Host ligands and oral bacterial adhesion Studies on phosphorylated polypeptides and gp-340 in saliva and milk
Liza Danielsson Niemi
Department of Odontology
Umeå University Umeå 2010
Responsible publisher under Swedish law: the Dean of the Medical Faculty © 2010 Liza Danielsson Niemi ISBN: 978-91-7264-969-9 ISSN: 0345-7532 Cover layout: Print & Media Printed by Print & Media Umeå, Sweden 2010
To my family
AABBSSTTRRAACCTT
Infectious diseases e.g. gastric ulcer, caries and perodontitis, are caused by bacteria in
a biofilm. Adhesion of bacteria to host ligands e.g. proteins, polypeptides and
glycoproteins, is a key event in biofilm formation and colonization of surfaces such as mucosa and tooth tissues. Thus, host ligands could contribute to the susceptibility to
infectious diseases. The general aim of this doctoral thesis was to study the effect of
phosphorylated polypeptides and gp-340 in saliva and milk on oral bacterial adhesion and aggregation.
Statherin is a non-glycosylated, phosphorylated polypeptide in saliva. The polypeptide inhibits precipitation and crystal growth of calcium phosphate and
mediates adhesion of microorganisms. By using a hybrid peptide construct, the domain for adhesion of Actinomyces isolated from human infections and from
rodents was found to reside in the C-terminal end, and the adhesion was inhibitable.
With alanine substitution the peptide recognition epitope in the C-terminal end was delineated to Q and TF, where QAATF was an optimal inhibitory peptide. In contrast,
human commensal Actinomyces bound to the middle region in a non-inhibitable
fashion. Gp-340 is another protein in saliva, and it is a large, multifunctional glycoprotein. Four novel size variants (I-IV) of salivary gp-340 were distinguished
within individuals, and their glycoforms were characterized. All four size variants
were identical in the N-terminal amino acid sequence and shared core carbohydrates. Low-glyco lung gp-340, high-glyco saliva gp-340, and size variants I-III aggregated
bacteria differently. Human milk, which shares many traits with saliva, could inhibit
adhesion of Streptococcus mutans to saliva-coated hydroxyapatite (s-HA), a model for teeth, in an individually varying fashion. Human milk caseins, lactoferrin,
secretory IgA, and IgG inhibited the binding avidly. By using synthetic peptides the
inhibitory epitope in β-casein was mapped to a C-terminal stretch of 30 amino acids.
Inhibition by human milk, secretory IgA and the β-casein-derived inhibitory peptide was universal among a panel of mutans streptococci.
The main conclusions are: (i) statherin mediates differential binding of commensal versus infectious Actinomyces strains with small conformation-dependent binding
epitopes, (ii) salivary gp-340 has individual polymorphisms that at least affect binding of bacteria, (iii) human milk inhibits S. mutans adhesion to s-HA in an
individually varying fashion, and the C-terminal end of human milk β-casein is one
inhibitory component. Together these results suggest that the studied host ligands can influence the composition of the oral biofilm. Statherin may protect the host from
colonization of bacteria associated with infections. Gp-340 size variants may affect
functions related to host innate immune defences such as interactions with a wide array of bacteria, and human milk may have a protective effect in infants from
colonization of mutans streptococci.
TTAABBLLEE OOFF CCOONNTTEENNTTSS
LIST OF PUBLICATIONS .............................................................. 9
ABBREVIATIONS ....................................................................... 10
INTRODUCTION.......................................................................... 11
INFECTIOUS DISEASES IN THE GASTROINTESTINAL TRACT..................................11 BACTERIAL BIOFILM ECOLOGY – A DETERMINANT FOR HEALTH OR DISEASE ..... 12 DENTAL CARIES – AN INFECTIOUS DISEASE INVOLVING BIOFILM ECOLOGY
BALANCE.......................................................................................................... 12 ADHESION AND BIOFILM FORMATION............................................................... 13 ORAL MICROBIAL ECOLOGY............................................................................. 14 HOST LIGANDS MEDIATE ADHESION OF ORAL BACTERIA ................................... 16 Host ligands in saliva ................................................................................ 17 Host ligands in milk ..................................................................................21
BIOREGULATORY PROTEINS AND PEPTIDES IN SALIVA AND MILK.......................23 HOST LIGAND GLYCOSYLATION AND SECRETOR STATUS ....................................24 MODEL MICROORGANISMS PRESENT IN ORAL BIOFILMS ...................................25 Actinomyces...............................................................................................25 Streptococci ...............................................................................................26
AIMS........................................................................................... 28
MATERIALS AND METHODS ..................................................... 29
BACTERIA AND FUNGUS STRAINS ......................................................................29 SALIVA AND MILK.............................................................................................29 PURIFICATION OF SALIVA PROTEINS .................................................................29 SIZE VARIANTS OF GP-340 ...............................................................................30 CARBOHYDRATE MAPPING OF GP-340 VARIANTS ..............................................30 SEPARATION AND IDENTIFICATION OF MILK PROTEINS ..................................... 31 ADHESION ASSAY ............................................................................................. 31 ADHESION INHIBITION ASSAY ..........................................................................32 PEPTIDES FOR MAPPING BACTERIA-BINDING EPITOPES.....................................33 BINDING OF BACTERIA IN SOLUTION (AGGREGATION ASSAY).............................34
RESULTS AND DISCUSSION...................................................... 35
PAPER I. BINDING EPITOPES FOR ACTINOMYCES ON SALIVARY STATHERIN .......35 PAPER II. VARIANT SIZE- AND GLYCOFORMS OF GP-340 WITH DIFFERENTIAL
BACTERIAL AGGREGATION................................................................................38
PAPERS III AND IV. EFFECTS OF HUMAN MILK AND MILK COMPONENTS ON
ADHESION OF MUTANS STREPTOCOCCI TO SALIVA-COATED HYDROXYAPATITE IN
VITRO .............................................................................................................. 42
CONCLUSIONS AND FUTURE PERSPECTIVES..........................48
ACKNOWLEDGEMENTS.............................................................50
REFERENCES ............................................................................. 52
List of Publications
9
LLIISSTT OOFF PPUUBBLLIICCAATTIIOONNSS
This thesis is based on the following papers, which will be referred to in the
text by their Roman numerals.
I. Danielsson Niemi L and Johansson I. Salivary statherin peptide-binding epitopes of commensal and potentially infectious
Actinomyces spp. Delineated by a hybrid peptide construct. Infect
and Immun. 2004 Feb;72(2):782-787.
II. Eriksson C, Frängsmyr L, Danielsson Niemi L, Loimaranta V,
Holmskov U, Bergman T, Leffler H, Jenkinsson H F and Strömberg
N. Variant size- and glycoforms of the scavenger receptor cysteine-
rich protein gp-340 with differential bacterial aggregation.
Glycoconj J. 2007 Apr;24(2-3):131-142.
III. Wernerson J, Danielsson Niemi L, Einarson S, Hernell O, Johansson I. Effects of human milk on adhesion of Streptococcus
mutans to saliva coated hydroxyapatite in vitro. Caries Res.
2006;40:412-417.
IV. Danielsson Niemi L, Hernell O, Strömberg N, Johansson I.
Human milk compounds inhibiting adhesion of mutans streptococci
to host ligand- coated hydroxyapatite in vitro. Caries Res.
2009;43:171-178.
Reprints of the original papers were made with permission from the
publishers.
Abbreviations
10
AABBBBRREEVVIIAATTIIOONNSS
aa Amino acid
AgI/II Antigen I/II
APRP Acidic proline-rich proteins
BSA Bovine serum albumin
BSSL Bile Salt-Stimulated Lipase
statherin-des-aax A compound obtained by the removal of an
amino acid (aa) residue from the polypeptide
statherin in the position x.
DMBT1 Deleted in malignant brain tumours 1
ELISA Enzyme-linked immunosorbent assay
gp-340 Glycoprotein 340, formerly called agglutinin
HA Hydroxyapatite
s-HA Hydroxyapatite coated with saliva
m-HA Hydroxyapatite coated with human milk
gp-340-HA Hydroxyapatite coated with gp-340
HIV Human immunodeficiency virus
IgG Immunoglobulin G
Lea-Ley Lewis a to Lewis y antigens
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
S-IgA Secretory immunoglobulin A
sLex sialyl-Lewis x
VNTR Variable number of tandem repeats
Abbreviations for amino acids used in the text:
Alanine Ala A Lysine Lys K
Arginine Arg R Phenylalanine Phe F
Asparagine Asn N Proline Pro P
Aspartate Asp D Serine Ser S
Glutamate Glu E Threonine Thr T
Glutamine Gln Q Tryptophan Trp W
Glycine Gly G Tyrosine Tyr Y
Isoleucine Ile I Valine Val V
Leucine Leu L
Introduction
11
IINNTTRROODDUUCCTTIIOONN
Microbes attach to host tissues by interactions with host ligand molecules
(receptors). These interactions are specific, and characteristics of the ligands
are crucial for recognition by the bacterial attachment adhesins. Lately,
bacterial resistance to antibiotics has increased, making it important to find
alternative treatments for bacterial infections. Identifying host ligands and
delineating host ligand epitopes for bacterial interactions may lead to
construction of peptidomimetic drugs that can prevent colonization of
infectious microorganisms and favour commensal ones. Knowledge of host
ligands and host ligand polymorphism could also provide explanations for
host resistance and host susceptibility towards infectious diseases.
Infectious diseases in the gastrointestinal tract
Bacterial infections affect people worldwide, and they constitute major
health problems with associated morbidity and mortality. Infectious diseases
are among the top ten causes of death both in low income, middle income,
and high income countries (World Health Organization, 2008). Many
species of pathogenic bacteria are capable of colonizing and infecting the
gastrointestinal tract and other areas in the body. These microbes cause
diseases such as lower respiratory infections (e.g. Haemophilus influenzae,
Moraxella catarrhalis and Streptococcus pneumoniae) (Guthrie, 2001),
diarrhoeal diseases (e.g. Escherichea coli, Salmonella, Shigella,
Campylobacter, Vibrio cholerae) (Niyogi et al., 1994), and tuberculosis
(Mycobacterium tuberculosis) (Zhang and Yew, 2009). These infections are
among the major causes of mortality over the world, and together they
caused almost eight million deaths in 2004 (World Health Organization,
2008). Other common infectious diseases in the gastrointestinal tract,
caused by less pathogenic bacteria, are peptic ulcers and gastritis caused by
Helicobacter pylori (Aspholm-Hurtig et al., 2004), periodontitis (e.g.
Phorphyromonas gingivalis and Aggregatibacter actinomycetemcomitans)
(Asikainen and Chen, 1999), and dental caries associated with Streptococci
and Lactobacilli (Marsh, 1994).
Introduction
12
Bacterial biofilm ecology – a determinant for health or disease
There are approximately ten times as many bacterial cells in the human body
as human cells (Cucchiara and Aloi, 2009). However, most of the bacteria
are harmless or beneficial to the host. For example, commensal bacteria
provide infection resistance, increase the overall immunological capacity of
the host, and act as a colonization barrier towards pathogenic
microorganism (Salminen et al., 1995; Tlaskalová-Hogenová et al., 2004).
Bacteria and other microorganisms form complex communities (biofilms),
which once they are established, have a highly specific ecology for the
habitat, i.e., the mouth, gut, etc. Many chronic diseases in humans are
claimed to involve bacterial biofilms and ecological shifts therein (Donlan
and Costerton, 2002). According to Fux et al. (2005), more than 60% of
chronic infectious diseases relate to ecology shifts in the associated bacterial
biofilms. Examples of such diseases are the oral diseases, dental caries and
periodontitis, and also endocarditis, prostatitis, and cystic fibrosis (Fux et
al., 2005). Understanding development and maintenance of a health-
associated biofilm would be a subject of major therapeutic potential.
Dental caries – an infectious disease involving biofilm ecology balance
Dental caries is a highly prevalent chronic infectious disease that involves
dental plaque which is a bacterial biofilm on teeth. Caries is prevalent in
most countries, but the distribution in the Western populations is skewed.
That is, a majority experience few symptoms whereas 15-20 % of the
populations are highly diseased. Dental caries may cause pain, bone
infection, eating problems and social stigmata. The disease is characterized
by demineralisation of the tooth tissues occurring at low pH due to bacterial
fermentation of carbohydrates. Disease determinants involve both genetic
(tooth tissue, taste preference, saliva polymorphism and immune functions)
and lifestyle (eating and oral hygiene habits and fluoride exposure) factors
(Vieira et al., 2008; Stenudd et al., 2001). In dental caries the tooth biofilm
ecology is characterized by a shift towards more acidogenic (acid producing)
and aciduric (acid resistant) bacteria, such as mutans streptococci
(Streptococcus mutans and Streptococcus sobrinus), lactobacilli, and
Candida species. Other somewhat less acidogenic and aciduric bacteria have
also been associated with dental caries, e.g., S. mitis biovar 1, Actinomyces
naeslundii, A. viscosus, A. odontolyticus, A. israelii, Veillonella species
(Brailsford et al., 1998; Tanner et al., 2002; Becker and Paster, 2002; Nyvad
Introduction
13
and Kilian, 1990a). Besides prerequisites for attachment of the cariogenic
bacteria, in vivo proliferation, e.g. of S. mutans and Lactobacillus, is
favoured at low pH, due to the bacteria’s ability to proliferate over other
species, at low pH (Marsh, 2006). Thus, frequent or prolonged episodes of
low pH, such as after intake of sucrose or other fermentable carbohydrates,
may lead to an ecology shift towards higher proportions of, e.g. mutans
streptococci (Bradshaw et al., 1989; Balakrishnan et al., 2000; Marsh,
2006). This process forms the basis for the present ecological plaque
hypothesis of caries, in which several species of bacteria could be classified
as “cariogenic” and contribute to the development of dental caries (Marsh,
2006). Thus, “cariogenic” bacteria are normally present in low numbers in
the dental plaque biofilm. However, they do not cause disease until major
environmental changes occur, such as reduced salivary flow or high and
frequent intake of sucrose that changes the ecology of the biofilm (Sbordone
and Bortolaia, 2003; Marsh, 2006).
Colonization of the oral cavity starts during birth (normal delivery) and then
completes by transmission from the caregiver to the child (Li et al., 2000;
Caufield et al., 1993). Early colonization of mutans streptococci, as well as
avid saliva-mediated adhesion and high numbers of mutans streptococci in
saliva or the tooth biofilm, are predictors for dental caries (Teanpaisan et al.,
2007; Straetemans et al., 1998), whereas acquisition of S. sanguinis is
associated with healthy teeth (Becker and Paster, 2002). Thus,
establishment of a commensal oral flora in early childhood seems highly
beneficial.
Adhesion and biofilm formation
A biofilm consists of highly structured communities of microorganisms that
form on various biotic surfaces, such as mucosa, epithelia, tooth tissues, and
implants. It also forms on non-human (abiotic) surfaces such as industrial
water pipelines and catheters (Costerton et al., 1999; Kolenbrander, 2000;
Whittaker et al., 1996; Dunne, 2002).
Biofilm formation starts with an initial contact between the bacterial cell and
the surface via hydrophobic, van der Waals, electrostatic interactions or
steric hindrance (Dunne, 2002). Adhesin molecules on the bacteria then lock
the contact by binding to specific host molecules (ligands) e.g. proteins,
peptides, carbohydrates or lipids. Thus, a key event in biofilm formation is
the initial adhesion of bacteria to matching host ligands or partner bacteria
(Dunne, 2002; Costerton et al., 1999). Once attached to the surface, other
Introduction
14
bacteria bind to the already attached bacteria (co-adhesion) (Costerton et al.,
1999; Dunne, 2002; Marsh, 2006). Bacteria in the biofilm produce
extracellular polysaccharides, which are a major component of the biofilm
matrix (glycocalyx). The glycocalyx is a slime layer, with high viscoelasticity,
which surrounds and aids in anchoring the biofilm bacteria (Dunne, 2002).
Microbes in the oral biofilm proliferate, and at later stages, different
microbial species, such as S. gordonii and Pseudomonas aeruginosa form
micro communities within the biofilm organized in mushroom-like or corn-
cob formations with channels for water and nutrient transportation and
removal of waste (Donlan and Costerton 2002; Costerton et al., 1999). In the
mature biofilm approximately 15% of the volume is composed of cells, and
85% is matrix (Donlan and Costerton, 2002). Within the biofilm, bacterial
ecology is further regulated by: (i) reduced availability of oxygen facilitating
anaerobic species, (ii) nutrient limitations, (iii) gene regulation due to cell-
to-cell signalling depending on cell density (quorom sensing), (iv) metabolic
communication where excreted metabolites are used as nutrition by a
different bacterial cell, such as Veillonella species metabolizing lactic acid
from mutans streptococci, and (v) proliferation and detachment from the
biofilm (Dunne, 2002; Davies et al., 1998; Costerton et al., 1999;
Kolenbrander et al., 2002). Thus, the biofilm reaches a dynamic equilibrium
dependent on water, nutrients, removal of waste, pH, oxygen levels,
osmolarity, and released cell components from dead bacteria.
