INTRODUCTION

Staphylococcus aureus is primarily an extracellular pathogen. In order to initiate infection it adheres to components of the host extracellular matrix (ECM). Adherence is mediated by surface protein adhesins called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) (Patti et al., 1994a). In most cases the MSCRAMMs are covalently bound to peptidoglycan in the cell wall. However, there are several examples of MSCRAMMs that are non-covalently associated with the wall. Coagulasenegative staphylococci also express MSCRAMMs. This chapter will discuss the mechanisms of attachment of proteins to the cell wall and will review the properties of MSCRAMM proteins that have been characterized at the molecular level.

ANCHORING OF PROTEINS TO THE CELL WALL

Cell-wall-anchored proteins that are covalently bound to peptidoglycan are recognizable by a motif located at the C-terminus (Navarre and Schneewind, 1999). This comprises the sequence LPXTG (Leu-Pro-X-Thr-Gly) followed by hydrophobic residues that span the cytoplasmic membrane and by several positively charged residues. The positively charged residues are required to hold the protein transiently in the membrane during secretion through the Sec secretome (Schneewind et al., 1993). The LPXTG sequence is recognized by an enzyme called sortase that cleaves LPXTG between the Thr and Gly residues (Navarre and Schneewind, 1994; Ton-That et al., 1999). The carboxyl group of the Thr is joined to the amino group of the branch peptide in nascent peptidoglycan. In the case of S. aureus, linkage occurs to the NH2 group of the fifth Gly residue of the branch peptide, which would otherwise form the interpeptide bridge of cross-linked peptidoglycan (Ton-That and Schneewind, 1999; Ton-That et al., 1999).

Except when in utero, every human being is colonized by approximately 1014 microbes (mainly bacteria) that constitute the normal microflora. Apart from those organisms that are present in the lumen of the intestinal tract, all of these microbes maintain an association with their host by adhering to some cell, tissue or secretion. It is increasingly being realized that not only is this normal microflora beneficial to its host (e.g. by providing vitamins and protection from exogenous pathogens) but it is, indeed, essential for the host's proper development, for example in the differentiation and maturation of the intestinal tract, immune system, etc. A knowledge of how members of the normal microflora adhere to their host is, therefore, important in understanding the mutualistic association we know as Homo sapiens.

Adhesion of a pathogenic organism to its host is also the first stage in any infectious disease and this truism provides another justification for studying bacterial adhesion. Interest in this aspect of bacterial virulence is increasing rapidly as our armamentarium of antibiotics dwindles in effectiveness owing to the development of resistance in major pathogens. It is believed (and strongly hoped) that research into bacterial adhesion mechanisms will identify new targets for therapeutic intervention.

This book is intended to update the reader in key areas of research in the field of bacterial adhesion to host cells and tissues. It is divided into three parts that deal with: (I) the mechanisms underlying the adhesion of bacteria to host structures, (II) the effect that adhesion to host cells has on bacteria and (III) the consequences for the host cell (or tissue) of bacterial adhesion.

BACTERIAL CAPSULES

The production of extracellular polysaccharide (EPS) molecules is a common feature of many bacteria (Whitfield and Valvano, 1993; Roberts, 1996). These molecules may be linked to the cell surface and organized into a discrete structure termed the capsule or, alternatively, may comprise an amorphous slime layer that is easily sloughed off from the cell surface. In essence, EPS provides a hydrated negatively charged gel that surrounds the bacterium and it is the physicochemical properties of this gel that account for the biological properties of bacterial capsules. A striking feature of bacterial capsular polysaccharides is their diversity, both in terms of component sugars and the glycosidic bond between repeating sugar residues. Even within a single bacterial species, there can be enormous structural diversity. For instance, in the case of Streptococcus pneumoniae there are in excess of 90 capsular serotypes. This diversity has important implications for the design of vaccine formulations that are based on capsular polysaccharides. Paradoxically, amongst this array of structural diversity, there are capsular polysaccharide molecules that are conserved across different bacterial species, such as the Escherichia coli K1 and Neisseria meningitidis serogroup B capsular polysaccharide (Jennings, 1990). Both the diversity of capsular polysaccharides and the conservation of certain polysaccharide structures across species barriers raise questions about the evolution of capsule gene clusters and the selective pressures that drive structural diversity.

For nearly 70 years, from the pioneering experiments of F. Griffith on the transformation of avirulent unencapsulated pneumococci to encapsulation and virulence (Griffith, 1928), it has been known that the expression of a capsule is an essential virulence factor.

