possible explanation of the
adherence assay results may be that lysostaphin was able to bind to the plastic
surface of the wells. However, this is unlikely to be the case as the wells were
blocked with BSA to ensure that proteins in the L. lactis cell extracts only came
in contact with the ECM proteins. It is also unlikely that lysostaphin bound to
BSA itself as 20µg keratin mL-1 resulted in saturation of the wells (as
interpreted by the maximal adhesion of S. aureus observed in Figure 3.3), thus
not leaving much surface area for BSA to block. Therefore, the results suggest
that lysostaphin has either bound to keratin and thus prevent S. aureus
adherence or has enzymatically degraded bound S. aureus cells, and that the
CyuC portion of the CyuC-Lss fusion protein reduced the ability of lysostaphin
to bind to keratin or reduced the enzymatic activity of lysostaphin. Irrespective
of either of these hypotheses, it is clear that lysostaphin has significant potential
in the prevention of S. aureus interaction on keratin rich surfaces. Therefore, to
enhance the production of lysostaphin by L. lactis, the aim of the next chapter
was to identify and characterise L. lactis mutants which are altered in their
lysostaphin producing abilities.
73
CHAPTER 4
LACTOCOCCUS LACTIS FACTORS INVOLVED IN THE
EXPRESSION AND SECRETION OF ANTIMICROBIAL
CELL WALL LYTIC ENZYMES
74
4.1 INTRODUCTION
The levels of heterologous proteins secreted by L. lactis and other lactic acid
bacteria are generally low, and efforts have been made to improve the secretion
efficiency by modifying secretion signal sequences (Dieye et al., 2001; Ravn et
al., 2003) inactivating proteases (Poquet et al., 2000; Miyoshi et al., 2002;
Cortes-Perez et al., 2006) or supplying heterologous protein secretion
machinery (Nouaille et al., 2006). Some insight into the heterologous protein
secretion mechanism of L. lactis was gained when Nouaille et al. (2004)
identified thirteen genes which affected the secretion efficiency of the
staphylococcal nuclease reporter enzyme (NucT) using random mutagenesis.
The inactivation of these genes resulted in either increased or decreased NucT
secretion efficiency. One gene of particular interest was dltA, which encoded a
protein that catalyses the incorporation of D-alanine residues into lipoteichoic
acids (LTA) which results in a greater net positive charge on the cell surface. It
was hypothesised that the inactivation of dltA modified the cell wall to become
negatively charged, thereby reducing the secretion efficiency of the positively
charged NucT by electrostatic interactions.
In this study, random transposon mutagenesis was used to identify genes which
affect the expression and/or the secretion of lysostaphin by L. lactis.
Interestingly, none of the genes identified in the Nouaille et al. (2004) study
were identified. Instead, four genes were identified which, when inactivated,
resulted in an increase in the amount of lysostaphin secreted by L. lactis. These
genes have not been previously characterised as associated with protein
secretion, and they are likely to be involve in the modification of the cell
envelope of L. lactis, much like dltA.
4.2 MATERIALS AND METHODS
4.2.1 Construction of a lysostaphin expressing L. lactis strain suitable for
random insertional mutagenesis
Previously, the pGhost9:ISS1 plasmid carrying a lysostaphin expression cassette
(lss) was used to express and secrete lysostaphin in L. lactis (Turner et al.,
2007b). In this cassette, the DNA sequence encoding the Sep promoter and the
75
Sep secretion signal was fused to the beginning of the gene encoding the mature
lysostaphin protein (Figure 4.1B). A 6xhis encoding DNA region was also
inserted into the expression cassette to allow the detection of the recombinant
protein. In order to stabilise the lysostaphin expression cassette and remove the
pGhost9 plasmid DNA from the cell, it was necessary to integrate it into the
chromosome (Figure 4.2C). The strategy to obtain the chromosomally modified
lysostaphin expressing L. lactis strain (MG1363[lss]) was as follows. The lss
expression cassette was ligated to two fragments of the inactive histidine
biosynthesis (his) operon (Delorme et al., 1993) with lss and his in the same
orientation (Figure 4.1B). Firstly, the upstream fragment (his1; 1-2-kb) was
amplified from chromosomal DNA extracts of wild-type L. lactis using the
primers His1Sal5 and His1Xba3 (Figure 4.1A, Table 4.1) and corresponds to
647-bp from the start of hisC to 816-bp from the start of hisZ. The downstream
fragment (his2; 1.2-kb) was amplified using His2Xho5 and His2Eco3 (Figure
4.1A, Table 4.1) and corresponds to 834-bp from the start of hisZ to 222-bp
from the start of hisD. Both his fragments were first cloned into plasmids (his1
into pBluescript II KS+, and his2 into pGEM-T Easy), and then transformed
into E. coli JM109 for routine cloning and sequencing. Once the his1 and his2
sequences were verified to contain the correct sequence, his1 was digested from
pBS-his1 using SalI and XbaI, and his2 from pGEMT-his2 using XhoI and
EcoRI. These fragments were then ligated to the lss expression cassette which
was isolated as a XbaI and XhoI digested fragment from pSep-6 x His-Lss
(Turner et al., 2007b). The ligation reaction was used as a template for
amplification by PCR using the primers His1Sal5 and His2Eco3. The resulting
3.9-kb PCR fragment was digested with SalI and EcoRI, and cloned into
pGEM-T Easy (pGEMT-his1-lss-his2). Once the whole fragment was verified
to contain the correct sequence, it was cloned into pGhost9:ISS1 (pGhost9-his1-
lss-his2), transformed into wild-type L. lactis and stable integration of lss into
the chromosome was performed in two steps. Firstly, L. lactis pGhost9-his1-
lss-his2 was incubated at 37°C for 18 h, and integrants were selected on a
GM17 agar plate containing 2µg erythromycin mL-1 (GM17+2Em). A colony
was isolated, and was confirmed as containing an integrated copy of pGhost9-
his1-lss-his in the his operon by PCR using the chromosomal DNA as template
and the primers lss-Pst and His2-DS (Figure 4.2B, Table 4.1). In addition, the
76
integrant was confirmed to have lysostaphin activity on GM17+SaB agar plates
containing 2g erythromycin mL-1 (GM17+SaB+2Em). Clones that express
and secrete lysostaphin will produce clearing zones (or halos) around the
colonies (Turner et al., 2007b). Next, to remove the pGhost9 plasmid from the
chromosome, the lysostaphin expressing strain was incubated at 30C in the
absence of erythromycin to stimulate recombination by allowing the plasmid to
replicate, and then incubated at 37°C to allow the loss of the excised plasmid
(Biswas et al., 1993). This recombination event can occur in two ways. The
first results in the recombination of the plasmid with the lss expression cassette
remaining in the chromosome (Figure 4.2C), whilst the second results in the
complete lost of the construct (Figure 4.2D). The resulting stable integrant,
MG1363[lss] (Figure 4.2C), was selected for its lysostaphin activity and
erythromycin sensitivity phenotype.
4.2.2 Construction of a L. lactis transposon library by random insertional
mutagenesis
The MG1363[lss] strain was transformed with pGhost9:ISS1 according to the
methods described in sections 2.2.2 and 2.2.6. A random transposon library was
prepared essentially as described by Maguin et al. (1996). Briefly, the
MG1363[lss] strain containing pGhost9:ISS1 was incubated for 18 h in
GM17+5Em medium. The saturated culture was diluted 100-fold in GM17
medium (without erythromycin), and incubated for 3 h at 30C, and then for 3 h
at 37C. The culture was then diluted so that approximately 50 colony forming
units (cfu) were plated on each GM17+SaB+2Em or GM17+SaU agar plates
containing 2µg erythromycin mL-1 (GM17+SaU+2Em), and incubated at 37C
for two days to select for pGhost9-transposed mutants (Figure 4.4B).
