Bioengineering Lantibiotics for

Therapeutic success

Dr Des Fielddes.field@ucc.ie

QuebecNot Quebec

Food Veterinary Medicine

Health & Pharma

Food Safety and Shelf life extension Anti-mastitis treatment

Potential for use as Therapeutics

Bacterially produced, small, heat-stable, gene-encoded

peptides that are active against other bacteria and to which

the producer has a specific immunity mechanism.

-More than100 lantibiotics identified

wide variation in structures

wide variety of post-translational

modifications

Ile SerIle Thr Ser Leu Cys Thr Pro Gly Cys

OH

OH OH

OH

IleIle Leu Cys Pro Gly CysDhaDhb Dha Dhb

SHSH Lanthionine

Ile Dhb Ala

Ile

Dha

Leu

Ala Abu

Pro Gly

Ala K

S

S

Lanthionine-containing antibiotics

β-methyl-

lanthionine

Class I bacteriocins

Undergo post-translational modification (by modification enzymes)

Dairy products: processed cheese and cheese spreads; direct acidified cheeses;pasteurised dairy desserts;fresh and recombined milk; •Liquid egg •Dressings and sauces • Not very effective against Listeriamonocytogenes

Membranephospholipids

Wallpeptidoglycan

Breukink lab; Sahl lab

Lipid II

1.

2.

• Multiple activities

• Two domains separated by a ‘hinge’

Transport/

Processing

Alan

Structural

B C

J

D

Lanthionine

synthesis

Further

modifications

M

in out

T P

Processing

Gene-encoded biosynthesis

Potential for bioengineering through manipulation of the

structural peptide

Generate vast population of variant derivatives

A

‘Unlike the mammalian immune system, the

bacterium has no mechanism for generating a

diverse population of (lantibiotics)’.

‘……can be viewed as a kind of primitive antibody,

even having constant and variable regions.’

Liu & Hansen J Biol Chem 1992 267:25078

‘the extent to which (lantibiotics) can be

structurally altered without destroying their

character is unknown’

‘Unlike the mammalian immune system, the

bacterium has no mechanism for generating a

diverse population of (lantibiotics)’.

Natural variants

However, natural variants could be

regarded as diverse populations

Nisin A

Nisin Z

Nisin Q

Nisin U

Nisin U2

Nisin F

Nisin P

Nisin H

Cotter, P.D., C. Hill and R.P. Ross. 2005. Curr. Prot. Pept. Sci. 6:61.

57 derivatives

• nisin – was only example >8 derivatives described in literature

• mutants have been very useful for fundamental analysis

• unfortunately there was little success in enhancing activity

b

a

Complete Alanine Scanning of Lacticin 3147

‘the extent to which (lantibiotics)

can be structurally altered without

destroying their character is

known!!!’

‘CAN be viewed as a kind of primitive antibody,

even having constant and variable regions.’

Cotter et al 2006 Mol Microbiol

Development of systems for Lantibiotic Mutagenesis

A B T C I P R K F E G

Random Mutagenesis Saturation Mutagenesis Directed Mutagenesis

Q

Bioengineering Nisin

X X X

Random

NZ9800

pPTPL

NZ9800

pPTPL-nisA

NZ9800

pPTPL-nisAK22T

NZ9700 NZ9800 NZ9800

nisA-K22S

A. B.

NZ9800

pPTPL

NZ9800

pPTPL-nisA

NZ9800

pPTPL-nisAK22T

NZ9700 NZ9800 NZ9800

nisA-K22S

A. B.

Nisin Mutant

High-throughput Robotics

systems

Bioengineering Nisin

Random Banks

Single X Banks

Double X Banks

NisF/Z/Q S29X

Triple X Banks

8,000variants

3,456 variants

6,432 variants

432 variants

19,200 variants

In total: 37,520 variants…and counting!

L. lactisHP

S. aureus RF122

S. aureus SA113

S. aureus

NCDO1499

S. agalactiae ATCC

L. mono

MRSA

VISA

hVISA

VRE

E. faecium

E. faecalis

B. cereus

S. pyogenes

S. mutans

S. dysgalactiae

S. mitis

E. durans

S.uberis

E. casseliflavus

I Dhb Ala

IDha

L

Ala Abu

P G

Ala N

AAbu

Ala

HG

Abu

A

L

G

Ala

M

M K Abu Ala Dha KS I H VK

A

BC D

E

5

10

15

20

2530

SS

S S

S

I4V S29A/OthersM21V

Food Veterinary Medicine

Health & Pharma

Food Safety and Shelf life extension Anti-mastitis treatment

Potential for use as Therapeutics

Veterinary Medicine

Health & Pharma

Anti-mastitis treatment

Potential for use as Therapeutics

350,000 Pounds of Hot Dogs, Corn Dogs Recalled Amid Fears of Listeria ContaminationPosted by CNN Wire