INTRODUCTION

The area of contact between the teeth and the gums (gingiva) is an anatomically unique region that comprises mineralized tissue embedded in epithelium and exposed to a microbially abundant environment. The small (1–4mm deep) gap between the surfaces of the tooth and the gingiva is known as the gingival sulcus or crevice. The gingiva is highly vascularized and the crevice is lined with sulcular epithelial cells that differ from oral epithelial cells by exhibiting less keratinization. Apically, sulcular epithelium becomes junctional epithelium that is characterized by a lack of keratinization, limited differentiation and a relatively permeable structure. It is this junctional epithelium that directly interposes between the gingiva and the tooth surface (Fig. 13.1). In destructive periodontal disease there is migration of the junctional epithelium resulting in enlargement of the crevice into a deeper periodontal pocket that contains inflammatory cells such as neutrophils and T-cells. The gingiva itself also contains immune cells including B-cells, T-cells and dendritic cells. The microbiota of the gingival area in both health and disease is complex, with at least 500 species of bacteria present in the gingival crevice. Although many of these have pathogenic potential, the strongest causal associations have been demonstrated between Porphyromonas gingivalis and severe adult periodontitis, and between Actinobacillus actinomycetemcomitans and localized juvenile periodontitis.

Many factors contribute to the maintenance or disruption of the ecological balance in the subgingival area. The immunological status of the host, the relative and absolute numbers of specific organisms or groups of organisms, and environmental parameters such as tobacco use, all play a role in determining gingival health or disease (Socransky and Haffajee, 1992).

INTRODUCTION

Mucosal surfaces continue where the skin leaves off, completing the boundaries between the environment and internal tissues. It is to the mucosal barrier that pathogens first attach, and the ensuing molecular interactions determine whether the pathogens will cause infection or if health will be maintained.

The different cell populations in the mucosal barrier are equipped to sense and respond to the molecular contents in the lumen and to translate this molecular information into signals that can reach local or distant tissue sites. Inflammatory cascades are often the first to be activated by bacteria or viruses, and explain many aspects of acute disease. It is not the presence of microbes in the tissues that makes us sick. Vast numbers of bacteria may colonize different sites in the body, and viruses may persist for long periods of time, while the host remains perfectly healthy. It is the host response to the infecting agents that causes the symptoms and tissue damage. Inflammatory mediators thus provide a direct link between the microbe and the host in disease pathogenesis (for a review, see Svanborg et al., 1999).

It is commonly accepted that the most virulent bacteria cause the most severe acute symptoms and long-term effects, but the mechanisms used to trigger acute disease manifestations are less clear. Pathogens elicit the strongest host response, even though members of the indigenous microflora contain many of the same molecules. This is quite puzzling, but at least two factors may explain the difference.

INTRODUCTION

The oral cavity

The human oral cavity is home to a large and diverse microbial population. The mouth provides a wide range of different habitats for bacterial colonization and growth, including hard, non-shedding surfaces (the teeth) as well as various epithelial surfaces such as the tongue dorsum and buccal mucosa. The composition of the bacterial flora at these different sites can vary considerably, reflecting the range of surfaces for attachment and environmental conditions that are available for growth. Nevertheless, streptococci, which comprise some 20% of the human normal oral flora, can be isolated from most oral sites (Nyvad and Kilian, 1987; Frandsen et al., 1991). Streptococci, together with Actinomyces species, are the predominant organisms found in the early stages of biofilm (i.e. dental plaque) formation on tooth surfaces, and the interactions of these initial species with the salivary pellicle may define the strength of biofilm adhesion (Busscher et al., 1995). Furthermore, the streptococci participate in a wide range of intraand inter-generic coaggregation interactions that help to establish early biofilm communities (Kolenbrander and London, 1993; Kolenbrander, 2000). Consequently, streptococci play an important role in plaque development.

Oral streptococcal species have recently undergone considerable taxonomic upheaval (see Whiley and Beighton, 1998). This has resulted not only in the reclassification of species and the description of new species, but also the grammatical correction of Latin species epithets (thus Streptococcus crista has been renamed S. cristatus, and Streptococcus parasanguis is now S. parasanguinis). For this chapter we have chosen to retain the epithets in long-term usage.