77
his1 (1.2-kb) his2 (1.2-kb)PSep
ssSep lss
6xhis
*B
hisC hisZ hisG hisD
His1Sal5 His1Xba3 His2Xho5 His2Eco3
A
pGhost9hisC hisZ hisZ hisG hisD
his1 (1.2-kb) his2 (1.2-kb)PSep
ssSep lss
6xhis
*B
hisC hisZ hisG hisD
His1Sal5 His1Xba3 His2Xho5 His2Eco3
A
pGhost9hisC hisZ hisZ hisG hisD
Figure 4.1. The regions of the his operon cloned from the chromosome (A) and the structure of pGhost-his1-lss-his2 (B).
The two his fragments were PCR amplified using the primers sets His1Sal5 and His1Xba3 (his1; white block arrows) and His2Xho5 and
His2Eco3 (his2; white block arrows). The lss expression cassette consists of the Sep promoter (PSep) and secretion signal (ssSep; hash box) with a
6xhis coding sequence (black box) fused to the 5’ of the lysostaphin gene (lss; dotted box). An asterisk indicates a stop codon at the end of the
lysostaphin gene.
78
hisC hisZ hisG hisD
hisD
A
B
C
pGhost9-his1-lss-his2
Ts repA
hisChisZ
lss
hisZhisG hisD
EmR
PSep
hisC hisZ hisZ
hisG
hisD
hisC hisZ hisG
hisC hisZ hisG hisD hisC hisZ hisG hisD
lss-Pst His2-DS
37°C
30°C
his1 his2
D
hisC hisZ hisG hisD
hisD
A
B
C
pGhost9-his1-lss-his2
Ts repA
hisChisZ
lss
hisZhisG hisD
EmR
PSep
hisC hisZ hisZ
hisG
hisD
hisC hisZ hisG
hisC hisZ hisG hisD hisC hisZ hisG hisD
lss-Pst His2-DS
37°C
30°C
his1 his2
D
79
Figure 4.2. Stable integration of the lss expression cassette into the L. lactis chromosome.
The construct pGhost9-his1-lss-his2 was integrated into the his operon (large block arrows) by single crossover homologous recombination
through the his2 region (A). The site of homologous recombination is marked with a cross. The integrant (B) was confirmed by amplification
by PCR using the primers lss-Pst and His2-DS (broken arrows). A shift of the integrant to 30°C stimulates recombination between the
duplicated his genes by allowing the plasmid to replicate, thus leading to plasmid excision. Excision through his1 gives rise to a stable
integration of the lss expression cassette (C), whereas excision through his2 restores the original chromosomal structure (D).
80
Table 4.1. Strains, plasmids, and oligonucleotides used in this chapter.
Details Source/Reference Strains E. coli JM109 L. lactis MG1363 L. lactis MG1363[lss] L. monocytogenes ATCC 19112 S. aureus ATCC 49476 Plasmids pBluescript II KS+ pGEM-3Zf pGEM-T Easy pGhost9:ISS1 pSep-6 x His-Lss pBS-his1 pGEMT-his2 pGEMT-his1-lss-his2 pGhost9-his1-lss-his2 pSep511sec Oligonucleotides His1Sal5 His1Xba3 His2Xho5 His2Eco3 lss-Pst His2-DS ISS1-seq1 ISS1-seq2
Cloning host Plasmid free L. lactis subsp. cremoris L. lactis carrying the lss cassette on the chromosome Ampr ; 3.0-kb Ampr ; 3.2-kb Ampr ; 3.0-kb Emr ori (Ts); 4.6-kb ; non-replicative in L. lactis at 37°C Ampr; pGEM-3Zf containing the PSep-ssSep-6xhis-lss expression construct (5.3-kb) Ampr ; pBS containing fragment of MG1363 his operon on the 5’ end of the lss cassette (4.2-kb) Ampr ; pGEM-T Easy containing fragment of MG1363 his operon on the 3’ end of the lss cassette (4.2-kb) Ampr ; pGEM-T Easy vector containing the lss cassette within the his operon (6.9-kb) Emr ; pGhost9:ISS1 containing the lss cassette within the his operon (7.6-kb) Emr ; pGhost:ISS1 containing the PSep-ssSep-6xhis-Ply511 expression construct (5.5-kb) 5’-CAGGTCGACCTTTGGGAGTCGCCTTTGGCT 5’-TACTCTAGACTGAAACATCAGCCCAGTATA 5’-AGACTCGAGGGCGCAAGCGACTATATCCGG 5’-TTCGAATTCTTCTTCGCGCTCCTTGCGGTG 5’-AACAGCTGCAGGAGCTGCAACACATGAACATTC 5’-TGTGATTTGGTACGACGCAGAATTCTAAAGT 5’-CACGATAGCTTAGATTGTAACG 5’-GAACCGAAGAAATGGAACGCTC
Promega
Gasson, 1983 This study
Promega Promega Promega
Maguin et al., 1996 Turner et al., 2007b
This study This study
This study This study
Turner et al., 2007b
This study This study This study This study
Turner et al., 2007b This study
Maguin et al., 1996 Maguin et al., 1996
81
Restriction enzyme recognition sites introduced by primers are underlined.
82
4.2.3 Screening the transposon library for mutants with altered
lysostaphin activity
The mutants were screened for the absence of halos or halos larger than that
produced by the other colonies on the agar plate, which would be indicative of
lower or higher lysostaphin activity, respectively. Any mutants which initially
appeared to be of interest were isolated and inoculated in GM17+2Em medium,
and then incubated at 37C. After 18 h of incubation, the mutants were plated
by 16-streak technique onto one side of a GM17+SaB+2Em agar plate and a
GM17+SaU+2Em agar plate, whilst the other side was similarly inoculated with
the control strain, MG1363[pGhost9-his1-lss-his2]. These plates were
incubated at 37C for two days after which the size of the halos produced by the
colonies of the mutant were then directly compared with the halos produced by
the colonies of the control strain. Mutants with different sized halos compared
with the control strain were retained for further investigation. The average
diameter of the halo of the control strain was 3.4 mm. Larger halos from the
selected mutants had diameters 0.8 mm to 2.8 mm greater than that produced by
the control strain.
4.2.4 Characterisation of the pGhost9:ISS1 insertion site and isolation of
stable ISS1-generated mutants
The plasmid pGhost9:ISS1 contains unique restriction enzyme recognition sites
adjacent to the ISS1 element (Figure 4.4A). These were used after transposition
to clone chromosomal DNA flanking the pGhost9:ISS1 insertion site as
previously described (Maguin et al., 1996). Briefly, chromosomal DNA was
isolated and subjected to either EcoRI or HindIII digestion. Following this, the
digested DNA was placed in a ligation reaction which allowed recircularisation
of the fragments, including the pGhost9:ISS1 plasmid containing chromosomal
DNA (Figures 5.3B and C). The ligated products were transformed into E. coli
JM109, and incubated at 30C for two days. The plasmids were purified and
submitted for sequencing using the primer ISS1-seq1 for the EcoRI
chromosomal junction or ISS1-seq2 for the HindIII junction (Figures 4.3B and
C; Table 4.1). The genes flanking the inserted transposon structure were
83
identified by sequence comparison with the sequenced genome of L. lactis
subsp. cremoris MG1363 strain (Wegmann et al., 2007).