By forming biofilms, bacteria are more likely to overcome external
influences, such as host immune responses and antibacterial therapeutics
(Costerton et al., 1999; Fux et al., 2005). It has been hypothesized that the
mechanism behind the resistance to antimicrobial agents involves: (i) failure
to penetrate the biofilm, (ii) presence of slow growing, less susceptible
bacteria, (iii) bacterial phenotype shift leading to enhanced resistance, and
(iv) production of enzymes (Fux et al., 2005; Marsh, 2006; Sbordone and
Bortolaia, 2003). Bacteria in biofilms are also more resistant towards
mechanical forces due to densely packed bacterial cells in the slime matrix of
extracellular polysaccharides produced by the bacteria (Fux et al., 2005).
Thus, it is beneficial for the bacteria to organize themselves in biofilms.
Oral Microbial Ecology
The oral cavity offers various types of surfaces for bacterial colonization, i.e.
the buccal, gingival, palatal, and tongue epithelium, the hard tooth tissues,
and various tooth restorative materials. Both the soft and hard tissues and
dental materials are coated by a pellicle, composed of salivary components,
Introduction
15
gingival exudate, and externally derived (e.g. from foods) proteins,
glycoproteins and glycolipids. The pellicle may also offer host ligands for
Figure 1. Oral biofilm formation. Components from both saliva and diet are included in the pellicle, which displays possible adhesion sites for bacteria colonization as well as clearance of bacteria from the oral cavity. Initial bacteria such as Streptococcus and Actinomyces species adhere to the pellicle or co-adhere to other bacteria.
bacterial binding. Formation of the first bacterial monolayer, e.g. on the
teeth, is determined by bacterial availability and which host ligands are
available for bacterial attachment (Figure 1). Soluble ligands can also help
clear bacteria from the oral cavity. Oral biofilms are polymicrobial
communities, and more than 700 bacterial taxa have been identified in the
oral cavity (Jenkinson and Lamont, 2005; Aas et al., 2005). Some species
colonize early and some late in the colonization succession. Early colonizers
include Streptococcus, Actinomyces, Veillonella and Neisseria species
(Jenkinson and Lamont, 2005). Streptococci, such as S. sanguinis, S. oralis,
and S. mitis (biovar 1) and A. naeslundii dominate among bacteria initially
colonizing the teeth. It is suggested that S. sanguinis, S. oralis, and S. mitis
(biovar 1) constitute 60-90 % of the early colonizing flora (Kolenbrander et
al., 2002; Nyvad och Kilian, 1987; Nyvad och Kilian, 1990b). Succeeding
bacteria attach to adhering host ligands or to the already attached bacteria in
a very specific manner (Costerton et al., 1999; Dunne, 2002). This latter
phenomenon, co-aggregation or co-adhesion, is described for, e.g.
Actinomyces co-aggregation groups A-F and Streptococcus co-aggregation
groups 1-6 (Kolenbrander, 1989; Egland et al., 2001). In the maturing
Introduction
16
biofilm there is an ecological shift from predominantly aerobic bacteria
towards more anaerobic bacteria. Fusobacterium nucleatum, which can co-
aggregate with a wide array of oral bacteria, is suggested to function as a
bridge between early and late colonizers unable to co-aggregate with each
other (Sbordone and Bortolaia, 2003; Kolenbrander et al., 2002; Jenkinson
and Lamont, 2005; Bradshaw et al., 1998). For example, F. nucleatum could
bridge binding between the aerob-anaerob pair S. mutans and P. gingivalis
(Bradshaw et al., 1998).
Although the oral flora is highly diverse, the teeth are predominately
colonized by certain bacteria, e.g. species of the Streptococcus and
Actinomyces families (Li et al., 2004; Rüdiger et al., 2002).
Host ligands mediate adhesion of oral bacteria
The bacterial tropism is mainly determined by the bacterial adhesin and the
host ligand pairs available at particular host tissues. Bacterial adhesin-host
ligand recognition-binding is often highly specific. However, many bacteria
express more than one adhesin that enables them to bind to several host
ligands. Host ligands (proteins, polypeptides, peptides, glycoproteins,
glycolipids or glycosphingolipids) are expressed or attached to the host
surface, and the specific binding epitopes can be expressed by several host
ligands. Microbe-host interactions have been explored and delineated to
minor carbohydrate and peptide recognition motifs. Many tissue cells carry
glycosylated structures, and accordingly, a variety of microorganism-binding
carbohydrate epitopes have been described. For example, both p-fimbriated
(PapG adhesin) E. coli (Strömberg et al., 1990) and S. suis bacteria (Haataja
et al., 1994) bind to the disaccharide Galα1-4Gal in blood group P-related
glycolipids. A. naeslundii bacteria bind to GalNAcβ in salivary pellicles
(Strömberg et al., 1992).
Peptide motifs also act as binding sites for microorganisms. C. albicans bind
to the ArgGlyAsp (RGD) peptide in iC3b on epithelial cells (Bendel and
Hostetter, 1993). The adhesin in type-1 fimbriae of A. naeslundii Ly7 binds
to ProGln (PQ) in the acidic proline-rich proteins (APRP) in saliva (Gibbons
et al., 1991), and A. viscosus 19246 binds to ThrPhe (TF) in salivary statherin
(Li et al., 1999).
This doctoral thesis focuses on a set of model ligands in human saliva and
milk, specifically the phosphorylated statherin in saliva, the highly
glycosylated gp-340 in saliva, and the phosphorylated β-casein in milk.
Introduction
17
Host ligands in saliva
Approximately, 0.5-1 litre of saliva, with an average of 1 mg/ml of proteins, is
produced per 24 hours. Thus, the teeth, and other oral surfaces, are
continuously “flushed” by saliva. Saliva contains proteins, peptides,
glycoproteins, and glycolipids conferring multiple functions, including
lubrication, fascilitation of swallowing and speech, buffering capacitiy, and
antimicrobial defence such as bacterial clearance. In healthy conditions the
content of free carbohydrates and lipids is low in saliva. Several proteins
(host ligands) in saliva carry receptor epitopes (binding sites) for bacterial
attachment, some of which bind to bacteria in the fluid phase (e.g. gp-340).
This leads to aggregation and clearance of the bacteria from the oral cavity.
Other host ligands bind certain bacteria only when surface bound (e.g.
statherin, APRPs) (Gibbons and Hay, 1988; Paper I), and other host ligands,
e.g. gp-340, bind bacteria both when free in solution and when bound to a
surface (Loimaranta et al., 2005). The most abundant proteins in saliva are
Table 1. Major proteins in saliva and human milk with protective functions.
Saliva and milk proteins Protective function ________ Saliva proteins
Amylase Bacterial binding Cystatins Protease inhibitor Gp-340 Bacterial and viral binding Histatin Bacteriocidal and fungicidal Lactoferrin Bacteriostatic, bacteriocidal, bacterial binding Lysozyme Gram-positive bacteria lysis Mucins Bacterial binding PRP Bacterial binding Salivary peroxidase Bacteriostatic S-IgA Bacterial adherence prevention Statherin Bacterial binding Milk proteins α-lactalbumin Tumour cells apoptosis induction BSSL Lipid digestion Caseins Bacterial binding, Ca2+ binding Cytokines Immunomodulatory Haptocorrin Bacteriostatic Lactoferrin Bacteriostatic, bacteriocidal, bacterial binding Lactoperoxidase Bacteriostatic Lysozyme Gram-positive bacteria lysis S-IgA Bacterial adherence prevention ____________________________________________________
the PRPs, which make up approx. 25-30% of the salivary proteins, and
amylase. Examples of other, less abundant, but biologically active salivary
proteins, identified in the pellicle and biofilm, are statherin, gp-340,
lactoferrin, secretory immunoglobulin A (S-IgA), and lysozyme (Table 1). Of
Introduction
18
these, statherin and gp-340 have been selected as model host ligands in the
present thesis.
Statherin
Salivary statherin is a non-glycosylated, 43-amino acid residue, tyrosine-rich
polypeptide with a charged phosphorylated N-terminal end (Figure 2). It is
expressed by secretory cells in the salivary glands and nose (Cole et al., 1999;
Schlesinger and Hay, 1977). It is encoded by the STATH gene on
chromosome 4q13.3 close to the genes for caseins and histatins, which all
belong to a “secretory calcium-binding phosphoprotein gene cluster” (Huq et
al., 2005). The STATH gene is highly homologous, in introns and noncoding
exons, to the HIS1 gene encoding the antifungal histatin 1 (Sabatini et al.,
1993). Statherin has only been identified in man and monkeys, which implies
a late evolutionary appearance. In fact, it is suggested that the statherin
ancestral gene could have evolved from the casein genes (Huq et al., 2005).
Statherin exhibits diverse biological roles in the oral cavity (Ramasubbu et
al., 1993; Schlesinger and Hay, 1977) (Figure 2).
Figure 2. Functional domains in statherin.
It (i) mediates binding of bacteria, F. nucleatum, P. gingivalis, Actinomyces,
and fungi, C. albicans (Johansson et al., 2000; Li et al., 1999; Amano et al.,
1996; Sekine et al., 2004). The binding sites for F. nucleatum reside in the
middle to C-terminal end (aa 21-26 and aa 31-39) (Sekine et al., 2004),
whereas Leu29Tyr30 and Tyr41Thr42Phe43 are the binding epitopes for
P. gingivalis (Amano et al., 1996), (ii) inhibits crystal growth and
precipitation of calcium phosphate salts from supersaturated saliva
(Schlesinger and Hay, 1977), (iii) acts as a lubricant (Ramasubbu et al.,
Introduction
19
1993), and (iv) binds with high selectivity to hydroxyapatite in the N-
terminal region (Gilbert et al., 2000).
In aqueous solution statherin has a disordered structure, but upon
adsorbtion to hydroxyapatite statherin folds into a secondary structure
(Goobes et al., 2006). It is proposed that adsorbed statherin consists of an α-
helix in the N-terminal, a poly-proline type II structure, and an α-helix in the
C-terminal (Goobes et al., 2006; Long et al., 2001; Naganagowda et al.,
1998).
Although statherin is a small unglycosylated polypeptide, it occurs in several
polymorphic variants (Jensen et al., 1991). Jensen et al. (1991) showed three
different forms of statherin due to alternative RNA splicing and
posttranslational modifications: statherin SV1 which lacks the C-terminal
phenylalanine residue (statherin-des-Phe43), SV2 which lacks residue 6-15
(statherin-des6-15), and SV3 which is identical to SV2 but lacks the C-terminal
phenylalanine (statherin-des6-15, -Phe43). Later Inzitari et al. (2006) analyzed
whole saliva from 23 subjects and showed that statherin occurs in even more
variants. SV1, SV2 and statherin lacking the N-terminal aspartic acid
(statherin-des-Asp1) were found in almost all samples, whereas SV3 was
found in one of the 23 saliva samples. Other variants found were mono-
phosphorylated and non-phosphorylated statherin, statherin lacking the C-
terminal threonine and phenylalanine residues (statherin-des-Thr42, -Phe43),
statherin-des1-9, statherin-des1-10, and statherin-des1-13. These variants could
be due to proteolytic activity among bacteria, which inevitably exist in whole
saliva. However, all variants were also found in parotid saliva (n=3) except
for non-phosphorylated statherin and SV3 (Inzitari et al., 2006), which
suggests that the variants may not be derived from proteolytic activity by
bacteria.
Glycoprotein-340 (gp-340)
Salivary gp-340 is a 300-400 kDa, highly glycosylated protein that is a
homologue to lung gp-340 (Prakobphol et al., 2000), which is an alternative
spliced form of DMBT1 (deleted in malignant brain tumours 1) (Holmskov et
al., 1999). Gp-340/DMBT1 is encoded by the DMBT1 gene on chromosome
10q25.3-26.1 and is expressed in salivary glands, lung, trachea, stomach,
small intestine, and tears (Holmskov et al., 1999; Mollenhauer et al., 1997;
Schulz et al., 2002). Low levels of RNA for gp-340/DMBT1 are found in
brain, uterus, testis, prostate, pancreas and mammary glands (Holmskov et
al., 1999; Braidotti et al., 2004).
Introduction
20
Figure 3. Schematic illustration of salivary gp-340 protein domains, glycosylation pattern, and biological functions.
Gp-340 belongs to the Scavenger receptor cystein-rich (SRCR) protein
superfamily (Holmskov et al., 1997), which is a family involved in the innate
immune system (Ligtenberg et al., 2001, Mollenhauer et al., 2000). Gp-340
contains 13 SRCR domains (involved in protein-protein interactions in the
innate immune system) (Bikker et al., 2002) separated by SID (SRCR
interspersed domain) except for SRCR domain 4 and 5, and two CUB
(Clr/Clq Uegf Bmp1) domains (involved in development) (Bork and
Beckmann, 1993) separated by a 14th SRCR domain (Figure 3). There is also
a short Thr-region, a Ser-Pro-Thr region and a ZP (Zona Pellucida) domain
(Ligtenberg et al., 2001). Gp-340 contains putative glycosylation sites for O-
linked carbohydrate chains, highly densed in the SIDs, and 14 possible sites
for N-linked carbohydrate chains mainly in the C-terminal CUB-/ZP
domains (Holmskov et al., 1999; Mollenhauer et al., 1997). In saliva, gp-340
forms an oligomeric complex with S-IgA (Ericson and Rundegren, 1983).
Gp-340 has many biological functions. It binds surfactant-protein D (SP-D)
and A (SP-A) via protein-protein interactions in a calcium-dependent
manner (Holmskov et al., 1997; Tino and Wright, 1999). SP-D and SP-A
belong to the collectin family, which promotes binding of specific
carbohydrates on pathogenic microorganisms, e.g. bacteria, viruses and
yeasts, and activates complement cascades (Ligtenberg et al., 2001). Gp-340
interacts with bacteria through carbohydrate and peptide receptors. It is a
major host ligand for S. mutans, which is both aggregated and attached by
gp-340 (Ericsson and Rundegren, 1983; Loimaranta et al., 2005; Prakobphol
et al., 2000; Lightenberg et al., 2001). A 16-amino acid peptide from the
Introduction
21
SRCR domain (SRCRP2) is suggested to be a receptor for S. mutans, and the
bacteria-binding motif has been delineated to VEVLXXXXW (Bikker et al.,
2002; Bikker et al., 2004). Gp-340 also binds S. agalactiae, which is
implicated in neonatal meningitis, H. pylori, implicated in gastritis, peptide
ulcer disease and stomach cancer, and several commensal streptococci
(Prakobphol et al., 2000; Loimaranta et al., 2005). Further, gp-340 could
interact with polymorphonuclear leukocytes (PMN) through sLex epitopes
(Prakobphol et al., 1998; Prakobphol et al., 2000), binds S-IgA (Ligtenberg
et al., 2004), lactoferrin (Mitoma et al., 2001), MUC5B (Thornton et al.,
2001), induces aggregation of influenza A virus (Hartshorn et al., 2003), and
inhibits human immunodeficiency virus (HIV) infectivity by binding viral
glycoprotein 120 in a calcium-dependent manner (Wu et al., 2003). Further,
gp-340 binds C1q and activates the first complement component (C1) via the
classical pathway (Boackle et al., 1993) and stimulates random migration
(chemokinesis) of alveolar macrophages (Tino et al., 1999). Homologues of
gp-340/DMBT1 have been identified in rat (Ebnerin), mouse (CRP-ductin)
and rabbit (Hensin) (Li and Snyder, 1995; Madsen et al., 2003; Takito et al.,
1999).
Variation of DMBT1 has been described in the normal population as well as
in tumours. Variable numbers of tandem repeats (VNTRs, variation in length
of a repeated nucleotide sequence), in the SRCR/SID region are found in
tumours from brain, lung, pancreas and male breast (Mollenhauer et al.,
2002). Single nucleotide polymorphisms (SNPs, variation in one single
nucleotide) in DBMT1 (C/T polymorphism in the 5´-region) decreases the
promotor activity and is thus hypothesized to be associated with increased
breast cancer risk (Tchatchou et al., 2010). However, it is unknown whether
these tumour-associated polymorphisms are a cause or effect of the
carcinomas.
