Itzhak Ofek

Bacterial adhesion to cells and tissues

Principles of adhesion. Methods, models, data analyses. Bacterial cell surfaces: characteristics of bacterial adhesion. The animal cell membranes and surfaces: substratum for bacterial adhesion. Lectins as adhesions. Lectinophagocytosis. The adhesion of pyogenic cocci. Oral adhesion. Molecular biology. Bacterial adhesion in the natural environment. Common themes in adhesion.

Principles of Bacterial Adhesion

Certain fundamental aspects of bacterial adhesion have been known for years (reviewed in Marshall, 1976; Ellwood et al., 1979; Beachey, 1980a, b; Berkeley et al., 1980; Bitton and Marshall, 1980; Beachey et al., 1982; Schlessinger, 1982; Jones and Isaacson, 1984; Marshall, 1984; Mergenhagen and Rosan, 1985; Savage and Fletcher, 1985; Lark et al., 1986). More discussions on various fundamental aspects can also be found in other selected books (Boedeker, 1984; Mirelman, 1986; Switalski et al., 1989; Doyle and Rosenberg, 1990; Hook and Switalski, 1992). During the first decade of intense research on the adhesion of microorganisms to various substrata a number of points had become clear. One, there is little doubt that the survival of microorganisms in various niches is dependent on their ability to adhere to surfaces or substrata. Second, the adhesion process involves an interaction between complementary molecules on the respective surfaces of the microbe and the substratum. Third, the expression by the organisms of the macromolecules that participate in the adhesion process is under a number of regulatory control mechanisms. During the second decade of research most of the efforts have been focused on molecular mechanisms (fine specificity and genetic control) and consequences of the adhesion phenomenon, with the premise that the process of microbial adhesion may be specifically manipulated. In this first chapter the fundamental principles that have emerged from studies performed during the last two decades are reviewed, with an emphasis on how bacteria adhere to biological substrata, the importance of adhesion in infectious processes, and the basic genetic and phenotypic variables affecting adhesion. A summary of common terms that are now widely used and have become prominent in the concepts developed in the study of bacterial adhesion is given in Table 1-1. The chapters that follow provide more in-depth discussion of the molecular nature of adhesins and their receptors. In addition, thorough reviews of the genetic regulation of adhesins are presented, along with an appraisal of the biological significance of the adhesion process. Finally, recent advances on adhesion of the most widely studied pathogens are provided.

Methods, Models, and Analysis of Bacterial Adhesion

It is axiomatic to consider that most living and nonliving surfaces have a tendency to be colonized by microorganisms. The importance of microbial adhesion and colonization to surfaces was not appreciated until molecular techniques were applied to analyze modes and mechanisms of cell—substratum interactions. As more and more techniques became available, new knowledge was gained that made it possible to understand the modulation of the adhesion and subsequent colonization of many microorganisms. To date, no single experimental system has been developed that can be used to adequately characterize all aspects of microbe—substratum interactions. It is therefore essential that the reliabilities, advantages, and limitations of the existing techniques be understood. Most techniques employed in the study of adhesion yield restricted amounts of information, usually about defined events in a complicated series of interactions. This chapter considers methods for the study of adhesion. Consideration is given to model systems, methods for separating adherent from nonadherent cells, controlled and uncontrolled variables in experimental design, and approaches used in analyzing adhesion data. Finally, methods related to the identification and regulation of expression of adhesins and their receptors are reviewed.

Bacterial Lectins as Adhesins

In 1977, Ofek et al. suggested that proteins with lectin-like properties on bacterial surfaces could serve as adhesins that bind the organisms to animal cells. It was found that E. coli, bearing type 1 fimbriae specific for mannose, could agglutinate red cells. The adhesins of many pathogenic bacteria are now thought to be carbohydrate-binding proteins, possibly lectins (Table 5–1). Although some members of the genera Staphylococcus and Streptococcus appear to express adhesins that lack lectin activity, other members of the same genera are known to possess surface lectins with adhesin functions. Lectins that serve as adhesins may be associated with the cell wall, the outer membrane, or with fimbrial structures. More detailed information on the molecular biology of these lectins is provided in Chapter 9. In this chapter information is presented concerning the occurrence and specificities of some bacterial surface lectins and their role in infection. Many bacterial lectins have not yet been defined as adhesins. A comprehensive review of bacterial lectins is found in the book edited by Mirelman (1986).