For further analyses, mutants which contained only the ISS1 element on its
chromosome (herein referred to as ISS1-generated mutants; Figure 4.4C) were
generated from pGhost-transposed mutants by excising pGhost9:ISS1 from the
chromosome. This was to eliminate the erythromycin resistance marker and to
allow stability of the integrated ISS1-generated mutants element to grow at
30C, the optimal temperature for L. lactis. This was accomplished as described
by Maguin et al. (1996). Briefly, the pGhost9-transposed mutants were
incubated in GM17 medium at 37C for 18 h. The culture was diluted 106-fold
into GM17 medium and incubated at 30C for 18 h. This step stimulates
recombination between the duplicated ISS1 elements (Figure 4.4B) as this is the
permissive temperature for pGhost9:ISS1 plasmid replication. This gave rise to
a chromosomal structure whereby a single ISS1 element remained, thus
disrupting the gene (Figure 4.4C). Cultures were then diluted and plated onto
GM17 agar plates at 37C to prevent plasmid replication. Colonies were replica
plated onto GM17 agar plates with and without 5µg erythromycin mL-1.
Colonies in which excision had occurred and the plasmid was lost were
phenotypically sensitive to erythromycin. Where more than one mutant was
identified with insertion in the same gene, only one was selected to create a
temperature-stable mutant and used as a representative for further analyses.
These mutants were then retested for their lysostaphin activities by streaking a
loopful of overnight cultures onto one side of a GM17+SaB and GM17+SaU
agar plate, whilst the other side was similarly inoculated with the control strain,
MG1363[lss]. These plates were incubated at 30C for two days, after which
the size of the halos of the mutant colonies were then directly compared with the
control strain.
84
EcoRI HindIII
ISS1 ISS1pGhost9
EcoRIHindIII
ISS1-seq1 ISS1-seq2
ISS1
fragment ofchromosome
EcoRI
EcoRI
ISS1-seq1ISS1
fragment of chromosome
HindIII
HindIII
ISS1-seq2
A
B C
EcoRI HindIII
ISS1 ISS1pGhost9
EcoRIHindIII
ISS1-seq1 ISS1-seq2
ISS1
fragment ofchromosome
EcoRI
EcoRI
ISS1-seq1ISS1
fragment of chromosome
HindIII
HindIII
ISS1-seq2
A
B C
Figure 4.3. Representation of the pGhost9-transposed mutant between
duplicated ISS1 elements (A).
The primer used to sequence the chromosomal DNA included in pGhost9:ISS1
digested from EcoRI (B), or HindIII (C) is as indicated (broken arrow).
Symbols: white box and white block arrows, chromosomal DNA; green block
arrows, ISS1 transposon element; solid lines, pGhost9 plasmid; dotted arrows,
primer. EcoRI and HindIII sites are indicated.
85
ISS1
EcoRI
HindIII
pGhost9:ISS1
MG1363[lss] pGhost9:ISS1at 30°C
Shift temperature to 37°C and select for transposants
pGhost9-transposed mutant
ISS1 ISS1pGhost9:ISS1
A
B
Shift temperature to 30°C to stimulate plasmid replication and recombination between duplicated ISS1 elements
ISS1ISS1-generated mutant
C
ISS1
EcoRI
HindIII
pGhost9:ISS1
MG1363[lss] pGhost9:ISS1at 30°C
Shift temperature to 37°C and select for transposants
pGhost9-transposed mutant
ISS1 ISS1pGhost9:ISS1
A
B
Shift temperature to 30°C to stimulate plasmid replication and recombination between duplicated ISS1 elements
ISS1ISS1-generated mutant
C
Figure 4.4. Schematic representation of the creation of a mutant by random
transposon mutagenesis using pGhost9:ISS1 and the excision of the plasmid
from the chromosome.
L. lactis with pGhost9:ISS1 was cultured at 30°C (A). A shift in temperature to
37°C selected for pGhost9-transposed mutants (B). This structure must be
maintained at 37°C. Changing the temperature back to 30°C stimulated a
recombination event between the duplicated ISS1 elements by allowing the
plasmid to replicate. The result is a temperature stable ISS1-generated mutant
whereby a single ISS1 element remained in the chromosome thus disrupting the
gene (C). Symbols: white block arrows, chromosomal DNA; green block
arrows, ISS1 transposon element; solid lines, pGhost9 plasmid.
4.2.5 Prediction of operon structures
To determine if the genes containing ISS1 insertions were cotranscribed with
downstream genes, two operon prediction methods were used which are
available at http://www.microbesonline.org/operons (Price et al., 2005) and
http://operondb.cbcb.umd.edu/cgi-bin/operondb/operons.cgi (Ermolaeva et al.,
2001). The accuracy of the Price et al. (2005) method, which has been
estimated based on the prediction of experimentally proven operons and from
86
microarray expression data, is 82% for most genomes. The identification of
likely co-transcribed genes was predicted by at least one of these two methods.
4.2.6 Prediction of subcellular locations of proteins
Protein sequences were analysed using three freely available online programs:
SOSUI, a predictor of transmembrane regions (http://bp.nuap.nagoya-
u.ac.jp/sosui/) (Hirokawa et al., 1998); PHD transmembrane helix predictor
(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_phd.html)
(Rost et al., 1995); and TMpred, a predictor of transmembrane regions and
protein orientation (http://www.ch.embnet.org/software/TMPRED_form.html)
(Hofmann and Stoffel, 1993). .
4.2.7 Cell fractionation, protein extraction, SDS-PAGE, and western blot
L. lactis strains of interest were incubated for 18 h, diluted 50-fold into 10mL of
fresh medium, and incubated at 30°C (or at 37°C as indicated) until the OD600nm
reached 1.0. The cells were pelleted by centrifugation at 3000 – 5000 x g for 10
min at 4°C. Proteins in the supernatant were precipitated using TCA as
described in section 2.6.2. The protein pellet was resuspended in either 20L or
100L of 50mM NaOH and equal volumes of 2x SDS loading buffer. The cell
pellet was resuspended in 50L of 2x SDS loading buffer giving a total volume
of 200L per 10mL of culture.
The protein samples were heated at 97C for 5 min and separated by SDS-
PAGE as described in section 2.6.1. The gel was then either stained with
Coomassie for the visualisation of protein bands or transferred to nitrocellulose
membrane and the proteins were detected as described in section 2.6.3.
To semi-quantify the amount of proteins, films which have not been over
exposed were scanned (CanoScan800F, Canon), and the total amount of his-
labelled proteins from the supernatant and cell associated fractions were
estimated with the GeneTools software (Syngene). An example is shown in
Figure 4.5 where a band from MG1363[lss] and the murA2[lss] mutant were
compared using the GeneTools software.
87
MG[seplss] 31397 (murA2)
37
29
kDa 900 450 90 45 900 450 200
MG[seplss] 31397 (murA2)
37
29
kDa37
29
kDa 900 450 90 45 900 450 200
Figure 4.5. Example of a western blot used in semi-quantification.
MG1363[lss] and the L. lactis mutant strain 31397 were grown at 37C. The
numbers above the western blot protein bands represent the amount of culture
supernatant loaded in each lane in µL. Bands which did not suffer from over
exposure from MG1363[lss] (lane 450) and murA2[lss] mutant (lane 45) were
then compared using the GeneTools software. Molecular mass standards are
expressed in kilodaltons (kDa). Previous work has shown that supernatant and
cell associated extracts of wild-type L. lactis do not contain any proteins which
react with anti-His6 antibodies (Turner et al., 2007b).