Host ligands in milk
Human milk is the main source of nutrients for the infant and contains also
numerous proteins, glycoproteins, glycolipids, polypeptides, and
oligosaccharides that could affect colonization of bacteria in the oral cavity
(Table 1). Human milk proteins are traditionally divided into caseins and
whey proteins. Caseins are found in the pellet when human milk is
precipitated with acid and whey proteins are the remaining soluble proteins.
Caseins comprise 10-50% of the total protein in human milk and whey
proteins comprise 50-90% depending on lactation length (Kunz and
Lönnerdal, 1992). Caseins are further described below. The major
Introduction
22
components of the whey fraction of milk are: (i) α-lactalbumin, (ii)
lactoferrin, (iii) lysozyme, and (iv) other proteins. (i) α-lactalbumin, which
comprises 10-20 % of the total protein, is a 14.1-kDa protein, which is
calcium-binding although it is non-phosphorylated and non-glycosylated. α-
lactalbumin, forms a protein complex with oleic acid in the infant’s stomach,
and that induces apoptosis in tumour cells while normally differentiated cells
are unaffected (Gustafsson, 2005). (ii) Lactoferrin is an iron-binding protein
with a molecular weight of 80 kDa. Human lactoferrin possesses antiviral
potency against HIV (Harmsen et al., 1995) and inhibits attachment of
various bacteria, such as H. pylori, to gastric epithelial cells (Orsi, 2004),
E. coli to urinary tract epithelium (Hanson, 2004), and S. mutans to dental
polymer disks (Berlutti et al., 2004). In addition, lactoferrin per se is
bacteriostatic (it inhibits growth of bacteria) by scavening iron (Lönnerdal,
2003; Soukka et al., 1991) and fungistatic towards C. albicans (Andersson et
al., 2000). (iii) Lysozyme is a 15-kDa protein and a major component of the
whey protein fraction. It causes lysis of Gram-positive bacteria by
hydrolyzing β-1,4 linkages of N-acetylmuramic acid and 2-acetylamino-2-
deoxy-D-glucose in the outer cell wall (Chipman and Sharon, 1969).
Interestingly, lysozyme and lactoferrin enhance each other’s effects, i.e.
lactoferrin binds and degrades lipopolysaccharides in the outer cell wall, and
lysozyme degrades the inner proteoglycan matrix leading to death of the
bacteria (Ellison and Giehl, 1991). Lysozyme also has an antiviral effect
against HIV (Harmsen et al., 1995). (iv) Other proteins present in the whey
fraction of human milk are the highly glycosylated bile salt-stimulated lipase
(BSSL) (Bläckberg and Hernell, 1981), albumin, and the immunoglobulins S-
IgA and IgG.
Caseins
Among the many proteins in human milk, caseins were selected for deeper
analyses. Caseins, which form micelles or submicelles in the native stage, are
present in three forms: α-, β-, and κ-casein. Human milk contains β- and κ-
casein, while bovine milk also contains two forms of α-caseins. β-casein
constitutes approximately 85% of the human milk caseins, but the β/κ-ratio
changes during lactation. β-casein is a 212-aa, 24-kDa, non-glycosylated
protein that is phosphorylated (0-5 phosphates) in the N-terminal end with
high affinity for hydroxyapatite and binding of Ca2+ (Lönnerdal, 2003). β-
casein is thought to be an unfolded or random coil structure protein, and
based on the aa sequence, it forms little secondary structure due to the many
proline residues that are evenly distributed in the protein that disrupt α-
helical and β-sheet structures (Bu et al., 2003). However, Syme et al. (2002),
found that β-casein contains approximately 10% α-helical structures and
20% β-structure. β-casein is encoded by the CSN2 gene on chromosome
Introduction
23
4q13.3 located within the same region as the STATH gene (Huq et al., 2005).
κ-casein is a glycosylated protein with a molecular weight of 37 kDa. It is
present in lower concentrations (<15% of the total caseins) in human milk. κ-
casein inhibits adhesion of H. pylori to gastric mucosa, probably due to
interactions with the carbohydrate moieties (Strömqvist et al., 1995;
Hamosh, 1998). It has also been reported that α-, β-, and κ-caseins of bovine
origin inhibit adhesion of S. mutans to saliva-coated hydroxyapatite (Vacca-
Smith et al., 1994).
Bioregulatory proteins and peptides in saliva and milk
Innate immunity is an immediate peptide-based host defence mechanism
that controls bacterial adhesion and growth before an antigen-specific
immune response has been developed (Boman, 1998; Gough and Gordon,
2000). Bioregulatory active peptides are released from “mother” proteins
with less biological effect, and their effect is rapid. Their effect is exerted by
various mechanisms, such as reduction of microbial growth, promotion of
clearance by aggregation, inhibition of adhesion of opportunistic microbes,
promotion of adhesion of commensal bacteria, normalization of pH, and
induction of bacterial phagocytosis.
The cecropines in insects, which lyse bacterial, but not eukaryotic cells, were
the first innate immunity peptides identified (Steiner et al., 1981). By now,
several hundred antimicrobial peptides have been identified. Many of the
identified innate immunity peptides are present in both saliva and milk, e.g.:
β-defensins (killing of Gram-negative and some Gram-positive bacteria and
C. albicans) (Mathews et al., 1999; Jia et al., 2001); LL37 (broad-spectrum
antimicrobial activity against bacteria, C. albicans and viruses) (Murakami
et al., 2002; Murakami et al., 2005); histatin 5 (from histatin 3, possessing
antifungal activity) (Oppenheim et al., 1988; Ruissen et al., 2001; Pollock et
al., 1984); and lactoferricin (from the amino terminal region of lactoferrin
has bacteriocidal, antiviral and cytotoxic activity against cancer cells) (Orsi,
2004; Gifford et al., 2005; Eliassen et al., 2006). In addition, both saliva and
milk contain bioregulatory proteins such as lysozyme (Aimutis, 2004) and
lactoperoxidase (Månsson-Rahemtulla et al., 1988; Aimutis, 2004). Salivary
gp-340 is an innate immunity glycoprotein with many functions such as
interactions with bacteria, viruses, yeasts (Lightenberg et al., 2001;
Prakobphol et al., 2000; Loimaranta et al., 2005; Hartshorn et al., 2003; Wu
et al., 2003) and stimulation of random migration of macrophages (Tino and
Wright, 1999). Salivary statherin is reported to be bacteriostatic (Kochanska
et al., 2000) and may contribute to the antimicrobial properties of nasal fluid
Introduction
24
(Cole et al., 1999). In addition, the bovine milk casein peptides β-
casomorphine-7 and kappacin possess opiate like activity and inhibit
bacterial growth, respectively (Kurek et al., 1992; Malkoski et al., 2001).
Thus, there are several proteins and peptides present in both saliva and milk
that possess bioregulatory activity and protect the host from infection.
Host ligand glycosylation and secretor status
Many host proteins in human saliva and milk are glycosylated, which is a
post-translational modification, in which monosaccharides are sequentially
added by glycosyltransferases in the Golgi apparatus and endoplasmic
reticulum. There are two main types of glycosylation, N-glycosylation and O-
glycosylation. In N-glycosylation the polysaccharide is linked to aspargine
and in O-glycosylation the polysaccharide is linked via N-
acetylgalactosamine (GalNAc) to serine or threonine. The carbohydrates are
highly diverse and differ in structure between tissues, individuals and animal
species (Lis and Sharon, 1993). Glycans have many functions, both physico-
chemical, such as control of protein folding, stabilization of protein
conformation, and resistance against proteases (Lis and Sharon, 1993). The
glycans also have various biological functions, such as participating in
microbial interactions (Aspholm-Hurtig et al., 2004; Strömberg et al., 1990),
interaction with viruses, and cell-cell communication (Varki, 2006). The
difference in glycosylation could explain the tropism of infections between
tissues, individuals and animal species.
Prominent examples of glycosylation diversity between individuals are the
ABH and Lewis blood group antigens, crucial to transfusion medicine,
among many others. In the H antigens (H type 1 and H type 2), Fucα1-2 is
added to the terminal Gal of the type 1 (Galβ1-3GlcNAcβ-R) or type 2 (Galβ1-
4GlcNAc-R) precursor chains. Antigens A and B are GalNAcα1-3 and Galα1-
3 added to the terminal Gal on the H-antigens. Lewis a antigen (Lea) and
Lewis x antigen (Lex) are Fucα1-3/4 added on the GlcNAc of the type 1
(Galβ1-3GlcNAcβ-R) or type 2 (Galβ1-4GlcNAc-R) precursor, respectively.
The difucosylated Lewis b antigen (Leb) and Lewis y antigen (Ley) are Fucα1-
3/4 added on the GlcNAc of H type 1 and H type 2, respectively. The secretor
phenotype, expressing ABH, Leb, and Ley in saliva and milk, is found in 80%
of the population of Europe and North America, while the non-secretor
phenotype, expressing Lea and Lex but not ABH, Leb, and Ley, is found in the
remaining 20% (Marionneau et al., 2001; Henry et al., 1995).
Introduction
25
Secretor status glycosylation has been associated with susceptibility towards
infectious diseases. Non-secretors are more prone to recurrent urinary tract
infections by E. coli because the addition of fucose by α-1,2-
fucosyltransferase in secretors masks the binding epitope (Stapleton et al.,
1992). It is also suggested that non-secretors are more susceptible to
candidosis as they harbour higher carriage of C. albicans (Ben-Aryeh et al.,
1995). Non-secretors are also more caries prone (Arneberg et al., 1976;
Holbrook and Blackwell, 1989). Secretors are susceptible to Norwalk virus
infection causing gastroenteritis, whereas non-secretors are not (Lindesmith
et al., 2003), and the H. pylori BabA adhesin binds to Leb present in
secretors (Ilver et. al., 1998).
Model microorganisms present in oral biofilms
Actinomyces
Actinomyces are Gram-positive, pleomorphic bacteria of which several
species colonize soft and hard tissues of the mouth. Thus, A. naeslundii,
A. viscosus, A. odontolyticus, A. israelii and A. gerencseriae are identified in
the mouth (Li et al., 2004; Aas et al., 2005). Actinomyces species are
considered to be part of the commensal oral flora. A. naeslundii are pioneer
colonizers and could be important in stabilizing the early biofilm together
with streptococci (Dige et al., 2009). Actinomyces species can remove sialic
acid epitopes by sialidases and change the environment in the biofilm by
modulation of pH (consuming lactic acid and degradation of urea) and
removal of oxygen (Dige et al., 2009). A. naeslundii genospecies 2 (and also
other species) mediate mutualistic growth of the anaerob P. gingivalis
(Periasamy and Kolenbrander, 2009). However, if invading a broken
epithelial cell lining, Actinomyces may cause severe infections (Smego and
Foglia, 1998). Further, Actinomyces has been discussed in relation to root
surface caries (Brailsford et al., 1998) and root canal infections (Collins et al.,
2000).
A. naeslundii, with the two sub-species genospecies 1 and 2, are the
predominant Actinomyces species in dental biofilms (Whittaker et al., 1996).
A. naeslundii expresses two antigenically distinct fimbriae (type-1 and type-
2) for attachment to host ligands. Type-1 fimbriae of A. naeslundii and
A. viscosus, encoded by the fimP gene, mediate binding through protein-
protein interaction to saliva APRP and statherin, respectively (Cicar et al.,
1991; Hallberg et al., 1998). The type-2 fimbriae, encoded by the fimA gene
and expressed by both genospecies of A. naeslundii, mediate binding to
Introduction
26
carbohydrate structures (β-linked galactose and galactosamine) (Hallberg et
al., 1998; Ruhl et al., 2004). Some A. naeslundii express both type-1 and
type-2 fimbriae (e.g. T14V, Ruhl et al., 2004), some only express type-2 (e.g.
ATCC 12104, Ruhl et al., 2004), whereas no strain expressing only type-1 has
been found.
The taxonomy of Actinomyces has changed in the last few decades. The
former separation of A. viscosus and A. naeslundii based on their catalase
reactions, was changed when Johnson et al. (1990) investigated DNA-DNA
relatedness among oral Actinomyces. Johnson et al. proposed that
A. naeslundii serotype II and III, A. viscosus serotype II, and Actinomyces
serotype NV should be included into A. naeslundii genospecies 2. Further,
Johnson et al. proposed that A. naeslundii genospecies 1 included
A. naeslundii serotype I and that A. viscosus serotype I (a strain isolated
from hamster mouth) was named A. viscosus. These taxonomy changes were
also supported by Putnins and Bowden (1993). Henssge et al., (2009) have
further proposed that A. naeslundii genospecies 2 should be named A. oris,
A. naeslundii genospecies WVA 963 should be named A. johnsonii, and
A. naeslundii genospecies 1 should be named A. naeslundii. In the present
thesis the taxonomy by Johnson et al. (1990) is followed.
Streptococci
Streptococci are Gram-positive, chain forming, and nearly spherical bacteria.
Most Streptoccocci that colonize the mouth are part of the commensal flora,
and several species are dominant in the first monolayer on teeth, such as
S. sanguinis, S. oralis and S. mitis (Nyvad and Kilian, 1990b). Still many
species of streptococci are linked to infections, such as tonsillitis, otitis, and
dental caries (Stenudd et. al, 2001; van Houte, 1993; Brook 1998).
Historically, streptococci have been classified into groups depending on their
carbohydrate composition in the cell wall (Lancefield, 1933). Streptococci
can also be classified into 6 groups (mutans, mitis, anginosus, pyogenic,
bovis and salivarius) based on relatedness of their 16S rRNA (Nobbs et al.,
2009). The mutans group streptococci (S. mutans and S. sobrinus) are
implicated in dental caries. Mutans streptococci are both aciduric and
acidogenic by producing lactic acid as a by product of carbohydrate
metabolism. However, other non-mutans groups (e.g. mitis- and salivarius-
group) are also thought to be acidogenic at low pH (van Houte, 1994). Still
others, such as S. sanguinis, are associated with health (Skovbjerg et al.,
2009; Becker and Paster, 2002).
Introduction
27
Multiple surface polypeptides (adhesins) are expressed by streptococci for
adhesion to host ligands and other microorganisms. Many of the adhesin
proteins contain a consensus sequence (LPxTz) in the C-terminal end,
through which the adhesin is linked to the cell wall (Nobbs et al., 2009). Oral
streptococci express three families of adhesins (Antigen I/II (AgI/II)-, Csh-,
and Fap1-family proteins) (Jakubovics et al., 2005). AgI/II is expressed by
most streptococci in the oral cavity. Proteins belonging to the AgI/II family
are e.g. SspA and SspB in S. gordonii (Loimaranta et al., 2005), Pas protein
in S. intermedius, and SpaP or Pac (also termed B, IF, P1, SR, MSL-1) in
S. mutans (Oho et al., 2004). The proteins in the AgI/II family are highly
conserved. Thus, SpaP and Pac are 97% identical in the amino acid sequence,
and SspA and SspB are >96% identical in the C- and N-terminal end but are
only 26% identical in the V-region (Jakubovics et al., 2005). Specifically, the
alanine-rich N-terminal region of the AgI/II adhesin of S. mutans interacts
with glycosylated proteins, such as the saliva gp-340 protein (Hajishengallis
et al., 1994; Oho et al., 2004). Several other streptococci, including
S. gordonii, have also been shown to adhere to salivary gp-340
(Kolenbrander, 2000; Prakobphol et al., 2000). In addition, AgI/II are
shown to interact with other host ligands (e.g. collagen, laminin, and
fibronectin) and other oral microorganisms such as A. naeslundii,
C. albicans, P. gingivalis (Nobbs et al., 2009).
Aims
28
AAIIMMSS
The overall aim of the present thesis was to study the effect of
phosphorylated polypeptides and gp-340 in saliva and milk on adhesion and
aggregation of oral bacteria. The specific aims were the following:
I. delineate the binding epitope in the calcium-binding phosphorylated
polypeptide statherin for A. naeslundii and A. viscosus.
II. investigate the presence of phenotypic variation of saliva gp-340.
III. explore if human milk promotes or prevents S. mutans adhesion to
saliva-coated hydroxyapatite.
IV. identify human milk components inhibiting adhesion of S. mutans
to saliva-coated hydroxyapatite and investigate whether gp-340 is
expressed by mammary glands.
Materials and Methods
29
MMAATTEERRIIAALLSS AANNDD MMEETTHHOODDSS
This section gives an overview of materials and major methods used in the
thesis. For more detailed information see the individual papers.
