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

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