4.2.8 Endolysin Ply511 expression and secretion in [lss] mutant strains
The L. monocytogenes bacteriophage endolysin Ply511 was transformed into
wild-type L. lactis, the control strain (MG1363[lss]), the lom[lss], murA2[lss],
and trmA[lss] mutant strains. The Ply511 gene was introduced into these strains
using the pGhost9:ISS1 plasmid under the control of the Sep expression system
(pSep511sec; Table 4.1) (Turner et al., 2007b). The transformed strains were
tested for Ply511 activity against L. monocytogenes cells on GM17+LmB agar
plates containing 5µg erythromycin mL-1 (GM17+LmB+5Em) (Turner et al.,
2007b). Ply511 was also detected on western blots using anti-His6 monoclonal
antibody as described in section 4.2.7.
4.2.9 Lysozyme resistance test
The lysozyme resistance test was performed as described by Veiga et al. (2007).
Briefly, solutions of chicken egg white lysozyme (Sigma Aldrich) in GM17
were freshly prepared and diluted 10-fold into molten GM17 agar at 45°C.
88
Then, 5µL of cultures incubated for 18 h were diluted 10-fold and then spotted
onto the GM17 agar containing different concentrations of lysozyme. The
plates were then incubated at 30°C or 37°C as indicated.
4.2.10 Transmission electron microscopy (TEM)
Cultures incubated for 18 h were diluted 100-fold and incubated at either 30ºC
or 37ºC. After 3, 6, and 20 h of incubation, 100µL of culture was fixed in 0.4%
v/v glutaraldehyde (ProSciTech), 100mM cacodylate buffer (pH 7.3). After 18
h of fixative, the cells were washed in 100mM cacodylate buffer. Cells were
postfixed in 1% w/v osmium tetroxide and embedded in Spurr expoxy resin.
Ultrathin sections (50 – 100 nm) were cut and stained with 2% w/v uranyl
acetate and 0.1% w/v lead citrate prior to examination and photography using
the JOEL 1200 EX transmission electron microscope. Ultrathin sectioning and
TEM photography were conducted by Dr Christina Theodoropoulos (Analytical
Electron Microscopy Facility, QUT).
4.2.11 Statistical data analysis of significance using Student’s t-test
Statistical data analysis was performed using unpaired Student’s t-test as
calculated by the software GraphPad QuickCalcs
(http://www.graphpad.com/quickcalcs/index.cfm).
4.2.12 Alignment and phylogenetic analysis
The protein sequences were aligned using the program CLUSTALX as
implemented by the MEGA4 software (Tamura et al., 2007) with the gap
opening and extension penalties of 10.0 and 0.2, respectively. Phylogenetic tree
was constructed by neighbour-joining method using MEGA4 based on pairwise
distances between amino acid sequences (Saitou and Nei, 1987).
4.3 RESULTS
4.3.1 Isolation and identification of mutants with altered lysostaphin
activity
In total, 35,881 MG1363[lss] mutant clones were screened for increase in or the
absence of lysostaphin activity on GM17+SaB+2Em and GM17+SaU+2Em
89
agar plates. On that basis, 124 mutants were initially selected and their
lysostaphin activity was directly compared to the appropriate control strain,
MG1363[pGh-his1-lss-his2], by growing both on the same agar plate. Ten of
the 124 initial mutants were confirmed to have a halo size greater than that of
MG1363[pGh-his1-lss-his2], whilst one mutant was confirmed as having no
halo. The locations of the transposed pGhost9:ISS1 were identified by
comparison of the flanking sequences with the L. lactis MG1363 genome
sequence (Wegmann et al., 2007) (Table 4.2). The mutant with no lysostaphin
activity resulted from the transposition of pGhost9:ISS1 into the lss expression
cassette. The other mutants were located in four separate genes: llmg_0609
(three mutants), murA2 (five mutants), trmA (one mutant), and llmg_2148 (one
mutant).
The plasmid pGhost9:ISS1 was excised from all mutants to create ISS1-
generated mutants (Figure 4.4C). These ISS1-generated mutants were streaked
onto GM17+SaB (without erythromycin) to reconfirm the lysostaphin activity
phenotype and also to compare against the control strain, MG1363[lss]. The
llmg_0609 and llmg_2148 mutants retained their increased lysostaphin activity
at 30°C, whilst the large zones of activity were only observed at 37°C in the
murA2 and trmA mutants. The amount of lysostaphin secreted, generation time
and stationary culture pH were measured for all mutants (Table 4.2). The
increase in lysostaphin activity of the ten mutants was determined by western
blot analysis to be due to an increase in the amount of lysostaphin as compared
with the control strain, MG1363[lss] (Figure 4.6, Table 4.2 shaded column). The
levels were determined by direct comparison to the amount secreted by
MG1363[lss] which was assigned a value of 1.0 (Table 4.2). The average
increases (± standard deviation) in the lysostaphin levels in the cell extracts and
supernatants of the three llmg_0609 mutants were 3.0-fold (±0.2-fold), and
12.0-fold (±3.6-fold), respectively; both levels were significantly higher than
those of the control strain (p < 0.01). The average increase (± standard
deviation) in the lysostaphin level in the supernatants of the five murA2 mutants
grown at 37C was 6.2-fold (±0.7-fold), and the level was significantly higher
than that of the control strain (p < 0.01).
90
4.3.2 Characterisation of the genes which affected lysostaphin secretion
4.3.2.1 The gene llmg_0609 is incorrectly annotated and is renamed lom
Three independent ISS1 insertions were found in the gene llmg_0609 (Figure
4.6A, Table 4.2) which is annotated in the L. lactis MG1363 genome to
putatively encode for an enzyme (PabC) which functions as a 4-amino-4-
deoxychorismate lyase (ADC lyase). It is unlikely that llmg_0609 is the pabC
gene as it has recently been proposed that the open-reading frame of the true
pabC gene in L. lactis MG1363 is contained within the llmg_1154 open-reading
frame as a fusion to pabB (Wegkemp et al., 2007). Furthermore, a comparative
alignment of llmg_0609 to the pabC gene sequences from E. coli (Green et al.,
1992) and B. subtilis (Slock et al., 1990) did not reveal any similarities.
Therefore, it is proposed that llmg_0609 be renamed as lom (lysostaphin
oversecreting mutant) to avoid confusion.
An in silico approach was taken in an attempt to gain an insight into what may
be the true function of lom. Searches through the Genbank database using the
blastp and tblastx algorithms (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi)
revealed significant protein sequence homologies to putative ADC lyase from
Streptococcus species as well as to streptococcal proteins with predicted solute-
binding functions. However, none of these homologous proteins have been
subjected to functional analyses. Protein sequence analysis of Lom revealed a
single transmembrane helix predicted to lie in the central region (amino acids
177 to 195; IITVIVVLLILVVGGTGWY) (Figure 4.6A) with the C-terminal
region of the protein extending outside of the membrane.
91
Table 4.2. Characteristics of mutants with lysostaphin activity greater than that of the wild-type.