Bacteria and fungus strains
The following bacteria and fungus strains were used in the studies:
A. viscosus strains R28, T6-1600, ATCC 19246, A. naeslundii gsp 1 strains
35334, 29952, 30267, ATCC 12104 and gsp 2 strains T14V, M4356,
A. radicidentis strain 42377, S. mutans strains Ingbritt, LT11, NG8, JBP,
S. suis KU5, E. coli MS 506, S. sobrinus strains OMZ176, 6715, H. pylori
strain 17-1, genetically modified Lactococcus lactis strains expressing Pac or
SpaP, and C. albicans strain GDH18.
Saliva and milk
Parotid saliva secretion was stimulated by an acidic lozenge and the secreted
saliva was collected into ice-chilled test tubes using sterile Lashley cups. Milk
was collected from mothers who had been breast-feeding for 6 to 13 months.
The milk was defatted by centrifugation and the pellet and upper cream layer
were discarded. Saliva and milk samples were stored at -80°C until used.
Depending on the purpose, saliva and milk were used from single donors or
pooled from several donors (see further below).
Purification of saliva proteins
Statherin was purified from pooled fresh parotid saliva. The saliva was
separated by anion-exchange chromatography, and fractions containing
statherin were concentrated before separation by gelfiltration. Identity and
purity were determined with sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) and bacteria binding specificity to A. viscosus
19246 (Li et al., 2001).
Materials and Methods
30
Gp-340 was purified from fresh parotid saliva, from individual donors
(Paper II) or pooled from multiple donors (Papers II and IV), utilizing the
ability of S. mutans strain Ingbritt to bind gp-340 in solution. Equal volumes
of parotid saliva and bacterial suspension of S. mutans Ingbritt were mixed
and incubated at 37°C for formation of aggregates. After centrifugation and
washing, gp-340 was released from the bacteria by repeated treatment with
ethylenediamine tetraacetic acid (EDTA). The pooled supernatants were
concentrated and proteins separated by gelfiltration. The gp-340-containing
fractions were pooled and concentrated. The identity and purity of gp-340
was determined with electrophoresis, on 4-20% Tris-HCl gel in reduced and
non-reduced form, and by confirming the aggregation and adhesion patterns
for three S. gordonii strains (DLI, M5, and SK184) (Loimaranta et al.,
2005).
Size variants of gp-340
Individual gp-340 variants were revealed by separating unreduced parotid
saliva proteins from healthy, young individuals (n=7) with SDS-PAGE on 5%
precast gels. Gp-340 was detected with mAb143 after immunoblotting. The
size variants were confirmed by separating purified gp-340 from the same
subjects and staining with mAb143 and glycan detection.
Carbohydrate mapping of gp-340 variants
Carbohydrates present on gp-340 size variants were investigated by antibody
and lectin binding in an overlay assay. Briefly, parotid saliva and gp-340
separated by SDS-PAGE were blotted onto a hydrophobic membrane
(PVDF). After blocking with dried milk or bovine serum albumin (BSA),
antibody or lectin was incubated with the membrane, and results were
detected using horseradish peroxidase-labeled secondary antibodies or
peroxidase-conjugated streptavidin and chemiluminiscence.
Enzyme-linked immunosorbent assay (ELISA) and ELISA-like lectin binding
were also used for mapping of carbohydrates on gp340. The wells of a
microtiter plate were coated with gp-340, and after blocking with BSA, the
wells were incubated with primary antibodies directed against blood group-
and Lewis antigens (A, B, H, Lea, Leb, Lex, Ley, and sLex), or biotinylated
lectins. Horseradish peroxidase-conjugated secondary antibodies or
Materials and Methods
31
peroxidase-conjugated streptavidin and ortho-phenylenediamine substrate
were added before measuring absorbance.
Secretor status of the participants was determined by using parotid saliva in
ELISA with antibodies against antigen H (cross-reacting with A and B
antigen), Lea, Leb, Lex, and Ley.
Separation and identification of milk proteins
Defatted milk was separated by gelfiltration, the fractions containing
proteins were collected, and dominant proteins were identitified by SDS-
PAGE and immunoblotting. Primary antibodies against IgG (γ-chain), IgA
(α-chain), albumin, lactoferrin, lysozyme, BSSL and gp-340 were used. The
fractions were further used to test for bacterial binding and binding
inhibition.
Adhesion assay
Adhesion of bacteria and C. albicans to different ligands was examined using
a hydroxyapatite (HA) assay (Gibbons and Hay, 1988). HA beads, a model
for tooth enamel, were coated with host ligands (e.g. statherin, hybrid
peptide, gp-340, milk or saliva), and the coated beads were washed and
blocked with BSA before incubated with radiolabelled microorganisms
(Figure 4). Thereafter unbound cells were washed away and the amount of
bound microorganisms was determined by scintillation counting in a β-
counter. BSA-coated beads (BSA-HA) were used as negative control and
Figure 4.
Priciple of
HA assay.
Radiolabelled
bacteria and
Candida cells
were bound
to test ligands
coated onto
HA-beads.
Materials and Methods
32
binding to standard saliva as positive control. Background binding, i.e. to
BSA-HA, was low (1-6%) and constant within the experiments.
Adhesion inhibition assay
The ability of test substances to inhibit microorganism binding was tested by
(a) simultaneous addition of bacteria and test substance to host ligand-
coated HA (Papers III and IV) (Figure 5, panel a), (b) pre-incubation of
microorganisms with test substance before adding to host ligand-coated HA
(Papers I and IV) (Figure 5, panel b), and (c) pre-incubation of host ligand-
coated HA with test substance before microorganisms were added
(unpublished results, Paper IV) (Figure 5, panel c). Binding after exposure to
Figure 5. Schematic illustration of inhibition experiments. (a) Bacteria and test substance are added simultaneously to HA-beads coated with host ligand (saliva/gp-340) and blocked with BSA. The test substance can inhibit bacterial binding by blocking bacterial adhesins or receptors on saliva/gp-340. (b) Bacteria were pre-incubated with test substance and washed once before added to HA-beads coated with saliva/statherin/gp-340 and blocked with BSA. If inhibition occurs, the test ligand blocks the bacterial adhesins. (c) HA-beads coated with host ligand (saliva/gp-340) and blocked with BSA are pre-incubated with test substance. Untreated bacteria are added to the washed pre-incubated HA-beads. If inhibition occurs, the test substance blocks the host receptor.
test substances was compared with binding of untreated microorganisms/s-
HA. Inhibition was calculated by using the equation 100x(bindingcontrol-
Materials and Methods
33
bindingtest)/bindingcontrol. The samples were run in triplicate and both a
positive and a negative control (BSA-HA) were included in each experiment.
In Paper I, inhibition of bacterial binding by test substances was also
investigated using calcium-phosphate-coated latex beads that were coated
with statherin and blocked with BSA. Microorganisms were incubated with
test substance (statherin or peptides) and added to the latex beads.
Aggregation was scored visually on a scale from 1 to 5 (Hallberg et al., 1998).
Latex beads coated with BSA were used as negative control and statherin-
coated latex beads with untreated microorganism were used as positive
control.
Peptides for mapping bacteria-binding epitopes
For mapping of bacteria-binding epitopes in statherin, synthetic custom
peptides and a hybrid peptide construct were used. The hybrid peptides had
a common hydroxyapatite binding (phosphorylated) N-terminal end,
Figure 6. Construction of hybrid peptides. A common phosphorylated N-terminal end with high affinity for hydroxyapatite, consisting of the first 15 aa of statherin, is linked to various test epitopes by a proline residue. The proline residue is flexible and facilitates presentation of the test epitopes when the hybrid peptide adheres to hydroxyapatite. Five hybrid peptides, which together covered aa 16-43 of statherin, were constructed.
Materials and Methods
34
consisting of the first 15 amino acids (aa) of statherin, linked to various test
epitopes of 5-6 aa via a proline residue (Figure 6) (Gilbert et al., 2000).
Together, the test epitopes covered aa 16 to 43 of statherin. Synthetic custom
peptides with unmodified endings covering the same amino acids as the test
epitopes of the hybrid peptides were used in inhibition experiments.
For mapping of binding epitopes in β-casein, syntetic peptides (30 aa)
together covering the sequence of β-casein, were used. In addition, a peptide
from lactoferrin, corresponding to a 13-aa stretch in bovine lactoferrin
inhibiting S. mutans binding to saliva and gp-340-coated HA (Oho et al.,
2002; Oho et al., 2004), was synthesized, and used for binding-inhibition
experiments.
Binding of bacteria in solution (aggregation assay)
Equal amounts of bacteria (Actinomyces, Streptococcus) or Candida cells
and test substance (saliva, milk, milk fractions, purified milk proteins,
statherin, or peptides) were incubated at room temperature or 37°C on a
glass slide. Aggregation was scored visually on a scale from 1 to 5 (Hallberg
et al., 1998).
Aggregation by gp-340 was done by adding gp-340 to washed S. mutans,
S. suis, H. pylori, L. lactis or E. coli and OD700 was recorded for 90 minutes.
Aggregation was expressed as the percentage of OD700 at 60 minutes
compared to OD700 at 0 minutes.
Results and Discussion
35
RREESSUULLTTSS AANNDD DDIISSCCUUSSSSIIOONN
Paper I. Binding epitopes for Actinomyces on salivary statherin
The adhesion of Actinomyces strains to statherin segments revealed that
strains with different origins bind to different statherin segments, which may
confer a role of statherin in biofilm diversity. The statherin molecule has
three distinct structural domains when bound to hydroxyapatite (Long et al.,
2001; Naganagowda et al., 1998; Goobes et al., 2006). An α-helix in the N-
terminal region, followed by a polyproline type II structure, and an α-helix
(Figure 7). Based on the binding specificity the bacteria could be divided into
three distinct binding types (types I, II, and III). Actinomyces isolated from
human infections and rat/hamster (type I adhesion) bound in the C-terminal
α-helix region, whereas commensal Actinomyces (type II adhesion) bound in
the middle polyproline type II region. The third binding type, Actinomyces
isolated from human infections and also C. albicans, bound to statherin but
not to any of the linear statherin segments. This suggests the need for the
whole statherin molecule for the correct conformation and presentation of
the binding epitope(s). None of the bacteria bound to the hydroxyapatite-
binding, helical N-terminal part of statherin.
Figure 7. Binding of Actinomyces strains to statherin. Commensal strains bound to the middle region of statherin whereas infectious Actinomyces strains bound to the C-terminal end. The binding of infectious strains could be inhibited whereas commensal strains could not be inhibited by statherin-derived peptides.
Results and Discussion
36
To further delineate the binding epitopes, hybrid peptides were used. The
hybrid peptides were constructed as a fusion between the statherin
hydroxyapatite-binding N-terminal end and various test epitopes, connected
via a proline residue. This directed the epitope out from the surface and
fascilitated presentation of the epitope. The N-terminal end (aa 1-15) did not
mediate binding of any of the tested microorganisms. Together the test
epitopes cover the middle- and C-terminal part of statherin. This type of
fusion peptide has been used to show that an osteopontin peptide, PGRGDS,
mediates dose-dependent adhesion of melanoma cells when bound to
hydroxyapatite (Gilbert et al., 2000). By using hybrid peptides, Drobni et al.
(2006a) showed that A. naeslundii, A. viscosus, and S. gordonii recognized
different peptide motifs in saliva PRP-1.
Actinomyces strains classified as type I bound to the most C-terminal test
epitopes, PYQPQY (aa 33-38), QQYTF (aa 39-43), and PYQPQYQQYTF (aa
33-43), whereas type-II strains bound to the two middle YQPVPE (aa 21-26)
and QPLYPQ (aa 27-32) epitopes. The only common amino acids among the
binding motifs are Y and Q, suggesting that these aa could be influencial for
binding. When substituting some aa in QQYTF with alanine, an amino acid
that does not add any features but removes them, it was discovered that the
TF dipeptide was crucial for inhibition and desorbtion of type-I strains, since
QQYTF, QAATF and TF, but not QQYAA and PYQPQY, inhibited binding.
However, the inhibition and desorbtion effect was even more efficient when
preceding the TF dipeptide with QAA (QAATF instead of QQYTF). This
might indicate that both Q and TF are binding sites or that QAA gives the
peptide an optimal configuration for the presentation of the TF epitope. The
importance of single amino acids flanking a binding epitope has been shown
for RGD peptides that inhibit appressorium formation in Uromyces
appendiculatus (Correa et al., 1996). Appressorium is a hypha structure by
which the plant pathogenic fungus, Uromyces appendiculatus, infects its
host plant. Also for RGRPQ, a peptide released from saliva PRP-1 that
inhibits adhesion of A. naeslundii to tooth enamel, it has been shown that
single amino acids are important for the binding inhibition (Drobni et al.,
2006b; Drobni et al., 2006c). In accordance with the findings in Paper I, the
Q residue in RGRPQ was crucial for binding inhibition, but the size and
hydrophobicity of the amino acids at positions 2 and 4, respectively, were
also influential (Drobni et al., 2006c). The latter result was accomplished
with QSAR (quantitative structure activity relationship) analyses. QSAR is a
multivariate mathematical model in which chemical properties are related to
biological activity. The model can help predict peptide design for optimal
biological response. This approach could also be useful for optimizing
inhibitory peptides from statherin.
Results and Discussion
37
Actinomyces strains with infectious and rat/hamster origin could be
inhibited by peptides in solution whereas commensal strains could not. It
has earlier been shown that the FomA porin protein of F. nucleatum, a
bacterium associated with periodontitis, adheres to the same middle section
of statherin as the commensal strains in this study (Nakagaki et al., 2010).
However, the binding of F. nucleatum could be inhibited by statherin-
derived peptides (Sekine et al., 2004). P. gingivalis, also associated with
periodontitis, is also reported to bind the C-terminal portion of statherin in
an inhibitable fashion (Amano et al., 1996). This implies that there is a
possible adhesion-regulating property residing within the statherin
molecule, where adhesion of commensals is promoted whereas adhesion of
potentially infectious strains is inhibited. Bacterial proteolytic activity in the
saliva might release peptides from statherin that could inhibit the adhesion
of various bacteria, and thus regulate adhesion of bacteria in the oral biofilm
in favour of commensals. It is therefore tempting to speculate that statherin
is an innate immunity polypeptide, as has been suggested by Cole and
coworkers (1999) and by Kochanska et al. (2000), with a possible role in
protecting the host from colonization of bacteria associated with infections.
Several different statherin variants (among others SV1, SV2, SV3, Jensen et
al., 1991) have been detected in saliva, and Vitorino et al. (2008) have further
detected small statherin fragments in the aquired enamel pellicle, among
others YPQPYQPQ (aa 29-36) and some N-terminal fragments. According to
Helmerhorst et al. (2008), statherin could be cleaved by bacteria-derived
glutamine endoprotease. This endoprotease preferentially cleaves after a Q
residue, with specificity for XPQ. Helmerhorst et al. (2008) found several
statherin peptides in whole saliva, among them a peptide cleaved after
Q35P36Q37. The remaining YQQYTF (aa 38-43) peptide should then be
released, which hypothetically could block adhesion of type I Actinomyces.
Thus, it would be of interest to test the different statherin variants and
released statherin peptides for binding and binding inhibition of the panel of
oral bacteria belonging to binding type I-III. Presumably type I Actinomyces
would not bind SV1, SV3 and statherin des-Thr42, Phe43, and thus individuals
with more of these statherin variants would possibly favour colonization of
commensal Actinomyces strains.
Statherin binds not only hydroxyapatite, but it is also thought to cross-link to
buccal epithelial cells (BEC) (Bradway et al., 1992), catalysed by oral
transglutaminase to form cross-links between glutamine (Q) and lysine (K)
residues (Yao et al., 1999). Statherin has one lysine residue (Lys6) in the N-
terminal and seven glutamine residues in the middle and C-terminal part,
where cross-linking can form. The reactivity of lysine (K) was higher (100%)
than of glutamine (Q) (14%) (Yao et al., 1999). For statherin it has been
Results and Discussion
38
shown previously that the C-terminal forms cross-links catalyzed by
transglutaminase (Bradway et al., 1992). Cross-linking of the C-terminal
would, hypothetically, alter the presentation of the C-terminal. Therefore, it
is hypothesized that Actinomyces type I from infectious origin and
rat/hamster might not be able to adhere to statherin cross-linked to BEC,
but still they could bind by co-aggregates. However, this remains to be
investigated. In addition, cross-linking between Lys6 and Gln37, facilitated by
transglutaminase, forms cyclo-statherin Q-37 detected in whole human
saliva (Cabras et al., 2006). How this cyclization affects bacterial binding is
unknown.