INTRODUCTION

Oral spirochaetes colonize the gingival crevice and periodontal pocket adjacent to inflamed gingival tissues as their primary habitat. They are found most often as part of a loosely adherent mass of motile bacteria at the interface of the biofilm known as subgingival dental plaque and the sulcular and junctional epithelium lining the pocket. This location offers the opportunity to co-adhere with other bacteria and to adhere to a variety of host cells and extracellular matrix (ECM) components. Spirochaetes are motile, and they can translocate between epithelial cells, through the basement membrane, and even into the viscous ECM of the lamina propria of the gingiva. En route, whole cells or fragments shed from their outer sheath may become attached to epithelial cells, gingival fibroblasts, endothelial cells, and leukocytes, and thereby may initiate cascades of cellular responses that affect the normal functions of host cells and the remodelling of the periodontal tissues. Thus the adhesion of oral spirochaetes to ECM components and cells probably contributes to the exacerbation of periodontal lesions and to the chronicity of lesions by interfering with wound healing in sites where infections are not sufficiently suppressed by host defences or by therapy.

Most oral spirochaetes have not yet been cultivated in the microbiology laboratory (Qui et al., 1994). Until the advent of methodology based on molecular genetics, there was little understanding of the great diversity represented by this group, which was traditionally classified by cell size and by the number of periplasmic flagella originating at the poles of these long, slender bacteria.

INTRODUCTION

Contact-dependent secretion pathways, also known as type III secretion pathways, have been identified in a large number of animal and plant pathogenic bacteria (Van Gijsegem et al., 1993; Cornelis and Van Gijsegem, 2000). The designation ‘contact dependent’ comes from the observation that proteins are secreted from bacteria after they contact their particular eukaryotic cell target (Ginocchio et al., 1994; Ménard et al., 1994; Rosqvist et al., 1994; Wolff et al., 1998; Vallis et al., 1999; van Dijk et al., 1999). The name type III differentiates this secretion pathway from the four other secretion pathways that have been identified thus far in Gram-negative bacteria (for recent reviews, see Henderson et al., 1998; Wandersman, 1998; Burns, 1999; Stathopoulos et al., 2000; Thanassi and Hultgren, 2000;). Many of the components of the type III secretion pathways of Gram-negative bacteria resemble components of the flagellar assembly apparatus found in these same organisms (Minamino and Macnab, 1999; Bennett and Hughes, 2000).

Yersinia enterocolitica, a pathogen of a variety of mammals, has three apparently independent type III secretion pathways that are involved in virulence. The best studied of these (and probably the best-studied type III secretion pathway in all bacteria), the plasmid-encoded Ysc secretion pathway, is found in all three pathogenic species of Yersinia – Y. enterocolitica, Y. pestis and Y. pseudotuberculosis (for a review, see Cornelis and Van Gijsegem, 2000). This contact-dependent secretion pathway is involved in the secretion of 11 proteins, termed Yops, most of which are translocated into host cells, where they alter host cell functions, resulting in the inhibition of antimicrobial activities of phagocytic cells (for a review, see Cornelis, 2000).

INTRODUCTION

The list of prokaryotic microorganisms able to cause illness or death in animals and plants is long. In fact, infectious diseases constitute the most common cause of human morbidity and death in the world. Our fight against these pathogens, however, started to become a serious scientific business only in our very recent history. The experience gathered during the past century has already demonstrated that even the initial hopes of the antibiotic era, namely to save uncountable lives by eradicating or limiting bacterial infections, turned out to be idealistic dreams of a scientific generation rather than deliverable promises. We now realize that the concept of either a pathogen-free world or the existence of a universally active therapeutic tool belongs to the realms of science fiction.

The treatment of infected patients has been rendered complicated by the constant emergence of multidrug-resistant strains. This is further impaired by the appearance of novel infectious agents that are not treatable by conventional therapies. Although it is expected that, as a result of the combined efforts of pharmaceutical companies, we will see new antibiotics coming onto the market, it has become clear that this strategy to combat pathogenic bacteria is insufficient and has limited value. In fact, a single point mutation in a gene from a member of the microbial community can destroy the outcome of many years of research and development. The identification of novel molecular targets for therapeutic intervention, in combination with high throughput screening for novel active compounds, will have a key role in the discovery of new tools to address these problems.