Levels of Lysostaphin
Mutant Genea
Homologous
proteins Predicted functionb
Downstream gene possibly affected by ISS1 insertion
(predicted function)c Cell
associatedd Supernatantd Generation
time (mins)e pHf MG1363[lss] 1.0 (1.0) 1.0 (1.0) 67 (91)g 4.3 2190 llmg_0609 Unknown (membrane location
predicted) greA (transcription elongation factor)
3.1 9.8 59 4.3
11135 “ “ 3.1 10.1 61 4.5 24185 “ “ 2.8 16.1 56 4.4 18662 llmg_0517 MurA2 Uridine diphosphate (UDP)-
N-acetylglucosamine enolpyruvyl transferase
llmg_0518 (unknown function) 3.0 (0.8) 3.4 (6.2)g 63 (75)g 4.3
24189 “ “ “ 0.7 (2.0)g 1.3 (5.4)g 62 (76)g 4.3 28801 “ “ “ 1.3 (nd)g 1.3 (7.2)g 59 (79)g 4.3 31394 “ “ “ nd (nd)g 2.4 (6.0)g 57 (63)g 4.3 31397 “ “ “ nd (nd)g 2.8 (6.4)g 64 (72)g 4.3 16270 llmg_0640 TrmA (Spx) Temperature resistance;
transcription regulation None 1.9 (1.2)g 4.3 (7.2)g 77 (55)g 4.3
2194 llmg_2148 UbiG 3-demethylubiquinone-9 3-methyltransferase
fmt (methionyl-tRNA fomyltransferase)
3.3 6.6 56 4.4
a Gene in which pGhost9:ISS1 integration occurred. b Predicted function as determined by annotation from MG1363 genome available on Genbank. c Downstream genes that are likely located in an operon with the gene containing the ISS1 element were predicted by computational methods (section 5.2.5). d The amount of lysostaphin secreted was determined by western blot analysis of the total amount of lysostaphin in the cell associated or supernatant fractions in comparison to MG1363[lss], which is assigned the index 1.0. e Generation time was calculated based on the exponential growth phase (Madigan et al., 2001). f pH was measured from overnight cultures grown in GM17 medium supplemented with a final concentration of 1% w/v glucose at 30°C. g Figures in parentheses are measurements taken from cultures grown at 37°C. h nd indicates that lysostaphin was not detected in the cell associated fraction by western blot analysis.
92
rpoE llmg_0609 greA
transmembrane helix
2418
5
1113
521
90
hyp protapt
hyp prot
suhB murA2 hyp prot tig
1866
224
189
2880
131
394
3139
7
clpP hyp prot trmA hyp protput prot
1627
0
A
C
E
2937
11135MG1363
20L200L
[lss][lss]
30°C30°C
2937
28801MG1363
90L450L
[lss][lss]
37°C37°C
16270MG1363
45L200L
[lss][lss]
37°C37°C
2937
B
D
F
rpoE llmg_0609 greA
transmembrane helix
2418
5
1113
521
90
hyp protapt
hyp prot
suhB murA2 hyp prot tig
1866
224
189
2880
131
394
3139
7
clpP hyp prot trmA hyp protput prot
1627
0
A
C
E
29372937
11135MG1363
20L200L
[lss][lss]
30°C30°C
2937
28801MG1363
90L450L
[lss][lss]
37°C37°C
16270MG1363
45L200L
[lss][lss]
37°C37°C
29372937
B
D
F
93
Figure 4.6. Identification of the insertion sites for the nine over-secreting mutants.
(A) Locations of the three mutants in the llmg_0609 gene at nucleotide positions 818 (mutant 24185), 1354 (mutant 11135), and 1408 (mutant
2190) from the start of the gene. The predicted single transmembrane helix is located from amino aids 177 to 195 (dotted box). (B) A
representative western blot of lysostaphin in the supernatant fractions from MG1363[lss] and mutant 1135[lss] grown at 30ºC. (C) The five
independent insertions in the murA2 gene are located at nucleotide positions 116 (mutant 28801), 753 (mutant 24189), 766 (mutant 31397), 1050
(mutant 31394), and 1220 (mutant 18662). (D) A representative western blot of lysostaphin in the supernatant fractions from MG1363[lss] and
mutant 28801[lss] grown at 37ºC. (E) The single mutation of the trmA gene (mutant 16270) is located 34-bp upstream of the start of the gene.
(F) A representative western blot of lysostaphin in the supernatant fractions from MG1363[lss] and mutant 16270[lss] grown at 37ºC. The
variable supernatant quantities loaded in all the western blots are indicated in the tables directly above the blots.
94
4.3.2.2 The murA2 gene encodes for the primary MurA in L. lactis
Five mutations were identified in the murA2 gene (Figure 4.6C, Table 4.2).
This gene putatively encodes UDP-N-acetylglucosamine enopyruvyl transferase
(MurA), which catalyses the first step in the biosynthesis of peptidoglycan
(Marquardt et al., 1992). As with other low-G+C Gram-positive bacteria (Du et
al., 2000), L. lactis has two genetic copies of the MurA enzymes (MurA1 and
MurA2) (Wegmann et al., 2007). MurA2 in L. lactis is more closely related to
enzymes in related species that are annotated as the primary MurA enzyme. L.
lactis MurA2 is 61% and 40% identical to the B. subtilis MurAA and MurAB
proteins, respectively (Figure 4.7).
L. lactis MurA2
S. pneumoniae MurA1
B. subtilis MurAA
E. coli MurA
B. subtilis MurAB
L. lactis MurA1
S. pneumoniae MurA2100100
10099
0.05
Primary MurA enzyme
L. lactis MurA2
S. pneumoniae MurA1
B. subtilis MurAA
E. coli MurA
B. subtilis MurAB
L. lactis MurA1
S. pneumoniae MurA2100100
10099
0.05
L. lactis MurA2
S. pneumoniae MurA1
B. subtilis MurAA
E. coli MurA
B. subtilis MurAB
L. lactis MurA1
S. pneumoniae MurA2100100
10099
0.05
Primary MurA enzyme
Figure 4.7. Phylogenetic tree showing L. lactis MurA2 is more closely related
to the primary MurA in other species.
The tree was constructed by the neighbour-joining method. The scale bar
represents 0.05 expected amino acid replacements per site. The percentage of
replicate trees in which the associated taxa clustered together in the bootstrap
test (1000 replicates) are shown next to the branches.
The lysostaphin over-secretion phenotype of the murA2 mutants was observed
under heat stress at 37C (Table 4.2). As the function of MurA is in
peptidoglycan biosynthesis, the thickness of the cell walls of the murA2 mutants
was examined under TEM. Examinations of the thickness of the cell walls
(Figure 4.8) did not reveal any gross changes between the murA2 mutants and
the MG1363[lss] strain at both 30°C and 37°C. Similarly, Coomassie-stained
SDS-PAGE analysis of proteins from supernatant fractions showed that the
murA2 mutants (and other mutants) did not release greater amounts of
intracellular proteins than the control strain (MG1363[lss]) (Figure 4.9).
95
100nm
A B
C D
100nm
100nm 100nm
Cell membrane
Outer edge of cell wall
100nm100nm
A B
C D
100nm100nm
100nm100nm 100nm100nm
Cell membrane
Outer edge of cell wall
Figure 4.8. Transmission electron micrographs of the control strain,
MG1363[lss] (A, B) and the murA2[lss] mutant (C, D).
Overnight cultures of both strains were diluted 100-fold and cultured at 30C
(A, C) and 37C (B, D) for 6 h. The thickness of the cell wall was determined
by measuring the distance between the cell membrane and the outer edge of the
cell wall on the TEM micrographs.
96
M 1 2 3 4 5 6 7 8 9
20
2935
4890
128
30C 37C
Usp45
Figure 4.9. Coomassie-stained SDS-PAGE of proteins from the supernatant
fractions.
The lanes on the SDS-PAGE are as follows: pre-stained SDS-PAGE standards
indicated in kDa (lane M); the control strain, MG1363[lss] (lanes 1 and 5),
trmA[lss] mutant (lanes 2 and 6), lom[lss] mutant (lane 3), and murA2[lss]
mutant (lanes 4 and 7). The level of Usp45, an abundantly secreted
extracellular protein of unknown function (Van Asseldonk et al., 1990), was
used as an approximate indicator of the total amount of proteins loaded onto the
gel. The control and mutant strains were incubated at either 30C or 37C for 18
h and the supernatant fraction was processed according to section 4.2.7.