In the present paper we have suggested that the statherin peptides could
adopt different conformations depending on which amino acids that preced
the binding epitope. Conformational changes in statherin (Gibbons et al.,
1990; Amano et al., 1996) and APRP (Gibbons, 1989) have been shown
previously. In particular, salivary proteins, such as APRP, may contain
cryptic segments (cryptitopes), i.e. hidden binding epitopes, which only
become exposed when adsorbed to hydroxyapatite (Gibbons, 1989).
Statherin also seems to adopt different conformations depending on whether
it is in solution or bound to hydroxyapatite, since statherin per se could not
inhibit bacterial binding to statherin or hybrid peptides coated on HA-beads,
and statherin was unable to aggregate bacteria in solution. It is reported that
statherin has a disordered structure in aqueous solution and that the α-helix
in the N- and C-terminal areas form upon adsorption to hydroxyapatite
(Goobes et al., 2006). This supports the finding of the different behaviour of
statherin in solution versus when attached to hydroxyapatite. Together,
these findings suggest that statherin might play a role in biofilm diversity,
possibly promoting colonization of commensal bacteria. Statherin per se also
exists in different polymorphisms and could give rise to bioactive peptides
that could bind bacteria differentially. Hypothetically, this could alter the
composition of the biofilm towards a more health-associated or more
disease-associated biofilm.
Paper II. Variant size- and glycoforms of gp-340 with differential bacterial aggregation
In contrast to statherin, gp-340 is a large, heavily glycosylated protein. It is a
mucin hybrid type of protein with its tandem repeats of O-glycosylated and
serine-, threonine-, and proline-rich SID domains (involved in bacterial
interactions) and cystein-rich SRCR domains (for protein-protein
interactions). The domains contribute to the complex biological innate
Results and Discussion
39
immunity behaviour of gp-340 and to the potential links to caries and
Crohn’s disease (inflammatory disease of the gastrointestinal tract).
In this paper, four novel size variants of gp-340 were revealed in parotid
saliva and in purified gp-340 from seven donors, as detected by SDS-PAGE
and Western blot using a monoclonal antibody (mAb143) directed towards
the gp-340 protein core. Variants I, II, and III had single broad bands (345,
375, and 389 kDa, respectively), whereas variant IV had a double band (345
and 287 kDa) (Figure 8). When purified gp-340 from the four variants was
stained for carbohydrates the same bands appeared confirming the gp-340
size variants and that they were glycosylated. Purified gp-340 from lung
lavage, of a person with pulmonary alveolar proteinosis, also displayed a
double band in Western blot with mAb143. Lung gp-340 seemed to have
lower carbohydrate content than parotid saliva gp-340 (donor with variant
II). A glycan detection kit and carbohydrate composition analysis by high-
performance anion-exchange chromatography with pulsed amperometric
detection (HPAEC-PAD) confirmed a lower and different composition for
lung gp-340.
a) b) c)
I II III IV I II III IV Lung I II III IV Lung
Figure 8. Gp-340 size variants. (a) Unreduced parotid saliva was separated by SDS-PAGE and gp-340 size variants were detected by monoclonal antibody (mAb143). (b) Glycan detection of purified gp-340 separated by SDS-PAGE, revealed the same banding patterns. (c) The different size variants were further confirmed by mAb143 detection of purified gp-340 from the same individuals. However, the lower band of variant IV was difficult to detect.
The four size variants were sequenced with Edman degradation and were
found to have an identical N-terminal sequence (TGGWIP), which suggests
that the N-terminal protein core could be similar among the different size
variants. However, differences/deletions could still reside in the middle or C-
terminal of gp-340, and thus explain the different size variants detected.
Mollenhauer et al. (1999 and 2002) have described a DMBT1 allelic variant
Results and Discussion
40
(6 kb) with 5 SRCR and 5 SID domains deleted in the middle of the protein.
It has been found that variable numbers of tandem repeats (VNTRs) within
the SRCR/SID region in DMBT1 form a multi-allele system that could be
involved in the variability of DMBT1 in tumours and in healthy subjects
(Mollenhauer et al., 2000 and 2002). The polymorphism of gp-340 could
thus be explained by allelic variation, VNTRs, or splice variation in the
protein core. Post-translational modifications, such as proteolysis or
differences in glycosylation, could also be possible explanations for the gp-
340 size variants, but this remains to be investigated. It could be speculated
that variant IV could be derived from proteolysis of variant I since one of the
bands had the same size as variant I. Additionally, variant IV was only
detected in one individual.
The different size variants of gp-340 could possibly be related to several
biological functions. Jonasson et al. (2007) showed that the gp-340 I protein
correlates positively with caries experience and adhesion of S. mutans
Ingbritt to s-HA. The phenotype is overrepresented in subjects with Db
protein, a caries-susceptibility factor, and subjects positive for both gp-340 I
and Db experienced more caries (Jonasson et al., 2007). Thus, it is
speculated that gp-340 phenotype I could be a caries-susceptibility factor.
The caries-associated gp-340 size variant I was less common (22-26%) than
II/III in both Jonasson’s and the present study. Renner et al. (2007)
observed that the DMBT1 deletion variant (6 kb) was overrepresented
among patients with Crohn’s disease. One may hypothesize that the small 6
kb DMBT1 variant corresponds to the smaller (faster migrating) gp-340 size
variant I, however this remains to be investigated further.
All of the size variants of gp-340 from the seven donors were glycosylated
but displayed some differences in their glycosylation. The individuals with
gp-340 size variants I and IV versus II and III had secretor or non-secretor-
like fucosylated patterns, respectively, as shown by fucose-specific lectin
(UEA-1), ABH blood group antigens and Lewis antigens binding patterns.
However, several core carbohydrates, such as sialylated Galβ1-3GalNAc,
α2-6 and α2-3-linked sialic acids, N-glycans, and type 2-lactosamine or
polylactosamine structures seemed to be carried by all four variants. The
differences in glycosylation between the size variants could be indirectly
linked to the size variations because post translational glycosylation
normally varies between individuals. However, it could also be directly
linked to the size variants based on different or deleted glycosylation sites.
Gp-340 mediates binding of a wide array of bacteria by carbohydrate-lectin
binding or protein-protein interactions. Thus, differences in glycosylation
could potentially influence the ability to aggregate bacteria. The low and
Results and Discussion
41
high-glycosylated lung and parotid saliva gp-340 aggregated bacteria
differently (Table 2). The low-glyco- and GlcNAc-lung gp-340 displayed very
low aggregation of S. mutans LT11, whereas parotid saliva gp-340 had high
aggregation, potentially indicating a polylactosamine receptor. By contrast,
both lung and saliva gp-340 aggregated S. mutans Ingbritt strongly,
indicating that the two S. mutans strains (Ingbritt and LT11) bind to
different receptors. Such a presence of adhesins with different receptor
specificities has been described for S. gordonii (Loimaranta et al., 2005). In
addition, the high-glyco- and GlcNAc-saliva gp-340 aggregated S. suis KU5
highly, with binding specificity to sialylpolylactosamine, while lung gp-340
did not aggregate S. suis KU5, whereas both lung and saliva gp-340
aggregated S. suis 836 highly. The gp-340 size variants I-III showed
differential aggregation of S. suis KU5 and Leb-binding H. polyri 17:1, with
preferential aggregation of the highly glycosylated variant II (Table 2).
However, S. mutans Ingbritt was aggregated by all three size variants to a
similar extent indicating that the gp-340 variants share some carbohydrate
and peptide receptor motifs. By contrast, adhesion of S. mutans Ingbritt to
gp-340 variants I-III varied (I>II>III) (Jonasson et al., 2007), suggesting
that different epitopes are involved in aggregation and adhesion (Loimaranta
et al., 2005).
Table 2. Bacteria aggregation with different gp-340 variants.
_____________Aggregationa______________ Species and High glyco Low glyco Saliva gp-340 variant Receptor strains saliva lung I II III sugar gp-340 gp-340 __________________________________________________________ S. mutans
Ingbritt +++ +++ +++ +++ +++ sialic acid LT11 +++ - unknown S. suis
KU5 +++ - + +++ ++ Sia2-3PLb 836 +++ +++ Galα1-Gal H. pylori
17:1 ++ - ++ + Leb ___________________________________________________________ a Aggregation is indicated with + = weak aggregation, ++ = moderate aggregation, +++ = high aggregation, and – = no or very low aggregation. b Sia2-3PL = sialylα2-3polylactoseamine
The conclusion is that saliva harbours different gp-340 size variants among
individuals. In addition, individual differences in gp-340 glycosylation were
found. These size- and glyco-polymorphisms could affect both S. mutans
adhesion, development of caries, and interaction with a wide array of
bacteria and viruses (including HIV and influenza virus). In this respect, it is
notable that the differences in glycosylation between the gp-340 variants
possibly could be related to secretor status, which for a long time has been
Results and Discussion
42
discussed in relation to infections, such as Norwalk virus infection
(Lindesmith et al., 2003), H. pylori infection (Ilver et al., 1998), candidosis
(Ben-Aryeh et al., 1995) and dental caries (Arneberg et al., 1976; Holbrook
and Blackwell, 1989). Moreover, gp-340 is a pattern-recognition protein that
binds both Gram-positive and Gram-negative bacteria through conserved
leucine-rich repeats (Loimaranta et al., 2009). Therefore, the different size
variants of gp-340 could affect other biological pattern-recognition functions
related to host innate immune defences. Recently, gp-340 polymorphisms
have been linked to Crohn’s disease (Renner et al., 2007), and other diseases
involving bacterial components, such as dental caries (Jonasson et al.,
2007).
Papers III and IV. Effects of human milk and milk components on adhesion of mutans streptococci to saliva-coated hydroxyapatite in vitro
Human milk is an exocrine secretion which is very similar to saliva in many
aspects. Several proteins and peptides in saliva with innate immunity
functions are also present in human milk; examples are lactoferrin,
lactoperoxidase, lysozyme, LL37, and defensins (Murakami et al., 2005;
Lönnerdal, 2003; Jia et al., 2001). Gp-340 is another protein present in
saliva that also possesses innate immunity properties and binds S. mutans
strongly when the protein is bound to hydroxyapatite. Gp-340 has been
found in tears (Schulz et al., 2002; Jumblatt et al., 2006), saliva, lung, small
intestine, trachea, and stomach (Holmskov et al., 1999). Low levels of
messenger ribonucleic acid (mRNA) of gp-340/DMBT1 have also been
detected in mammary glands of non-lactating women (Holmskov et al.,
1999), and our own preliminary results suggested that gp-340 might be
present in human milk leading to the hypothesis that gp-340 is expressed in
lactating women, i.e. in human milk. However, further testing showed that
none of four tested antibodies directed against gp-340 could detect gp-340
in human milk separated with SDS-PAGE and Western blotting. This lead to
rejection of the hypothesis and the conclusion that the gp-340 protein is not
present in human milk or that the levels are too low to be detected by the
assay. This finding is supported by the fact that neither milk nor any milk
fractions mediated avid binding of S. mutans. However, human milk
proteins in the size interval of S-IgA, BSSL, lactoferrin, albumin, β-casein,
and lysozyme did adhere to hydroxyapatite as determined with SDS-PAGE.
In contrast, human milk could aggregate S. mutans weakly, but the extent of
aggregation varied between the individuals’ milk and strains. This might
Results and Discussion
43
indicate that milk proteins bound to hydroxyapatite have an unfavourable
conformation that does not present the binding epitope for S. mutans,
whereas the epitope is exposed in solution. This phenomenon, but reverse
when the epitope is exposed in solid phase (adsorbed to hydroxyapatite), but
not in solution, has been shown for statherin (Paper I; Amano et al., 1996)
and APRP (Gibbons and Hay 1988; Gibbons et al., 1991). In addition, gp-340
contains cryptitopes and binds different bacteria in solution as opposed to
when bound to hydroxyapatite (Loimaranta et al., 2005).
The effect of human milk on adhesion of cariogenic S. mutans to tooth
enamel coated with saliva protein (s-HA) was studied. It has previously been
shown that S. mutans adhere to certain salivary proteins (e.g. gp-340 and
PRP) coated on hydroxyapatite (Ericson and Rundegren, 1983; Gibbons and
Hay, 1989), and in this study the binding of S. mutans Ingbritt was 19-69%
to parotid saliva samples from 21 mothers. The mothers could be divided
into three groups depending on S. mutans binding to s-HA and binding
inhibition by their milk. The mothers in group I displayed high binding of
S. mutans to s-HA and high inhibition by milk; group II had moderate
binding to s-HA and moderate to low inhibition; whereas group III had
lower binding to s-HA and enhanced binding by milk (see Figure 2 in Paper
III). Milk with high inhibition effect also had a higher aggregation score, and
milk that increased binding aggregated S. mutans poorly. In the binding
experiments described in this paragraph, milk and saliva were from the same
mother.
The results above show that there are individual variations in the expression
of epitopes for S. mutans binding and binding inhibition. To further examine
the individual variation, saliva from three mothers (two avidly-binding
salivas and one low-binding saliva) were used to cross-test inhibition with
milk from all mothers. The adhesion of S. mutans to avidly-binding saliva
was inhibited by milk from all groups (group I, II, and III). However, milk
from all three groups increased S. mutans binding to hydroxyapatite coated
with low-binding saliva. Thus, the ability to inhibit binding of S. mutans
seems to depend on individual variations in the composition of the saliva.
Therefore, gene variation, alternative splicing, variation in
glycosyltransferases, or number of tandem repeats in saliva proteins might
contribute to the inhibiting effect. To further examine whether different
glycosylation phenotypes were present in groups I-III, blood group of the
mothers were determined from the medical records. In group I, 57 % of the
mothers had blood group antigen A, whereas 83% in group III had blood
group antigen O. Thus, there seems to be a difference in the most common
blood group among mothers with avidly-blocking milk (group I) and
enhanced-binding milk (group III). It has been reported that blood group
Results and Discussion
44
antigens are involved in susceptibility towards infections, e.g. binding of
H. pylori BabA adhesin to A, B, and O antigens in gastric epithelium (Aspholm-Hurtig et al., 2004). However, due to the small number of subjects
involved in this study, these findings only suggest that the blood group
glycosylation might be involved, but a more thorough study must be
performed to draw any firm conclusions.
These results suggest that the individual properties of saliva, such as
polymorphism in the heavily glycosylated S. mutans Ingbritt ligand gp-340
in saliva, determine the degree of S. mutans inhibition caused by human
milk. We have previously shown that gp-340 is a blood group- and Lewis-
antigen bearing protein (Paper II). In fact, a pilot evaluation suggested that
gp-340 variant I was over represented among mothers belonging to group I
(unpublished results). This is in line with the findings that gp-340 variant I
correlates positively with caries experience in 12-year-old children and that
adhesion of S. mutans Ingbritt to saliva-coated hydroxyapatite correlates
positively with their parotid saliva (Jonasson et al., 2007). This hypothesis is
presently being evaluated in a larger group.
To further investigate which components in human milk that are responsible
for the inhibition, human milk from two mothers with high binding/high
inhibition (group I) milk was fractionated with gelfiltration. Human milk
fractions coated onto hydroxyapatite did not mediate binding of S. mutans,
which further confirms that gp-340 or any other hydroxyapatite-binding
ligand for S. mutans is not present in human milk. However, fractions from
peaks containing IgG and S-IgA, BSSL, albumin, caseins, lactoferrin, and α-
lactalbumin inhibited binding of S. mutans to s-HA by more than 50%, and
thus were hypothesized as inhibitory components. Purified human milk
proteins confirmed that lactoferrin, caseins, S-IgA, and IgG reduced binding
of S. mutans Ingbritt avidly whereas BSSL and α-lactalbumin reduced the
binding moderately to low. However, lysozyme and albumin had no
inhibitory effect, suggesting that some other compound(s) in these peaks
were responsible for the inhibition. These findings are in accordance with
earlier studies, since an inhibitory effect of bovine lactoferrin and bovine
caseins on S. mutans adhesion to salivary proteins has been shown
previously (Vacca-Smith et al., 1994; Oho et al., 2002). Additionally,
S. mutans-specific S-IgA is present in human milk (Camling et al., 1987) and
an inhibitory effect of S-IgA on S. mutans adherence to s-HA has been
shown (Hajishengallis et al., 1992). However, it is speculated that bacterial
IgA1 proteases in the oral cavity might interfere with this anti-adherence
effect (Hajishengallis et al., 1992).