Hot dog meat trial

(Lux-labelled Listeria)

Field et al 2008 Mol Microbio[

Field et al 2010 Microbiol Biotechnol

Enhanced anti-listeria activityM21V WT

Listeria monocytogenes

Model Food studies with Nisin V (M21V) fermentate in

combination with essential oils e.g. cinnamaldehyde,

carvacrol

Field et al., 2015 AEM

In food, Nisin is used as a fermentate containing 2.5% nisin (e.g. nisaplin® )

Chocolate Milk Commercial Soup

AORI Ts

A B T C I P R K F E G

A B T C I P R K F E GV

Mutate nisA to nisV

1 nt change

A B T C I P R K F E G

V B T C I P R K F E G

50%

50%

VORI Ts

Cotter et al., 2002 AEMCotter et al., 2006 Mol Microbiol

L. monocytogenes LO28

Nisin A Nisin V

Ruling by EU/EPA in 2011 that Nisin V is excluded from Directive2009/41/EC

Not regulated as a GMM!

Food

Health & Pharma

Food Safety and Shelf life extension

Potential for use as Therapeutics

Veterinary MedicineAnti-mastitis treatmentFood Safety and Shelf life extension

Health & Pharma

Food

Potential for use as Therapeutics

Antibiofilm activity /Synergy with antibiotics

Methicillin-resistant S. pseudintermedius (MRSP):opportunistic animal pathogen responsible for skin, soft tissue, and surgical site infections.Multi-drug resistant, ability to form Biofilms

Biofilms of S. pseudintermedius DSM 21284

Wt I4V

Field et al 2015 PLoS ONE

Field et al 2016 Frontiers in Micro

Peptide S. aureus 54072

Mg/L

S. aureus 20777

Mg/L

S. aureus NCDO 1499

Mg/L

S.aureusRF122

Mg/L

L. lactisMG1363

Mg/L

L. lactisHP

Mg/L

B. LongumUCC

Mg/L

L. acidophilusUCC

Mg/L

Nisin A 1 8 0.25 2 0.06 <0.125 0.06 0.03

Variant 1 0.25 0.5 0.06 0.25 0.06 0.125 0.06 0.06

Variant 2 4 0.25 1 2 0.5 0.5 0.12 0.12

Variant 3 0.25 0.125 0.03 1 0.125 0.125 0.24 0.12

The BAD The GOOD

Nisin versus Bovine Mastitis pathogens

Veterinary MedicineAnti-mastitis treatment

Food

Health & Pharma

Food Safety and Shelf life extension

Potential for use as Therapeutics

Veterinary MedicineAnti-mastitis treatment

FoodFood Safety and Shelf life extension

The Outer membrane (OM) of the Gram

negative cell wall which acts as a barrier for

the cell, restricting the access of the peptides

to the cytoplasmic membrane

Enhanced derivatives against Gram Negatives

5 10 15 20 25

0.2

0.6

1.0

1.4 Untreated

Nisin S29A 15M

Colistin 0.75g/ml

Nisin S29A 15M + Col

0.75g/ml

Time (Hrs)

OD

60

0n

m

P. aeruginosa PA-01

Field et al., 2012 PLoS ONE

Field et al .,2016 Frontiers in Micro

But…..some bacteria have evolved nisin resistance mechanisms!!!!!

• Nisin Resistance Protein (NSR) degrades nisin by cleaving the peptide bond

between MeLan28 in ring E and Ser29

• NSR protein provides a 20-fold increased resistance towards nisin

The Nisin Resistance Protein (NSR)

632 Da

2720 Da

+

Khosa et al 2016 Frontiers in Micro

NSR homologues are found in many pathogenic and non-pathogenic strains including:

-E. faecium-S. epidermidis-Strep. sanguinis-Strep. dysgalactiae-S. agalactiae-Corynebacterium casei-C. ammoniagenes

More widespread than we thought!!!

Ser29X derivatives

Molecular modelling provides strong evidence of a rigidity and inflexibility in the region around the nisincleave point for the S29P derivative compared to the wild type Nisin A.

37,520

340000000000000000000

Cork Bacteriocin Group

University College Cork

Prof Colin HillProf Paul RossDr Des FieldDr Ciorsdan CampionKevin EganJenna EllisAngeliki AngelopolouFanny CharlotInes BaghouPhiliswa Mbandlwa

Teagasc

Dr Paul CotterDr Sheila MorganDr Mary ReaPaula O’ConnorHarsh MathurElaine LawtonCleriane Andre

The Irish Government under the National Development Plan

Funding