4.3.2.3 More lysostaphin is secreted by the trmA[lss] mutant strain under
high temperature stress
A single insertion was identified 34-bp upstream of the trmA gene (Figure 4.6E,
Table 4.2). TrmA has homology to Spx, an oxidative stress regulator in B.
subtilis (Nakano et al., 2003), and there are seven genes in the trmA family
encoded by the MG1363 genome (Wegmann et al., 2007). It is interesting to
note that the trmA mutant in this study secreted more lysostaphin under heat-
stress conditions at 37C (Table 4.2), despite a previous report that the absence
of TrmA stimulated an increase in the proteolytic activity of the intracellular
protease, ClpP (Frees et al., 2001)
4.3.2.4 Basis for lysostaphin secretion in llmg_2148[lss] mutant is unclear
Finally, one of the mutants selected from the screening had an inactivation in
the gene annotated as llmg_2148 (Table 4.2). The basis for the increased
97
lysostaphin secretion remains unclear. This gene is annotated as a putative
enzyme, 3-demethylubiquinone-9 3-methyltransferase (UbiG; Wegmann et al.,
2007), which in E. coli is involved in ubiquinone biosynthesis (Stroobant et al.,
1972). However, this annotation may be inaccurate as llmg_2148 has no
similarity to functionally characterised UbiG enzymes in the databases.
4.3.3 The lom, murA2, and trmA mutant strains secrete higher levels of
the cell wall hydrolytic enzyme, Ply511, compared to wild-type
To examine whether the increase in lysostaphin secretion is specific to
lysostaphin, the ability of the wild-type L. lactis, the control strain
(MG1363[lss]), and the lysostaphin secreting mutant strains (lom[lss],
murA2[lss], and trmA[lss]) to express another heterologous protein, Ply511, was
compared. Ply511 is a peptidoglycan hydrolytic endolysin (N-acetylmuramoyl-
L-alanine amidase) from L. monocytogenes bacteriophage (Loessner et al.,
1995a). All strains transformed with the Ply511 expression cassette
(pSep511sec; Table 4.1) demonstrated activity against both L. monocytogenes
and S. aureus, as evidenced by zones of clearing on agar plates containing
autoclaved cells. The zones of activity of Ply511 on GM17+LmB+5Em agar
plates were compared, and the diameters of the activity zones between the
control and the mutant strains were found to be the same. Zones of clearing of
the mutant and the control strains on agar plates could not be adequately
captured to an adequate resolution using a digital camera or laboratory gel
documentation system and therefore data could not be shown. The amount of
Ply511 secreted into the supernatant was examined using western blot analysis.
The amounts of recombinant Ply511 found on the cell associated fraction and
secreted into supernatant by the mutant strains, lom[lss][511], murA2[lss][511],
and trmA[lss][511] were greater than the control strain, MG1363[lss][511]
(Figure 4.10).
98
293750
293750
lysostaphin293750
293750
293750
293750
Cell associated Supernatant
Ply511
[511][511][511][511]
1ml1ml1ml1ml
[lss][lss][lss][lss]
trmAmurA2lomMG1363
450µl450µl450µl900µl
[lss][lss][lss][lss]
[511][511][511][511]
trmAmurA2lomMG1363
293750
293750
lysostaphin293750
293750
293750
293750
Cell associated Supernatant
Ply511
[511][511][511][511]
1ml1ml1ml1ml
[lss][lss][lss][lss]
trmAmurA2lomMG1363
450µl450µl450µl900µl
[lss][lss][lss][lss]
[511][511][511][511]
trmAmurA2lomMG1363
Fgure 4.10. Western blot detection of L. lactis strains secreting lysostaphin and Ply511 in the cell associated and supernatant fractions.
The different strains and the quantities loaded on the western blot are indicated in the table. Molecular mass standards are expressed in kDa.
The amount of cell associated protein is equivalent to 1mL of late-exponential phase culture. The amount of supernatant protein loaded in each
lane is equivalent of 900L of late-exponential phase culture for the control strain (MG1363[lss][511]) and 450L for mutant strains. The
Ply511 protein resolved at approximately 40kDa. The lysostaphin protein resolved at approximately 31kDa.
99
4.3.4 The murA2 and trmA mutants were more resistant to lysozyme
hydrolysis
A recent study has shown that a mutation in trmA results in lysozyme resistance
in MG1363 (Veiga et al., 2007). In this study, the trmA mutant which expresses
lysostaphin (trmA[lss]) was also more resistant to lysozyme than the wild-type
expressing lysostaphin (MG1363[lss]) (Figure 4.11). Interestingly, the control
strain, MG1363[lss], was observed to be much more sensitive to lysozyme
hydrolysis compared with the non-lysostaphin expressing wild-type strain,
MG1363 (Figure 4.11B). It was also observed that the murA2[lss] mutant strain
was moderately more resistant to lysozyme than the control strain
(MG1363[lss]) (Figure 4.11B).
4.4 DISCUSSION
The aim of this study was to identify L. lactis factors which affect the secretion
efficiency of the peptidoglycan hydrolase lysostaphin, and mutants which
overproduce this important antimicrobial enzyme. A previously described
method was adapted in this study whereby random mutagenesis (using the ISS1
transposon in the plasmid pGhost9:ISS1) was used to identify factors which
affected the secretion of the staphylococcal nuclease reporter (NucT) in L. lactis
(Nouaille et al., 2004). In this study, a L. lactis strain was constructed in which
the lysostaphin gene was integrated into the chromosome under the control of
the Sep promoter and secretion signal. Transposon mutagenesis was then
performed on this strain to generate mutants which produced higher or lower
lysostaphin activities.
In total, 35,881 MG1363[lss] transposon mutants were screened and eleven
clones were identified which had altered lysostaphin activity. As ISS1 insertion
is reportedly random (Maguin et al., 1996), the number of mutants screened
theoretically corresponds to approximately 14-fold coverage of the L. lactis
MG1363 genome, approximately 2.53-Mbp (Wegmann et al., 2007). Nouaille
et al. (2004) expressed reservations that pGhost9:ISS1 insertional transposition
was totally random as they had screened over 35,000 mutants, and yet did not
obtain any mutants with an insertion in the nucT expression cassette thus
causing the abolition of NucT expression. In contrast, this study identified a
100
A0 10-1 10-2 10-3 10-4
B
MG1363
MG1363[lss]
lom[lss]
murA2[lss]
trmA[lss]
MG1363[lss]
murA2[lss]
trmA[lss]
C
dilutions of cultures
MG1363
MG1363
0.25mg lysozyme mL-1
at 30C
0.25mg lysozyme mL-1
at 37C
A0 10-1 10-2 10-3 10-4
B
MG1363
MG1363[lss]
lom[lss]
murA2[lss]
trmA[lss]
MG1363[lss]
murA2[lss]
trmA[lss]
C
dilutions of cultures
MG1363
MG1363
A0 10-1 10-2 10-3 10-4
B
MG1363
MG1363[lss]
lom[lss]
murA2[lss]
trmA[lss]
MG1363[lss]
murA2[lss]
trmA[lss]
C
dilutions of cultures
MG1363
MG1363
0.25mg lysozyme mL-1
at 30C
0.25mg lysozyme mL-1
at 37C
Figure 4.11. Dilutions of cultures incubated for 18 h spotted onto GM17 agar
with various concentrations of lysozyme.