INTRODUCTION

Expression of the majority of proteins and other cellular constituents within bacteria such as Escherichia coli appears to be homogeneous within the bacterial population, changing only in response to specific alterations in the environment. Moreover, for most cellular properties, environmental change appears to elicit a uniform response from each cell. However, for a subset of genes, expression is restricted to a fraction of the population. In this more unusual mode of gene regulation, termed phase variation, individual cells switch between expressing (ON) and non-expressing (OFF) states reversibly at frequencies that can be as high as one switch per cell per generation to one switch per 10,000 cells per generation. Thus, at the level of an individual cell, it appears that, in molecular terms, one or more of the steps that are required for phase switching occurs at a low frequency. Furthermore, phase variation appears to be a random or stochastic process. According to this hypothesis, the molecular events leading to phase switching occur with equal probability for each cell in a given phase over the course of the cell cycle. As described in more detail below, phase variation is nevertheless regulated in most (if not all) phase variable systems in E. coli in response to specific environmental signals. Where regulation does occur, it seems likely that switching still occurs at random, even though the regulation results in switching at a different frequency. In the related phenomenon of antigenic variation, different cells express variants of the same protein or other macromolecule.

Extreme environments affect the responses of organisms at many levels, from the structure of their proteins and membranes, to the ways in which they carry out their lives, their interactions with their environment and other organisms, and their life history, behaviour and evolution. While it would be an unhelpful truism to say that organisms are adapted to live in the places in which they live, there are some particular features of the biology of organisms in extreme environments. The extreme conditions which they face affect all aspects of their biology – theirs is an extreme biology which may give us some important insights into the nature of life in general. In this chapter, I will explore whether we can see any general patterns for life in extreme environments. But, first, let us revisit what we mean by an extreme organism.

WHAT IS EXTREME, REVISITED

In Chapter 1, I proposed a definition of extreme organisms by reference to the life boxes of organisms: the range of physical conditions under which they exist (the non-biological features of their ecological niche). I also proposed that we could recognise two groups of extreme organisms. Extremophiles have life boxes which are beyond the limits of those of the majority of organisms. Cryptobiotes have life boxes which are the same as, or substantially overlap, those of the majority of organisms, but, when the conditions of their environment deteriorate beyond those under which they can maintain their life processes (metabolism), rather than dying (as do most organisms), they cease metabolism and await the return of favourable conditions.

As a child, I lived in the Midlands in the centre of England. Family holidays meant a long car journey and I can still remember the excitement of that first rare glimpse of the sea. We feel a similar sense of elation on discovering a waterfall or a lake during a walk in the country or on hearing the patter of rain after a long dry period. Perhaps the emotional response we feel to water is a recognition of how important it is to us and to life in general. For we all know that water is essential for life. However, although it is true that life cannot exist without at least periodic access to water, organisms can live in some very dry places and some can survive the almost total loss of water from their bodies.

Why is water so important? Water comprises by far the largest proportion of the chemical make-up of the bodies of most organisms, usually from 60 to 90 per cent. Indeed, as JBS Haldane once remarked, ‘Even the Pope is 70 per cent water’. Living organisms need metabolic processes which maintain their structure and enable them to move, grow and reproduce. Metabolism involves chemical reactions and these reactions need a medium in which they can take place in a controlled manner. Consider, for example, how organisms get their energy. Energy comes mainly from the oxidation or burning of sugars.

… there's no place like home (in the words of the poem by John Howard Payne). Although the environments inhabited by some organisms seem humble to us, to these organisms they are ‘home’. Extreme habitats abound on Earth. They seem extreme to us because of high temperatures (hot springs, hydrothermal vents, hot deserts), low temperatures (polar regions, alpine environments, winter temperate environments, cold deserts), lack of water (deserts), high pressures (ocean depths), acidic or alkaline conditions (acid mine waste, the stomach, soda lakes), high salt concentrations (salt lakes) and lack of oxygen (decomposing organic material, estuarine muds, vertebrate intestine). Other extreme situations include toxic chemicals and exposure to ultraviolet radiation. Many extreme habitats challenge the organisms that live there with some combination of these stresses. Let us look at the characteristics of some of these extreme habitats and the adaptations which enable organisms to call them home.

DESERTS

Not all deserts are hot and sandy. A desert is defined by its lack of water rather than its temperature. This does not mean that there is no water but that rainfalls, and other inputs of water, are irregular and infrequent. Deserts cover one-third of the Earth's land surface (Figure 2.1). This includes semi-arid areas (annual precipitation of less than 600 millimetres), arid areas (less than 200 millimetres) and hyper-arid areas (less than 25 millimetres). Deserts form in the leeward side of mountain ranges, in inland areas which are remote from oceans and where dry stable air masses form, resisting convective currents which would bring rain.