Results and Discussion
45
By preincubating either s-HA or bacteria with purified milk proteins in the
adhesion inhibition assay it was found that lactoferrin, β-casein,
α-lactalbumin and IgG all had an effect on the salivary host ligand side,
whereas S-IgA and BSSL had an effect on the bacterial side (Figure 9). The
effect on the salivary host ligand side could depend on binding of the milk
protein to the host ligands. That binding could prevent the bacteria to
adhere, or exchange bound salivary host ligands on hydroxyapatite, as
described for bovine casein derivatives (caseinoglycomacropeptide and
caseinophosphopeptides) that displace albumin from hydroxyapatite (Neeser
et al., 1994). As mentioned earlier, the milk proteins per se did not mediate
adhesion of S. mutans to hydroxyapatite. However, for lactoferrin, β-casein,
and α-lactalbumin, the binding was enhanced when preincubated with
bacteria, but the net effect (simultaneous addition) was inhibitory.
Figure 9. Adhesion of S. mutans to s-HA or gp-340-HA when milk proteins
are preincubated with either s-HA/gp-340-HA or the bacteria. Percent S. mutans binding is compared with control binding of non inhibited S. mutans binding to s-HA or gp-340-HA. (a) Milk proteins with effect on the saliva/gp-340, (b) milk proteins with effect on the bacteria.
For S-IgA and BSSL, the inhibitory effect was on the bacterial side, possibly
binding to the adhesin and preventing attachment to salivary host ligand
adsorbed to hydroxyapatite. The increased binding when preincubated with
s-HA or gp-340-HA was not easily explained. One feasible explanation could
be that S-IgA and BSSL alter the presentation of exposed binding epitopes of
the hydroxyapatite-adsorbed host ligands, giving a more favourable
Results and Discussion
46
presentation of the epitope to the bacterial adhesins. However, the net effect
was inhibitory.
Human milk contains both β- and κ-casein, but β-casein constitutes almost
85% of the total caseins in human milk. Therefore, it was hypothesized that
β-casein was the strong inhibitor among the caseins. Seven synthetic
peptides covering the sequence of β-casein were synthesized and tested for
inhibition. A peptide covering the 30 most C-terminal amino acids of
β-casein had an inhibitory effect of 87% at a concentration of 0.375 µM,
whereas none of the other peptides had any effect, suggesting that a
S. mutans inhibitory binding epitope resides in the C-terminal portion of β-
casein. Lactoferrin was one of the other strong inhibitory components, and it
has earlier been shown that a lactoferrin fragment (Lf411, aa 473-538) of
bovine milk inhibits aggregation of S. mutans to gp-340 (Mitoma et al.,
2001). Further, it has been shown that a particular peptide Lf (480-492) of
the Lf411 fragment is responsible for the inhibition of PAc (AgI/II adhesin of
S. mutans) to the PAc-binding domain in salivary agglutinin (Oho et al.,
2004). Therefore, a lactoferrin peptide covering aa 501-513, corresponding
to aa 480-492 (85% similarity in aa sequence) in lactoferrin from bovine
milk, was also tested for inhibition, and inhibited binding of S. mutans
Ingbritt to s-HA by 66% at a concentration of 500 µM.
Further, adhesion inhibition was compared among six strains of mutans
streptococci (S. mutans Ingbritt, LT11, NG8, JBP and S. sobrinus 6715,
OMZ176). All strains were inhibited by human milk, although S. sobrinus
6715 was only slightly inhibited. All strains were also inhibited by the most
C-terminal β-casein peptide and S-IgA, whereas lactoferrin only inhibited
S. mutans Ingbritt and no other strain (see Table 1 in Paper IV). It was not
expected that the lactoferrin inhibition of the strains would be so diverse.
The first teeth emerge when the infant is around 6 months old, and
colonisation of S. mutans is thought to occur when the teeth have erupted.
This has been suggested to occur in a discrete “window of infectivity” around
19-31 months of age (Caufield et al., 1993). However, by using molecular
methods, Tanner et al. (2002) have observed that S. mutans and S. sobrinus
can be detected from tongue and tooth samples in children younger than 18
months (n=57). A relationship between early acquisition of mutans
streptococci and dental caries has been seen (Straetemans et al., 1998). The
relevance of anti-adhesive mechanisms of human milk is relevant in
developing countries where the children usually are breast fed for a longer
time than in the Western world. In Sweden full or partial breast feeding of
infants is common, but decreases with age (69% at 6 months, 39% at 9
months, and 17% at 12 months in 2006) (The National Board of Health and
Results and Discussion
47
Welfare, 2006). Interestingly, human milk coated on hydroxyapatite
promotes adhesion of commensal bacteria such as A. naeslundii and
S. sanguinis (unpublished results). Thus, human milk could have a
protective effect in infants also in Western countries, since it could still
favour acquisition of a commensal bacterial flora, and it also could prevent
or delay adhesion of S. mutans on the initial primary teeth.
Concluding Remarks
48
CCOONNCCLLUUSSIIOONNSS AANNDD FFUUTTUURREE PPEERRSSPPEECCTTIIVVEESS
This doctoral thesis has focused on studying the interactions between saliva
and milk host ligands and oral bacteria with relevance for biofilm formation.
Dental caries, a chronic infectious disease, has been thought of as a target
disease for selection of model ligands and bacteria for studying such
interactions. The main conclusions are the following:
• Bacteria from different origins bind to different segments of
statherin. The binding motif for strains with infectious and rodent
origin was delineated to a small, linear epitope that seemed to be
dependent on conformational presentation. Adhesion of commensal
bacteria was promoted whereas adhesion of infectious strains was
inhibited. Thus, statherin seems to possess adhesion-regulating
properties that may protect the host from colonization of bacteria
associated with infections. Since statherin is reported to occur in
different forms and proteolytically cleaved peptides are released
from statherin, a future aim of interest would be to delineate a
possible role of statherin in innate immunity including how the
different statherin variants affect adhesion.
• There are individually different size variants of gp-340 in saliva, and
in addition there are differences in glycosylation that may be related
to secretor status of the host. The size- and glycoforms could
aggregate bacteria differently and may affect many biological
functions related to host innate immune defences. This would be a
highly interesting topic for further studies.
• Human milk could inhibit mutans streptococci adhesion to s-HA in
an individually varying fashion, dependent on individual properties
of the saliva. Several milk proteins could inhibit adhesion and
especially a C-terminal peptide of β-casein was a potent inhibitory
component together with a lactoferrin peptide. Thus, human milk
may have a protective effect against early acquisition of the caries-
associated mutans streptococci in infants. We also have unpublished
data showing that human milk mediates binding of health-
associated species. Therefore, it would be interesting to explore the
role of human milk, and specific components therein, in a complex
biofilm model. A possible long-term aim would be to compose baby
Concluding Remarks
49
formulas mediating such milk protective characteristics together
with nutrition.
To sum up, the studied host ligands may affect bacterial colonization and
biofilm formation and could possibly also alter the susceptibility towards
infections of the host.
Acknowledgements
50
AACCKKNNOOWWLLEEDDGGEEMMEENNTTSS
Det är många personer som har stöttat, uppmuntrat och peppat mig under
årens lopp, både arbetskamrater, familj och vänner. Jag vill därför skicka ett
stort och varmt TACK till alla som på ett eller annat sätt har hjälpt mig under
den här tiden. Speciellt vill jag dock tacka mina arbetskamrater på
tandläkarhögskolan:
Min handledare Ingegerd Johansson, som har handlett mig i
forskningens värld. Tack för ditt engagemang och din breda kunskap som du
har delat med dig av, givande diskussioner och allehanda hjälp samt krav på
tidsplaner. Utan dig hade det inte blivit någon avhandling!
Pernilla Lif Holgersson, min bihandledare, för all hjälp i samband med
slutförandet av avhandlingen.
Nicklas Strömberg, för brett kunnande, diskussioner och hjälp, särskilt i samband med gp-340 artikeln, samt bra synpunkter på delar av
avhandlingen.
Josephine Wernersson för gott samarbete, skicklighet på lab, hjälp och
trevligt sällskap. Ulla Öhman för teknisk hjälp, stor kunskap och hjälp att hitta saker på lab. Ann-Charlott Idahl för hjälp med diverse saker på lab
och även administrativ hjälp.
Anette Jonasson, Mirva Drobni, Christer Eriksson, det har varit trevligt att lära känna er och dela lab med er. Mirva, tack också för trevligt
rumssällskap och ventilationskanal i mitten av resan. Lotta Harnevik, för bra kommentarer på delar av avhandlingen.
Nina Forsgren och Margareta Holmgren, ni var riktigt trevliga rumskompisar!
Hjördis Olsson, tack för diverse administrativ hjälp.
Till alla övriga personer på forskarvåningen som har gjort lab,
korridorerna, fika- och lunchrasterna trevliga: Elisabeth, Maria, Rolf, Pamela, Inger, Eva, Anna L-B, Karina, Emma, Kjell, Anna B, Mari, Voukko, Lars, Maggan, George, Vincent m.fl. Jag vill även tacka
Acknowledgements
51
Thomas Borén och alla medlemmar i hans grupp som under de första åren
bidrog till att muntra upp tillvaron.
Tack även till mamma och pappa (och er förståelse över inställd skidresa - ställa in det som verkligen är livet!) och till mina älsklingar Johan och Edvin för att ni har stått ut med mig under de här sista månaderna.
/Liza
References
52
RREEFFEERREENNCCEESS
Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the Normal Bacterial Flora of the Oral Cavity. J Clin Microbiol.
2005;43:5721-5732.
Aimutis WR. Bioactive properties of milk proteins with particular focus on
anticariogenesis. J Nutr. 2004;134:989S-995S.
Amano A, Kataoka K, Raj PA, Genco RJ, and Shizukuishi S. Binding sites of salivary statherin for Porphyromonas gingivalis recombinant
fimbrillin. Infect Immun. 1996;64:4249-4254.
Andersson Y, Lindquist S, Lagerqvist C, Hernell O. Lactoferrin is responsible for the fungistatic effect of human milk. Early Hum Dev.
2000;59:95-105.
Arneberg P, Kornstad L, Nordbö H, Gjermo P. Less dental caries among secretors than among non-secretors of blood group substance.
Scand J Dent Res. 1976;84:362-366.
Asikainen S, Chen C. Oral ecology and person-to-person transmission of
Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.
Periodontol. 2000. 1999;20:65-81.
Aspholm-Hurtig M, Dailide G, Lahmann M, Kalia A, Ilver D, Roche N, Vikström S, Sjöström R, Lindén S, Bäckström A, Lundberg C, Arnqvist A, Mahdavi J, Nilsson UJ, Velapatiño B, Gilman RH, Gerhard M, Alarcon T, López-Brea M, Nakazawa T, Fox JG, Correa P, Dominguez-Bello MG, Perez-Perez GI, Blaser MJ, Normark S, Carlstedt I, Oscarson S, Teneberg S, Berg DE, Borén T. Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin. Science. 2004;23;305:519-522.
Balakrishnan M, Simmonds RS, Tagg JR. Dental caries is a
preventable infectious disease. Australian Dental Journal.
2000;45:235-245.
Becker MR, Paster BJ. Molecular analysis of bacterial species associated
with childhood caries. J Clin Microbiol. 2002;40:1001-1009.
Ben-Aryeh H, Blumfield E, Szargel R, Laufer D, Berdicevsky I. Oral Candida carriage and blood group antigen secretor status. Mycoses.
1995;38:355-358.
Bendel CM, Hostetter MK. Distinct mechanisms of epithelial adhesion
for Candida albicans and Candida tropicalis. Identification of the
participating ligands and development of inhibitory peptides. J Clin
Invest. 1993;92:1840-1849.
References
53
Berlutti F, Ajello M, Bosso P, Morea C, Petrucca A, Antonini G, Valenti P. Both lactoferrin and iron influence aggregation and biofilm
formation in Streptococcus mutans. Biometals. 2004;17:271-278.
Bikker FJ, Ligtenberg AJ, End C, Renner M, Blaich S, Lyer S, Wittig R, van't Hof W, Veerman EC, Nazmi K, de Blieck-Hogervorst JM, Kioschis P, Nieuw Amerongen AV, Poustka A, Mollenhauer J. Bacteria binding by DMBT1/SAG/gp-340 is confined
to the VEVLXXXXW motif in its scavenger receptor cysteine-rich
domains. J Biol Chem. 2004;279:47699-47703.
Bikker FJ, Ligtenberg AJ, Nazmi K, Veerman EC, van't Hof W,
Bolscher JG, Poustka A, Nieuw Amerongen AV, Mollenhauer J. Identification of the bacteria-binding peptide domain on salivary
agglutinin (gp-340/DMBT1), a member of the scavenger receptor
cysteine-rich superfamily. J Biol Chem. 2002;277:32109-32115.
Bläckberg L, Hernell O. The bile-salt-stimulated lipase in human milk.
Purification and characterization. Eur J Biochem. 1981;16:221-225.
Boackle RJ, Connor MH, Vesely J. High molecular weight non-
immunoglobulin salivary agglutinins (NIA) bind C1Q globular heads and
have the potential to activate the first complement component. Mol
Immunol. 1993;30:309-319.
Boman HG. Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scand J Immunol. 1998;48:15-25.
Bork P, Beckmann G. The CUB domain. A widespread module in
developmentally regulated proteins. J Mol Biol. 1993;231:539-545. Bradshaw, DJ, Marsh PD, Watson GK, Allison C. Role of
Fusobacterium nucleatum and coaggregation in anaerobe survival in
planktonic and biofilm oral microbial communities during aeration.
Infect. Immun. 1998;66:4729-4732.
Bradshaw DJ, McKee AS, Marsh PD. Effects of carbohydrate pulses and pH on population shifts within oral microbial communities in vitro.
J Dent Res. 1989;68:1298-1302.
Bradway SD, Bergey EJ, Scannapieco FA, Ramasubbu N, Zawacki S, Levine MJ. Formation of salivary-mucosal pellicle: the role of
transglutaminase. Biochem J. 1992;284:557-564. Braidotti P, Nuciforo PG, Mollenhauer J, Poustka A, Pellegrini C,
Moro A, Bulfamante G, Coggi G, Bosari S, Pietra GG. DMBT1
expression is down-regulated in breast cancer. BMC Cancer.
2004;9:4:46.
Brailsford SR, Lynch E, Beighton D. The isolation of Actinomyces
naeslundii from sound root surfaces and root carious lesions. Caries Res.
1998;32:100-106.
Brook I. Microbial factors leading to recurrent upper respiratory tract
infections. Pediatr Infect Dis J. 1998;17:S62-67.
References
54
Bu H, Sood SM, Slattery CW. The effect of conserved residue charge
reversal on the folding of recombinant non-phosphorylated human beta-
casein. Arch Biochem Biophys. 2003;15:244-250.
Cabras T, Inzitari R, Fanali C, Scarano E, Patamia M, Sanna MT, Pisano E, Giardina B, Castagnola M, Messana I. HPLC-MS
characterization of cyclo-statherin Q-37, a specific cyclization product of
human salivary statherin generated by transglutaminase 2. J Sep Sci.
2006;29:2600-2608.
Camling E, Gahnberg L, Krasse B. The relationship between IgA antibodies to Streptococcus mutans antigens in human saliva and breast
milk and the numbers of indigenous oral Streptococcus mutans. Arch
Oral Biol. 1987;32:21-25.
Caufield PW, Cutter GR, Dasanayake AP. Initial acquisition of mutans
streptococci by infants: evidence for a discrete window of infectivity. J
Dent Res. 1993;72:37-45.
Chipman DM, Sharon N. Mechanism of lysozyme action. Science
1969;165:454-465.
Cisar JO, Barsumian EL, Siraganian RP, Clark WB, Yeung MK, Hsu SD, Curl SH, Vatter AE, Sandberg AL. Immunochemical and
functional studies of Actinomyces viscosus T14V type 1 fimbriae with
monoclonal and polyclonal antibodies directed against the fimbrial
subunit. J Gen Microbiol. 1991;137:1971-1979.
Cole AM, Dewan P, Ganz T. Innate antimicrobial activity of nasal
secretions. Infect Immun. 1999;67:3267-3275.
Collins MD, Hoyles L, Kalfas S, Sundquist G, Monsen T, Nikolaitchouk N, Falsen E. Characterization of Actinomyces isolates
from infected root canals of teeth: description of Actinomyces
radicidentis sp. nov. J Clin Microbiol. 2000;38:3399-3403.
Corrêa A, Staples RC, and Hoch HC. Inhibition of thigmostimulated
cell differentiation with RGD-peptides in Uromyces germlings.
Protoplasma 1996;194:91-102.
Costerton, JW, Stewart PS, Greenberg EP. Bacterial biofilms: a
common cause of persistent infections. Science 1999;284:1318-1322.