All strains showed identical growth on GM17 agar plates without lysozyme at
30°C and 37°C. Therefore, only the growth of MG1363 is shown. (A) 0mg
lysozyme mL-1 at 30°C, (B) 0.25mg lysozyme mL-1 at 30°C, (C) 0.25mg
lysozyme mL-1 at 37°C. The trmA[lss] mutant strain is more resistant to
lysozyme hydrolysis than the control strain, MG1363[lss].
single mutant in which the transposon had inserted into the lysostaphin
expression cassette. The other ten mutants were all confirmed to secrete higher
level of lysostaphin than the control, MG1363[lss] (Table 4.2). Examination of
the L. lactis MG1363 genome showed that it is possible that the llmg_0609,
murA2, and llmg_2148 gene could be part of different operons. Downstream
genes predicted to be located in these operons by at least one of two
computational prediction programs (section 4.2.5) are described in Table 4.2.
Transcript analysis by Dupont et al. (2004) has shown that, due to the presence
of a promoter in the ISS1 element, ISS1 (and pGhost9:ISS1) insertion does not
lead to polar effects on downstream genes. Such effects are observed, however,
101
when the ISS1 sequence, which encodes a putative transposase, is orientated in
the same direction as the interrupted genes. In this study, the ISS1 elements
were orientated in the same direction as the interrupted genes in two llmg_0609
mutants (2190 and 24185) and two murA2 mutants (18662 and 31397). In these
mutants, it would be expected that the genes downstream would be transcribed
and that the phenotypes observed would not be due to polar effects. In the
llmg_2148 mutant, the ISS1 element is in the opposite orientation to the
interrupted gene and therefore may affect the transcription of the downstream
fmt gene (Table 4.2). It should also be noted that the ISS1 element in mutant
16270 has inserted 34bp upstream of the trmA gene and in the opposite direction
and is therefore expected to prevent its transcription.
This study then focused on the further characterisation of the lom, murA2, and
trmA mutants, as lom and murA2 had several independent insertional mutants
and the inactivation of trmA had been identified in other random mutagenesis
studies (Duwat et al., 1999; Frees et al., 2001; Turner et al., 2007a).
Three mutants had independent insertions in a gene encoding an uncharacterized
putative transmembrane protein (llmg_0609). This gene is annotated in
Genbank as pabC, and putatively encodes for an enzyme which functions as an
ADC lyase. In other bacterial systems, ADC lyase catalyses the third step in the
biosynthesis of para-aminobenzoate acid (pABA) and exists as part of an operon
with pabA and pabB. A recent study has proposed that the true L. lactis pabC is
fused to pabB (Wegkemp et al., 2007). Therefore, to avoid confusion,
llmg_0609 is herein referred to as lom, a gene of unknown function. Analyses
of the Lom protein sequence by three independent computer programs predicted
a single transmembrane helix spanning amino acids 177 to 195 which is located
in the central region of the protein (Figure 4.6A). This is further evidence that
lom has no similarities to the cytosolic PabC, and that its role in lysostaphin
secretion may be as a transmembrane protein. Therefore, the protein Lom is
likely to be localised in the membrane.
Five mutants had independent insertions in the murA2 gene, which encodes for
a putative UDP-N-acetylglucosamine enopyruvyl transferase (MurA). This
102
enzyme catalyses the first step in the peptidoglycan biosynthesis pathway
(Marquardt et al., 1992). As with other low-G+C Gram-positive bacteria (Du et
al., 2000), L. lactis has two MurA enzymes (MurA1 and MurA2) (Wegmann et
al., 2007). Despite the designation, MurA2 showed greater homologies to B.
subtilis MurAA (61%) and S. pneumoniae MurA1 (70%). Additionally, all
three are more similar to the MurA in E. coli and other bacteria with just one
MurA enzyme, than are the other MurA paralogues in the Gram-positive species
(Figure 4.7). The simplest explanation for this is that the L. lactis MurA2, B.
subtilis MurAA, and S. pneumoniae MurA1 are orthologues of E. coli MurA
and may represent the primary MurA. Previous studies have identified MurA2
proteins in the cytoplasm of wild-type L. lactis subsp. lactis IL1403 (Guillot et
al., 2003), and in particular, up-regulated in response to acid stress in two L.
lactis MG1363 mutants (Budin-Verneuil et al., 2007). Lysostaphin over-
production at high temperature in the murA2 mutants is most likely due to cell
wall modifications. Du et al. (2000) demonstrated that MurA1 and MurA2 were
both enzymatically functional and could substitute for each other in S.
pneumoniae. In contrast, MurAB could not substitute for MurAA in B. subtilis
(Kock et al., 2004). As the lysostaphin-secreting mutants did not have any
growth defects, it is likely that the murA1 paralogue is able to substitute
functionally at 30C, but not completely at 37C. Mutations in murA2 did not
have any obvious affect on the morphology or the thickness of the cell walls
(Figures 4.8 and 4.9).
One mutant contained an insertion upstream of the trmA gene. It is interesting
to note that two previous studies using pGhost9:ISS1 random mutagenesis in L.
lactis also identified insertional mutants upstream of trmA, strongly indicating
that this gene may not contain any ISS1-specific recognition sites (Duwat et al.,
1999; Frees et al., 2001; Turner et al., 2007a). The inactivation of this gene has
been reported to confer various stress-resistant phenotypes. Various random
mutagenesis studies identified trmA mutants as able to relieve temperature
sensitivity in recA (Duwat et al., 1999) and clpP (Frees et al., 2001) mutant
strains, and conferred resistance to tellurite and oxidative stress in the wild-type
strain (Turner et al., 2007a). In addition, a trmA mutant has increased resistance
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against the hydrolytic activity of lysozyme in the wild-type strain, due to
regulation of genes involved in the modification of cell wall acetylation (Veiga
et al., 2007). Veiga et al. (2007) hypothesised that TrmA competes with SpxB,
one of its paralogues, for the binding of RpoA, the α-subunit of RNA
polymerase. The inactivation of trmA may allow SpxB to access RpoA, and this
interaction activates the expression of oatA, encoding the lactococcal
peptidoglycan O-acetylase, thus modifying the cell wall to become lysozyme
resistant (Veiga et al., 2007). In this study, it was observed that the wild-type
strain expressing lysostaphin (MG1363[lss]) was more sensitive to lysozyme
compared with the non-lysostaphin secreting wild-type, MG1363 (Figure
4.12B). This result suggests that lysostaphin may be attacking lactococcal
peptidoglycan during its passage through the cell wall, thereby making it weaker
and more susceptible to lysozyme. According to Veiga et al., (2007) the
resultant interactions within a trmA mutant give rise to changes in acetylation
and thickness of peptidoglycan, thereby making it more resistant to lysozyme
hydrolysis. As such, the trmA mutant may also be more resistant to the non-
specific degradation caused during the secretion of lysostaphin and as a result
may be able to tolerate higher levels of secretion of the damaging lysostaphin.
Four genes capable of affecting the secretion of lysostaphin in L. lactis were
identified. These genes are different from those previously identified in the
NucT study (Nouaille et al., 2004), suggesting that lactococcal host factors that
affect the secretion of heterologous proteins differ depending on the protein of
interest. Whilst the mechanisms remain speculative, the inactivation of these
four genes led to increased amounts of lysostaphin secreted without any obvious
detrimental effects to the host cell morphology and growth rate. This study also
described the construction of novel L. lactis strains that are able to secrete two
kinds of peptidoglycan hydrolases, lysostaphin and Ply511. The identification
of these L. lactis strains with the capacity to over-secrete two heterologous
proteins of therapeutic interest enhances the consideration of L. lactis as an
antimicrobial agent. The results of this study also provide new insights into
lactococcal factors which are important for the secretion efficiency of
heterologous proteins, which may have applications in the food and
pharmaceutical industries.
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105
CHAPTER 5
GENERAL DISCUSSION
106
LAB are a group of Gram-positive, non-sporulating bacteria that include the
genera Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, and
Lactococcus. For thousands of years, certain LAB have been used in food- and
feed-fermentation processes, and they are also important members of the human
endogenous microflora that are associated with different mucosal surfaces of the
body. Therefore, LAB have had a long and safe association with humans and
their food. In the past 20 years, there has been increasing interest in the use of
LAB as protein delivery vehicles and for the production of heterologous
proteins of therapeutic interest. Of specific interest to this project is L. lactis, a
widely used bacterium in the food industry. The strain used in this study, the
plasmid-free L. lactis subsp. cremoris MG1363, is extensively used as a model
strain in LAB genetics and molecular biology research, and the knowledge
gained from fundamental research on this strain has been exploited for a wide
variety of biotechnological applications. The importance of developments for
biotechnology and microbiology research is enormous, as many molecular tools
initially developed for the model L. lactis, such as genetic manipulations and
heterologous protein expression, have also been shown to be useful in other
LAB (Maguin et al., 1996, Kleerebezem et al., 1997, Turner et al., 2004a).
The aim of this thesis was to develop L. lactis as an antimicrobial agent. To
achieve this purpose, two aims were developed: (i) the engineering of two
recombinant proteins as a fusion and the investigation of the ability of this
chimeric protein to inhibit the interaction of S. aureus with host ECM proteins,
(ii) and the identification of L. lactis factors which increased the secretion of
recombinant lysostaphin.
In the investigation of the first aim, L. lactis strains were constructed that
expressed and secreted two proteins of interest (CyuC and lysostaphin)
separately and as a single fusion protein using the Sep expression system. CyuC
was hypothesised to have binding activities to ECM proteins and thus might be
able to competitively inhibit the adherence of S. aureus to ECM proteins. It was
also hypothesised that the combination of CyuC and lysostaphin as a single
fusion protein (CyuC-Lss) would increase the inhibition effect due to its dual
functionality. This concept of fusion proteins is not unique. Previously,
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lysostaphin has been fused to the S. agalactiae bacteriophage endolysin B30 and
expressed in E. coli (Donovan et al., 2006). The fusion protein was shown to
have S. aureus and Sp. agalactiae lytic activities, suggesting that lysostaphin
can tolerate the addition of extra protein sequences on the N-terminus. In this
study, it was found that the CyuC and lysostaphin were both active as part of a
fusion protein. Crude cell extracts of the L. lactis strain with the pGhost9:ISS1
vector only was able to significantly inhibit S. aureus adherence to fibronectin,
whilst the L. lactis strain secreting lysostaphin was able to inhibit adherence to
keratin. Future research may be able to identify lactococcal protein(s) with
fibronectin binding activities. This may be achieved by western ligand blotting
of proteins which have been separated by 2D SDS-PAGE, and to identify the
dominant proteins by N-terminal sequencing. Other avenues for future research
may be to further investigate the mechanism by which the L. lactis secreting
lysostaphin strain is able to inhibit S. aureus adhesion to keratin. A
lactococcal intermediary protein between lysostaphin and keratin may be
identified by use of, for example, Biacore’s Flexchip technology (Biacore Life
Sciences) to screen for lactococcal proteins which exhibit binding affinities to
both lysostaphin and keratin.
The results of the first aim demonstrated the utility of L. lactis to express and
secrete heterologous lysostaphin. However, the levels of recombinant proteins
produced by L. lactis and other LAB are generally low when compared with that
produced by E. coli (Jana and Deb, 2005) and B. subtilis expression hosts
(Schallmey et al., 2004). Levels are also very much dependent upon the
expression system used and the heterologous protein of interest. Heterologous
protein production in E. coli is often intracellular and involves expensive and
problematic downstream purification processes to remove endotoxin or
lipopolysaccharide. Although B. subtilis is endotoxin free, heterologous
proteins are degraded by its complex extracellular proteolytic system (Westers
et al., 2004). In comparison, L. lactis is considered a desirable alternative for
heterologous protein production, and as such, attention in recent times has been
turned toward the modification of L. lactis to improve the export of recombinant
proteins. Examples of this can be found in the inactivation of htrA and clpP to
improve the secretion of NucT (see section 1.1.2), and the Nouaille et al. (2004)
108
study, which identified thirteen lactococcal genes which either increased or
decreased NucT secretion when inactivated (see section 4.1). The study in
chapter 4 identified four genes (lom, murA2, trmA, and llmg_2148) that were
different from the Nouaille et al. (2004) study. When these genes were
inactivated, the resultant mutants produced increased amounts of lysostaphin
protein and the L. monocytogenes bacteriphage endolysin, Ply511. This
discovery clearly demonstrated that the factors involved in the secretion of
recombinant proteins in L. lactis is very much dependent on the recombinant
protein of interest and may also relate to the expression and/or secretion system
used. It may also raise doubts regarding previous studies which investigated the
optimisation of protein secretion using only a single reporter protein, such as
NucT (e.g. Dieye et al., 2001; Ravn et al., 2003), and did not verify the efficacy
of the secretion system with a different recombinant protein. Of the four genes
identified, perhaps the most interesting is lom, both from the genetic and
biotechnological points of view. Its inactivation resulted in the greatest increase
in the amount of lysostaphin secreted compared with the other mutants, yet its
function remains unknown. In silico analyses revealed that the Lom protein has
similarities to other uncharacterised proteins with predicted solute-binding
functions, and that it has a single transmembrane helix predicted to lie in the
central region. Based on these in silico analyses, it is hypothesised that Lom
may function as part of a multi-component pore-forming complex, and may
therefore directly affect the secretion of lysostaphin out of the cell. Another
hypothesis is that Lom may act as a signalling protein similar to anti-sigma
factors, which also have one transmembrane spanning domain, and which
respond to a signal and accordingly modify protein export patterns or
proteolysis (Yoshimura et al., 2004). Whilst these are purely speculative, to
functionally characterise Lom may prove more challenging. A starting point
may be to assess the downstream effect of a L. lactis lom mutation by analysing
gene expression levels by microarray or protein expression levels by 2D SDS-
PAGE. From the perspective of developing L. lactis as an antimicrobial agent,
the inactivation of lom has generated a L. lactis strain capable of increased
lysostaphin production without detriment to cellular growth. A previous study
was able to produce lysostaphin using the NICE system intracellularly in L.
lactis (Mierau et al., 2005a). The advantage conferred by the Sep expression
109
system is that the production of lysostaphin is constitutive, whereas in the NICE
system, the induction of lysostaphin production has to be made at the
appropriate growth phase, and the growth of the culture is limited upon
induction (Mierau et al., 2005a). Unlike, the NICE system, the Sep expression
system (which includes a secretion signal) allows for lysostaphin to be readily
exported into the supertatant. Although the amounts of recombinant proteins
produced is low, the Sep expression system has been a consistent performer in
expressing functional recombinant proteins in LAB (Turner et al., 2004a;
2007b; Liu et al., 2007).
This body of research has advanced the knowledge base of L. lactis genetics and
its biotechnological applications. Of particular significance are the observations
that wild-type L. lactis can inhibit S. aureus adhesion to fibronectin, and
similarly, that extracts from a lysostaphin-secreting strain can inhibit S. aureus
adhesion to keratin. In addition, novel L. lactis strains were identified which
have increased production of lysostaphin, and also able to secrete more of a
secondary cell wall lytic enzyme. These results may be applied to the further
development of L. lactis as an antimicrobial agent, and to the development of
heterologous protein production in lactic acid bacteria.
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CHAPTER 6
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