Cucchiara S, Aloi M. Role of microflora in disease. In: Nutrition and
Health: Probiotics in pediatric medicine. Eds S. Michail and P.M.
Sherman, Humana Press, Totowa, NJ, USA. 2009.
Davies, DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP. The involvement of cell-to-cell signals in the
development of a bacterial biofilm. Science. 1998;280:295-298.
Dige I, Raarup MK, Nyengaard JR, Kilian M, Nyvad B. Actinomyces
naeslundii in initial dental biofilm formation. Microbiology.
2009;155:2116-2126.
References
55
Donlan RM, and Costerton JW. Biofilms: Survival mechanisms of
clinically relevant microorganisms. Clin Microbiol Rev. 2002;15:167-193.
Drobni M, Jonasson A, Strömberg N, Johansson I. Identification of a multiplicity of acidic proline-rich protein peptide epitopes among
Actinomyces and Streptococcus with different protein ligand binding
patterns. In Adhesion-related interactions of Actinomyces and
Streptococcus biofilm bacteria by Mirva Drobni. Odontological
dissertation, Umeå University, Umeå, Sweden. 2006a;ISSN 0345-7532,
ISBN 91-7264-111-8.
Drobni M, Li T, Krüger C, Loimaranta V, Kilian M, Hammarström L, Jörnvall H, Bergman T, Strömberg N. Host-derived
pentapeptide affecting adhesion, proliferation, and local pH in biofilm
communities composed of Streptococcus and Actinomyces species.
Infect Immun. 2006b;74:6293-6299.
Drobni M, Olsson IM, Eriksson C, Almqvist F, Strömberg N. Multivariate design and evaluation of a set of RGRPQ-derived innate
immunity peptides. J Biol Chem. 2006c;281:15164-15171.
Dunne WM Jr. Bacterial adhesion: Seen any good biofilms lately? Clin
Microbiol Rev. 2002;15:155-166.
Egland PG, Dû LD, Kolenbrander PE. Identification of independent Streptococcus gordonii SspA and SspB functions in coaggregation with
Actinomyces naeslundii. Infect Immun. 2001;69:7512-7516.
Ericson T, Rundegren J. Characterization of a salivary agglutinin reacting with a serotype c strain of Streptococcus mutans. Eur J
Biochem. 1983;133:255-261.
Eliassen LT, Berge G, Leknessund A, Wikman M, Lindin I, Lokke C, Ponthan F, Johnsen JI, Sveinbjornsson B, Kogner P, Flaegstad T, Rekdal O. The antimicrobial peptide, lactoferricin B, is
cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in
vivo. Int J Cancer. 2006;119:493-500.
Ellison RT 3rd, Giehl TJ. Killing of gram-negative bacteria by lactoferrin
and lysozyme. J Clin Invest. 1991;88:1080-1091.
Fux CA, Costerton JW, Stewart PS, Stoodley P. Survival strategies of infectious biofilms. Trends Microbiol. 2005;13:34-40.
Gibbons RJ. Bacterial adhesion to oral tissues: A model for incectious
diseases. J Dent Res 1989;68:750-760.
Gibbons RJ, Hay DI. Human salivary acidic proline-rich proteins and statherin promote the attachment of Actinomycec viscosus LY7 to apatic
surfaces. Infect Immun. 1988;56:439-445.
Gibbons RJ, Hay DI. Adsorbed salivary acidic proline-rich proteins contribute to the adhesion of Streptococcus mutans JBP to apatic
surfaces. J Dent Res. 1989;68:1303-1307.
References
56
Gibbons RJ, Hay DI, Childs WC 3rd, Davis G. Role of cryptic receptors (cryptitopes) in bacterial adhesion to oral surfaces. Arch Oral Biol.
1990;35 Suppl:107S-114S.
Gibbons RJ, Hay DI, Schlesinger DH. Delineation of a segment of
adsorbed salivary acidic proline-rich proteins which promotes adhesion
of Streptococcus gordonii to apatic surfaces. Infect Immun.
1991;59:2948-2954.
Gifford JL, Hunter HN, Vogel HJ. Lactoferricin: a lactoferrin-derived peptide with antimicrobial, antiviral, antitumor and immunological
properties. Cell Mol Life Sci. 2005;62:2588-2598.
Gilbert M, Shaw WJ, Long JR, Nelson K, Drobny GP, Giachelli CM, Stayton PS. Chimeric peptides of statherin and osteopontin that
bind hydroxyapatite and mediate cell adhesion. J Biol Chem.
2000;275:16213-16218.
Goobes G, Goobes R, Schueler-Furman O, Baker D, Stayton PS, Drobny GP. Folding of the C-terminal bacterial binding domain in
statherin upon adsorption onto hydroxyapatite crystals. Proc Natl Acad
Sci USA. 2006;103:16083-16088.
Gough PJ, Gordon S. The role of scavenger receptors in the innate immune system. Microbes Infect. 2000;2:305-311.
Gustafsson L, Hallgren O, Mossberg AK, Pettersson J, Fischer W, Aronsson A, Svanborg C. HAMLET kills tumor cells by apoptosis:
structure, cellular mechanisms, and therapy. J Nutr. 2005;135:1299-
1303.
Guthrie R. Community-acquired lower respiratory tract infections: etiology
and treatment. Chest. 2001;120:2021-2034.
Haataja S, Tikkanen K, Nilsson U, Magnusson G, Karlsson KA, Finne J. Oligosaccharide-receptor interaction of the Gal alpha 1-4Gal binding adhesin of Streptococcus suis. Combining site architecture and
characterization of two variant adhesin specificities. J Biol Chem.
1994;269:27466-27472.
Hajishengallis G, Koga T, Russell MW. Affinity and specificity of the interactions between Streptococcus mutans antigen I/II and salivary
components. J Dent Res. 1994;73:1493-1502.
Hajishengallis G, Nikolova E, Russell MW. Inhibition of Streptococcus
mutans adherence to saliva-coated hydroxyapatite by human secretory
immunoglobulin A (S-IgA) antibodies to cell surface protein antigen I/II:
reversal by IgA1 protease cleavage. Infect Immun. 1992;60:5057-5064.
Hallberg K, Hammarström KJ, Falsen E, Dahlen G, Gibbons RJ, Hay DJ, Strömberg N. Actinomyces naeslundii genospecies 1 and 2
express different binding specificities to N-acetyl-β-D-galactosamine,
whereas Actinomyces odontolyticus expresses a different binding
References
57
specificity in colonizing the human mouth. Oral Microbiol Immunol.
1998;13:327-336.
Hallberg K, Holm C, Öhman U, Stromberg N. Actinomyces naeslundii
displays variant fimP and fimA fimbrial subunit genes corresponding to
different types of acidic proline-rich protein and beta-linked
galactosamine binding specificity. Infect Immun. 1998;66:4403-4410.
Hamosh M. Protective function of proteins and lipids in human milk. Biol
Neonate. 1998;74:163-176.
Hanson LÅ. Protective effects of breastfeeding against urinary tract infection. Acta Paediatr Scand. 2004;3:154-156.
Harmsen MC, Swart PJ, de Béthune MP, Pauwels R, De Clercq E, The TH, Meijer DK. Antiviral effects of plasma and milk proteins:
lactoferrin shows potent activity against both human immunodeficiency
virus and human cytomegalovirus replication in vitro. J Infect Dis.
1995;172:380-388.
Hartshorn KL, White MR, Mogues T, Ligtenberg T, Crouch E, Holmskov U. Lung and salivary scavenger receptor glycoprotein-340 contribute to the host defense against influenza A viruses. Am J Physiol
Lung Cell Mol Physiol. 2003;285:1066-1076.
Helmerhorst EJ, Sun X, Salih E, Oppenheim FG. Identification of Lys-Pro-Gln as a novel cleavage site specificity of saliva-associated
proteases. J Biol Chem. 2008;283:19957-19966.
Henry S, Oriol R, Samuelsson B. Lewis histo-blood group system and
associated secretory phenotypes. Vox Sang. 1995;69:166-182.
Henssge U, Do T, Radford DR, Gilbert SC, Clark D, Beighton D. Emended description of Actinomyces naeslundii and descriptions of
Actinomyces oris sp. nov. and Actinomyces johnsonii sp. nov., previously
identified as Actinomyces naeslundii genospecies 1, 2 and WVA 963. Int
J Syst Evol Microbiol. 2009;59:509-516.
Holbrook WP, Blackwell CC. Secretor status and dental caries in Iceland. FEMS Microbiol Immunol. 1989;1:397-399.
Holmskov U, Lawson P, Teisner B, Tornoe I, Willis AC, Morgan C, Koch C, Reid KB. Isolation and characterization of a new member of
the scavenger receptor superfamily, glycoprotein-340 (gp-340), as a lung
surfactant protein-D binding molecule. J Biol Chem. 1997;272:13743-
13749.
Holmskov U, Mollenhauer J, Madsen J, Vitved L, Grønlund J, Tornøe I, Kliem A, Reid KB, Poustka A, Skjødt K. Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D. Proc Natl
Acad Sci U S A. 1999;96:10794-10799.
van Houte J. Microbiological predictors of caries risk. Adv Dent Res.
1993;7:87-96.
References
58
van Houte J. Role of micro-organisms in caries etiology. J Dent Res. 1994;73:672-681.
Huq NL, Cross KJ, Ung M, Reynolds EC. A review of protein structure and gene organisation for proteins associated with mineralised tissue
and calcium phosphate stabilisation encoded on human chromosome 4.
Arch Oral Biol. 2005;50:599-609.
Ilver D, Arnqvist A, Ögren J, Frick IM, Kersulyte D, Incecik ET, Berg DE, Covacci A, Engstrand L, Borén T. Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by
retagging. Science. 1998;279:373-377.
Inzitari R, Cabras T, Rossetti DV, Fanali C, Vitali A, Pellegrini M, Paludetti G, Manni A, Giardina B, Messana I, Castagnola M.
Detection in human saliva of different statherin and P-B fragments and
derivatives. Proteomics. 2006;6:6370-6379.
Jakubovics NS, Strömberg N, van Dolleweerd CJ, Kelly CG, Jenkinson HF. Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands. Mol
Microbiol. 2005;55:1591-1605.
Jenkinson HF, Lamont RJ. Oral microbial communities in sickness and
in health. Trends Microbiol. 2005;13:589-595.
Jensen JL, Lamkin MS, Troxler RF, Oppenheim FG. Multiple forms
of statherin in human salivary secretions. Arch Oral Biol. 1991;36:529-
534.
Jia HP, Starner T, Ackermann M, Kirby P, Tack BF, McCray PB Jr. Abundant human beta-defensin-1 expression in milk and mammary
gland epithelium. J Pediatr. 2001;138:109-112.
Johnson JL, Moore LV, Kaneko B, Moore WE. Actinomyces georgiae
sp. nov., Actinomyces gerencseriae sp. nov., designation of two
genospecies of Actinomyces naeslundii, and inclusion of A. naeslundii
serotypes II and III and Actinomyces viscosus serotype II in A.
naeslundii genospecies 2. Int J Syst Bacteriol. 1990;40:273-286.
Johansson I, Bratt P, Hay DI, Schluckebier S, Strömberg N.
Adhesion of Candida albicans, but not Candida krusei, to salivary
statherin and mimicking host molecules. Oral Microbiol Immunol.
2000;15:112-118.
Jonasson A, Eriksson C, Jenkinson HF, Källestål C, Johansson I, Strömberg N. Innate immunity glycoprotein gp-340 variants may
modulate human susceptibility to dental caries. BMC Infect Dis.
2007;11;7:57.
Jumblatt MM, Imbert Y, Young WW Jr, Foulks GN, Steele PS, Demuth DR. Glycoprotein 340 in normal human ocular surface tissues
and tear film. Infect Immun. 2006;74:4058-4063.
References
59
Kochańska B, Kedzia A, Kamysz W, Maćkiewicz Z, Kupryszewski G. The effect of statherin and its shortened analogues on anaerobic bacteria isolated from the oral cavity. Acta Microbiol Pol. 2000;49:243-251.
Kolenbrander PE. Surface recognition among oral bacteria: multigeneric
coaggregations and their mediators. Crit Rev Microbiol. 1989;17:137-
159.
Kolenbrander PE. Oral microbial communities: Biofilms, interactions and
genetic systems. Annu Rev Microbiol. 2000;54:413-437.
Kolenbrander PE, Andersen RN, Blehert DS, Egland PG, Foster JS, Palmer RJJr. Communication among oral bacteria. Microbiol Mol
Biol Rev. 2002;66:486-505.
Kunz C, Lonnerdal B. Re-evaluation of the whey protein/casein ratio of human milk. Acta Paediatr. 1992;81:107-112.
Kurek M, Przybilla B, Hermann K, Ring J. A naturally occurring opioid peptide from cow's milk, beta-casomorphine-7, is a direct
histamine releaser in man. Int Arch Allergy Immunol. 1992;97:115-120.
Lancefield RC. A serological differentiation of human and other groups of
haemolytic streptococci. J Exp Med. 1933;57:571-595.
Ligtenberg AJ, Bikker FJ, De Blieck-Hogervorst JM, Veerman EC, Nieuw Amerongen AV. Binding of salivary agglutinin to IgA. Biochem J. 2004;383:159-164.
Ligtenberg TJ, Bikker FJ, Groenink J, Tornoe I, Leth-Larsen R, Veerman EC, Nieuw Amerongen AV, Holmskov U. Human
salivary agglutinin binds to lung surfactant protein-D and is identical
with scavenger receptor protein gp-340. Biochem J. 2001;359:243-248.
Li J, Helmerhorst EJ, Leone CW, Troxler RF, Yaskell T, Haffajee AD, Socransky SS, and Oppenheim FG. Identification of early microbial colonizers in human dental biofilm. J Applied Microbiology.
2004;97:1311-1318.
Lindesmith L, Moe C, Marionneau S, Ruvoen N, Jiang X, Lindblad L, Stewart P, LePendu J, Baric R. Human susceptibility and
resistance to Norwalk virus infection. Nat Med. 2003;9:548-553.
Lis H, Sharon N. Protein glycosylation. Structural and functional aspects.
Eur J Biochem. 1993;218:1-27.
Li T, Johansson I, Hay DI, Strömberg N. Strains of Actinomyces
naeslundii and Actinomyces viscosus exhibit structurally variant fimbrial
subunit proteins and bind to different peptide motifs in salivary
proteins. Infect Immun. 1999;67:2053-2059.
Li T, Khah MK, Slavnic S, Johansson I, Strömberg N. Different type 1 fimbrial genes and tropisms of commensal and potentially pathogenic
Actinomyces spp. with different salivary acidic proline-rich protein and
statherin ligand specificities. Infect Immun. 2001;69:7224-7233.
References
60
Li XJ, Snyder SH. Molecular cloning of Ebnerin, a von Ebner's gland
protein associated with taste buds. J Biol Chem. 1995;270:17674-17679.
Li Y, Wang W, Caufield PW. The fidelity of mutans streptococci
transmission and caries status correlate with breast-feeding experience
among Chinese families. Caries Res. 2000;34:123-132.
Loimaranta V, Hytönen J, Pulliainen AT, Sharma A, Tenovuo J, Strömberg N, Finne J. Leucine-rich repeats of bacterial surface proteins serve as common pattern recognition motifs of human
scavenger receptor gp340. J Biol Chem. 2009;284:18614-18623.
Loimaranta V, Jakubovics NS, Hytönen J, Finne J, Jenkinson HF, Strömberg N. Fluid- or surface-phase human salivary scavenger
protein gp-340 express different bacterial recognition properties. Infect
Immun. 2005;73:2245-2252.
Long JR, Shaw W, Stayton PS, Drobny GP. Structure and dynamics of
hydrated statherin on hydroxyapatite as determined by solid-state NMR.
Biochemistry. 2001;40:15451-15455.
Lönnerdal B. Nutritional and physiologic significance of human milk
proteins. Am J Clin Nutr. 2003;77:1537S-1543S.
Madsen J, Tornøe I, Nielsen O, Lausen M, Krebs I, Mollenhauer J, Kollender G, Poustka A, Skjødt K, Holmskov U. CRP-ductin, the mouse homologue of gp-340/deleted in malignant brain tumors 1
(DMBT1), binds gram-positive and gram-negative bacteria and interacts
with lung surfactant protein D. Eur J Immunol. 2003;33:2327-2336.
Malkoski M, Dashper SG, O’Brien-Simpson NM, Talbo GH, Macris M, Cross KJ, Reynolds EC. Kappacin, a novel antibacterial peptide from bovine milk. Antimicrob Agents Chemother. 2001;45:2309-2315.
Marionneau S, Cailleau-Thomas A, Rocher J, Le Moullac-Vaidye B, Ruvoën N, Clément M, Le Pendu J. ABH and Lewis histo-blood group antigens, a model for the meaning of oligosaccharide diversity in
the face of a changing world. Biochemie. 2001;83:565−573.
Marsh PD. Microbial ecology of dental plaque and its significance in health
and disease. Adv Dent Res. 1994;8:263-271.
Marsh PD. Dental plaque as a biofilm and a microbial community –
implications for health and disease. BMC Oral Health. 2006;6:S14.
Mathews M, Jia HP, Guthmiller JM, Losh G, Graham S, Johnson GK, Tack BF, McCray PB Jr. Production of beta-defensin
antimicrobial peptides by the oral mucosa and salivary glands. Infect
Immun. 1999;67:2740-2745.
Mitoma M, Oho T, Shimazaki Y, Koga T. Inhibitory effect of bovine milk lactoferrin on the interaction between a Steptococcal surface
protein antigen and human salivary agglutinin. J Biol Chem.
2001;276:18060-18065.
References
61
Mollenhauer J, Herbertz S, Holmskov U, Tolnay M, Krebs I, Merlo A, Schrøder HD, Maier D, Breitling F, Wiemann S, Gröne HJ, Poustka A. DMBT1 encodes a protein involved in the immune defense
and in epithelial differentiation and is highly unstable in cancer. Cancer
Res. 2000;60:1704-1710.
Mollenhauer J, Holmskov U, Wiemann S, Krebs I, Herbertz S, Madsen J, Kioschis P, Coy JF, Poustka A. The genomic structure of
the DMBT1 gene: evidence for a region with susceptibility to genomic
instability. Oncogene. 1999;18:6233-6240.
Mollenhauer J, Müller H, Kollender G, Lyer S, Diedrichs L, Helmke B, Holmskov U, Ligtenberg T, Herbertz S, Krebs I, Madsen J, Bikker F, Schmitt L, Wiemann S, Scheurlen W, Otto HF, von Deimling A, Poustka A. The SRCR/SID region of DMBT1
defines a complex multi-allele system representing the major basis for its
variability in cancer. Genes Chromosomes Cancer. 2002;35:242-255.
Mollenhauer J, Wiemann S, Scheurlen W, Korn B, Hayashi Y, Wilgenbus KK, von Deimling A, Poustka A. DMBT1, a new
member of the SRCR superfamily, on chromosome 10q25.3-26.1 is
deleted in malignant brain tumours. Nat Genet. 1997;17:32-39.
Murakami M, Dorschner RA, Stern LJ, Lin KH, Gallo RL. Expression and secretion of cathelicidin antimicrobial peptides in
murine mammary glands and human milk. Pediatr Res. 2005;57:10-15.
Murakami M, Ohtake T, Dorschner RA, Gallo RL. Cathelicidin antimicrobial peptides are expressed in salivary glands and saliva. J
Dent Res. 2002;81:845-850.
Månsson-Rahemtulla B, Rahemtulla F, Baldone DC, Pruitt KM, Hjerpe A. Purification and characterization of human salivary
peroxidase. Biochemistry. 1988;27:233-239. Naganagowda GA, Gururaja TL, Levine MJ. Delineation of
conformational preferences in human salivary statherin by 1H, 31P NMR
and CD studies: sequential assignment and structure-function
correlations. J Biomol Struct Dyn. 1998;16:91-107.
Nakagaki H, Sekine S, Terao Y, Toe M, Tanaka M, Ito HO, Kawabata S, Shizukuishi S, Fujihashi K, Kataoka K. Fusobacterium nucleatum Envelope Protein FomA Is Immunogenic and
Binds to the Salivary Statherin-Derived Peptide. Infect Immun.
2010;78:1185-1192.
Neeser JR, Golliard M, Woltz A, Rouvet M, Dillmann ML, Guggenheim B. In vitro modulation of oral bacterial adhesion to
saliva-coated hydroxyapatite beads by milk casein derivatives. Oral
Microbiol Immunol. 1994;9:193-201.
Niyogi SK, Saha MR, De SP. Enteropathogens associated with acute diarrhoeal diseases. Indian J Public Health. 1994;38:29-32.
References
62
Nobbs AH, Lamont RJ, Jenkinson HF. Streptococcus adherence and colonization. Microbiol Mol Biol Rev. 2009;73:407-450.
Nyvad B and Kilian M. Microbiology of the early colonization of human
enamel and root surfaces in vivo. Scand J Dent Res. 1987;95:369-380.
Nyvad B and Kilian M. Microflora associated with experimental root
surface caries in humans. Infect Immun. 1990a;58:1628-1633.
Nyvad B and Kilian M. Comparison of the initial streptococcal microflora
on dental enamel in caries-active and in caries-inactive individuals.
Caries Res. 1990b;24:267-272.
Oho T, Bikker FJ, Nieuw Amerongen AV, Groenink J. A peptide domain of bovine milk lactoferrin inhibits the interaction between
streptococcal surface protein antigen and a salivary agglutinin peptide
domain. Infect Immun. 2004;72:6181-6184.
Oho T, Mioma M, Koga T. Functional domain of bovine milk lactoferrin
which inhibits the adherence of Streptococcus mutans cells to a salivary
film. Infect Immun. 2002;70:5279-5282.
Oppenheim FG, Xu T, McMillian FM, Levitz SM, Diamond RD, Offner GD, Troxler RF. Histatins, a novel family of histidine-rich
proteins in human parotid secretion. J Biol Chem. 1988;263:7472-7477.
Orsi N. The antimicrobial activity of lactoferrin: Current status and
perspectives. Biometals. 2004;17:189-196.
Periasamy S, Kolenbrander PE. Mutualistic biofilm communities
develop with Porphyromonas gingivalis and initial, early, and late
colonizers of enamel. J Bacteriol. 2009;191:6804-6811.
Polllock JJ, Denepitiya L, MacKay BJ, Iacono VJ. Fungistatic and fungicidal activity of human parotid salivary histidine-rich polypeptides
on Candida albicans. Infect Immun. 1984;44:702-707.
Prakobphol A, Thomsson KA, Hansson GC, Rosen SD, Singer MS, Phillips NJ, Medzihradszky KF, Burlingame AL, Leffler H, Fisher SJ. Human low-molecular-weight salivary mucin expresses the
sialyl lewisx determinant and has L-selectin ligand activity.
Biochemistry. 1998;37:4916–4927.
Prakobphol A, Xu F, Hoang VM, Larsson T, Bergstrom J, Johansson I, Frangsmyr L, Holmskov U, Leffler H, Nilsson C, Boren T, Wright JR, Strömberg N, Fisher SJ. Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung
scavenger receptor cysteine-rich protein gp-340. J Biol Chem.
2000;275:39860-39866.
Putnins EE, Bowden GH. Antigenic relationships among oral
Actinomyces isolates, Actinomyces naeslundii genospecies 1 and 2,
Actinomyces howellii, Actinomyces denticolens, and Actinomyces slackii.
J Dent Res. 1993;72:1374-1385.
References
63
Ramasubbu N, Thomas LM, Bhandary KK, Levine MJ. Structural characteristics of human salivary statherin: a model for boundary
lubrication at the enamel surface. Crit Rev Oral Biol Med. 1993;4:363-
370.
Renner M, Bergmann G, Krebs I, End C, Lyer S, Hilberg F, Helmke B, Gassler N, Autschbach F, Bikker F, Strobel-Freidekind O, Gronert-Sum S, Benner A, Blaich S, Wittig R, Hudler M, Ligtenberg AJ, Madsen J, Holmskov U, Annese V, Latiano A, Schirmacher P, Amerongen AV, D'Amato M, Kioschis P, Hafner M, Poustka A, Mollenhauer J. DMBT1 confers
mucosal protection in vivo and a deletion variant is associated with
Crohn’s disease. Gastroenterology. 2007;133:1499-1509.
Rüdiger SG, Carlén A, Meurman JH, Kari K, Olsson J. Dental biofilms at healthy and inflamed gingival margins. J Clin Periodontol.
2002;29:524-530.
Ruhl S, Sandberg AL, Cisar JO. Salivary receptors for the proline-rich protein-binding and lectin-like adhesins of oral actinomyces and
streptococci. J Dent Res. 2004;83:505-510.
Ruissen AL, Groenink J, Helmerhorst EJ, Walgreen-Weteringns E, Van’t Hof W, Veerman EC, Nieuw Amerongen AV. Effects of histatin 5 and derived peptides on Candida albicans. Biochem J.
2001;356:361-368.
Sabatini LM, Ota T, Azen EA. Nucleotide sequence analysis of the human
salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS
gene family. Mol Biol Evol. 1993;10:497-511.
Salminen S, Isolauri E, Onnela T. Gut flora in normal and disordered
states. Chemotherapy. 1995;41:5-15.
Sbordone L and Bortolaia C. Oral microbial biofilms and plaque-related
diseases: microbial communities and their role in the shift from oral
health to disease. Clin Oral Invest. 2003;7:181-188.
Schlesinger DH, Hay DI. Complete covalent structure of statherin, a
tyrosine-rich acidic peptide which inhibits calcium phosphate
precipitation from human parotid saliva. J Biol Chem. 1977;252:1689-
1695.
Schulz BL, Oxley D, Packer NH, Karlsson NG. Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-
molecular-mass glycoproteins in human tears. Biochem J.
2002;366:511-520.
Sekine S, Kataoka K, Tanaka M, Nagata H, Kawakami T, Akaji K, Aimoto S, Shizukuishi S. Active domains of salivary statherin on
apatitic surfaces for binding to Fusobacterium nucleatum cells.
Microbiology. 2004;150:2373-2379.
References
64
Skovbjerg S, Roos K, Holm SE, Grahn Håkansson E, Nowrouzian F, Ivarsson M, Adlerberth I, Wold AE. Spray bacteriotherapy decreases middle ear fluid in children with secretory otitis media. Arch
Dis Child. 2009;94:92-98.
Smego RA Jr, Foglia G. Actinomycosis. Clin Infect Dis. 1998;26:1255-
1261.
Soukka T, Lumikari M, Tenovuo J. Combined inhibitory effect of
lactoferrin and lactoperoxidase system on the viability of Streptococcus
mutans, serotype c. Scand J Dent Res. 1991;99:390-396.
Stapleton A, Nudelman E, Clausen H, Hakomori S, Stamm WE. Binding of uropathogenic Escherichia coli R45 to glycolipids extracted
from vaginal epithelial cells is dependent on histo-blood group secretor
status. J Clin Invest. 1992;90:965-972.
Steiner H, Hultmark D, Engström A, Bennich H, Boman HG. Sequence and specificity of two antibacterial proteins involved in insect
immunity. Nature. 1981;292:246-248. Stenudd C, Nordlund A, Ryberg M, Johansson I, Källestål C,
Strömberg N. The association of bacterial adhesion with dental caries.
J Dent Res. 2001;80:2005-2010.
Straetemans MM, van Loveren C, de Soet JJ, de Graaff J, ten Cate JM. Colonization with mutans streptococci and lactobacilli and the
caries experience of children after the age of five. J Dent Res.
1998;77:1851-1855.
Strömberg N, Borén T, Carlén A, Olsson J. Salivary receptors for GalNAc beta-sensitive adherence of Actinomyces spp.: evidence for
heterogeneous GalNAc beta and proline-rich protein receptor properties.
Infect Immun. 1992;60:3278-3286.
Strömberg N, Marklund BI, Lund B, Ilver D, Hamers A, Gaastra W, Karlsson KA, Normark S. Host-specificity of uropathogenic Escherichia coli depends on differences in binding specificity to Gal
alpha 1-4Gal-containing isoreceptors. EMBO J. 1990;9:2001-2010.
Strömqvist M, Falk P, Bergström S, Hansson L, Lönnerdal B, Normark S, Hernell O. Human milk kappa-casein and inhibition of
Helicobacter pylori adhesion to human gastric mucosa. J Pediatr
Gastroenterol Nutr. 1995;21:288-296.
Syme CD, Blanch EW, Holt C, Jakes R, Goedert M, Hecht L, Barron LD. A Raman optical activity study of rheomorphism in
caseins, synucleins and tau. New insight into the structure and
behaviour of natively unfolded proteins. Eur J Biochem. 2002;269:148-
156.
Takito J, Yan L, Ma J, Hikita C, Vijayakumar S, Warburton D, Al-Awqati Q. Hensin, the polarity reversal protein, is encoded by DMBT1,
References
65
a gene frequently deleted in malignant gliomas. Am J Physiol.
1999;277:277-289.
Tchatchou S, Riedel A, Lyer S, Schmutzhard J, Strobel-Freidekind O, Gronert-Sum S, Mietag C, D'Amato M, Schlehe B, Hemminki K, Sutter C, Ditsch N, Blackburn A, Hill LZ, Jerry DJ, Bugert P, Weber BH, Niederacher D, Arnold N, Varon-Mateeva R, Wappenschmidt B, Schmutzler RK, Engel C, Meindl A, Bartram CR, Mollenhauer J, Burwinkel B. Identification of a DMBT1 polymorphism associated with increased
breast cancer risk and decreased promoter activity. Hum Mutat. 2010;31:60-66.
Tanner AC, Milgrom PM, Kent R Jr, Mokeem SA, Page RC, Riedy CA, Weinstein P, Bruss J. The microbiota of young children from
tooth and tongue samples. J Dent Res. 2002;81:53-57.
Teanpaisan R, Thitasomakul S, Piwat S, Thearmontree A, Pithpornchaiyakul W, Chankanka O. Longitudinal study of the presence of mutans streptococci and lactobacilli in relation to dental
caries development in 3-24 month old Thai children. Int Dent J.
2007;57:445-451.
The National Board of Health and Welfare (2006). Breast-feeding, children born 2006 (in Swedish), Hälsa och sjukdomar 2008:7,
www.socialstyrelsen.se.
Thornton DJ, Davies JR, Kirkham S, Gautrey A, Khan N, Richardson PS, Sheehan JK. Identifiacation of a nonmucin
glycoprotein (gp-340) from a purified respiratory mucin preparation:
evidence for an association involving the MUC5B mucin. Glycobiology.
2001;11:969-977.
Tino MJ, Wright JR. Glycoprotein-340 binds surfactant protein-A (SP-A) and stimulates alveolar macrophage migration in an SP-A-independent
manner. Am J Respir Cell Mol Biol. 1999;20:759-768.
Tlaskalová-Hogenová H, Stepánková R, Hudcovic T, Tucková L, Cukrowska B, Lodinová-Zádníková R, Kozáková H, Rossmann P, Bártová J, Sokol D, Funda DP, Borovská D, Reháková Z, Sinkora J, Hofman J, Drastich P, Kokesová A. Commensal
bacteria (normal microflora), mucosal immunity and chronic
inflammatory and autoimmune diseases. Immunol Lett. 2004;93:97-
108.
Vacca-Smith AM, Van Wuyckhuyse BC, Tabak LA, Bowen WH. The effect of milk and casein proteins on the adherence of Streptococcus
mutans to saliva-coated hydroxyapatite. Arch Oral Biol. 1994;39:1063-
1069.
References
66
Varki A. Nothing in glycobiology makes sense, except in the light of
evolution. Cell. 2006;126:841-845.
Vieira AR, Marazita ML, Goldstein-McHenry T. Genome-wide scan
finds suggestive caries loci. J Dent Res. 2008;87:435-439. Vitorino R, Calheiros-Lobo MJ, Duarte JA, Domingues PM,
Amado FM. Peptide profile of human acquired enamel pellicle using
MALDI tandem MS. J Sep Sci. 2008;31:523-537.
Whittaker JC, Klier MC, Kolenbrander EP. Mechanisms of adhesion
by oral bacteria. Annu Rev Microbiol. 1996;50:513-552.
World Health Organization. The top ten causes of death. Fact sheet No 310, November 2008.
Wu Z, Van Ryk D, Davis C, Abrams WR, Chaiken I, Magnani J, Malamud D. Salivary agglutinin inhibits HIV type 1 infectivity through interaction with viral glycoprotein 120. AIDS Res Hum Retroviruses.
2003;19:201-209.
Yao Y, Lamkin MS, Oppenheim FG. Pellicle precursor proteins: acidic proline-rich proteins, statherin, and histatins, and their crosslinking
reaction by oral transglutaminase. J Dent Res. 1999;78:1696-1703.
Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium
tuberculosis. Int J Tuberc Lung Dis. 2009;13:1320-1330.