Gram-Positive Pyogenic Cocci

The pyogenic Gram-positive cocci include members of the genera Streptococcus and Staphylococcus. It appears that there are no general rules regarding the adhesion of the pyogenic cocci. A few, including strains of S. pyogenes, S. agalactiae, S. pneumoniae, and S. aureus, express cell surface lectins, whereas others depend on lipoteichoic acids, cell-wall-bound proteins, secreted polysaccharides, and hydrophobins. There is a growing body of evidence to suggest that all of the pyogenic Gram-positive cocci rely on multiple adhesins in order to adhere avidly to substrata (Hasty et al., 1992). Most adhesion studies have been carried out on Gram-positive cocci, the normal habitat of which is the animal host. The successful pyogenic cocci have the ability to adhere to and grow on mucosal tissues without causing symptoms. This is commonly known as the carrier state.

Relationship Between Bacterial Cell Surfaces and Adhesins

The central dogma of bacterial adhesion requires that the adhesin(s) function from the bacterial surface. In most cases, the adhesins are assembled on the surface, but in a few cases, the adhesins are initially secreted in the soluble form and then associate with the bacterial surface (Tuomanen, 1986; Baker et al., 1991; Wentworth et al., 1991). In either case, the adhesin must dock or anchor on the bacterial surface before it can participate in adhesive processes. Because adhesion is a property of most bacteria, especially of tissue-colonizing bacteria, it follows that evolution has selected specific structures that function as adhesins or onto which adhesins can assemble. In this chapter, a concise review of bacterial surface structures, with special emphasis on macromolecules involved in adhesion, is given. More comprehensive discussions of the bacterial cell surface are provided elsewhere (Rogers et al., 1980; Nikaido and Vaara, 1985; Krell and Beveridge, 1987; Doyle and Sonnenfeld, 1989; Handley, 1990; Hancock, 1991; Irvin, 1990; Gilbert et al., 1991).

Adhesion of Bacteria to Oral Tissues

The oral environment contains many kinds of bacteria, including both Gram-negative and Gram-positive cocci, bacilli, and spirochetes. More than 300 distinct species of bacteria may exist in the oral cavity. Some of these can be readily cultured and identified, whereas others can be cultured only with difficulty. Some may not be cultured at all. In ecological terms, the oral environment is a perfect niche for some bacteria. Frequently, the bacteria encountered in the oral environment are not found elsewhere in the body. In the mouth, bacteria are challenged by the turbulent effects of saliva; antibacterial proteins in saliva, such as lysozyme and lactoferrin; immunoglobulins; products from other microorganisms; and dietary constituents. Some of these factors influence adherent reactions, details of which will be discussed below.

Regulation and Expression of Bacterial Adhesins

There are various levels of complexity in bacterial cell surfaces. In Chapter 4, the main features of the Gram-negative and Gram-positive surfaces were reviewed. It was seen for both cell types that their surfaces were composed of components that associate noncovalently to form a supramolecular structure. These noncovalently associated molecules may be essential for survival of the bacteria in a particular environment. In general, it seems that adhesins are of the “associated” molecules. For example, in the case of S. pyogenes, M-proteins possess a domain that enables the proteins to be anchored to the wall matrix, whereas another domain is exposed and capable of interacting with lipoteichoic acid (LTA). In this sense then, the adhesin (LTA) is “carried” by M-protein or other surface proteins in order for the bacterium to adhere to an appropriate substratum (see also Chapter 6 for details). Similarly, in the Gram-negative Enterobacteriaceae, the adhesin(s) molecules are associated with fimbrial subunits forming a complex in which the fimbrial subunit is the major protein (Figure 9–1) (see Chapter 5). This is especially true in E. coli and may be true for other Enterobacteriaceae as well. It may, therefore, be a common strategy for bacteria to assemble the adhesin onto some other surface structure that serves as a carrier thereby forming an adhesive complex. This strategy would ensure that the adhesin is one of the most exposed molecules on the bacterial surface. The adhesin complex not only must be expressed in functional form on the bacterial surfaces but the organisms must also be able to regulate the expression or presentation of the adhesins in a functional form during the infectious process. Indeed, in all systems studied, one can always find adhesive and nonadhesive phenotypes of the same bacterial clone, and in many cases growth conditions that favor growth of one phenotype over the other have been defined.

Interaction of Bacteria with Phagocytic Cells

Phagocytic cells, unlike other cells of soft and hard tissue, are preordained to engulf organisms. The interaction of bacteria with phagocytic cells may be either beneficial or harmful to the bacteria. In some cases, the bacteria survive in phagocytes, thereby escaping from environmental challenge, whereas in other cases the outcome is lethal. In order for the phagocyte to recognize and ingest a bacterium it must possess receptors complementary to the bacterial surface. Several bacterial species have been found to express adhesins for which receptors are accessible on the phagocytic membrane. Three major nonopsonic mechanisms of interaction of bacteria with phagocytic cells in a serum-free system have been described (Table 7–1). One of these, termed lectinophagocytosis, is based on recognition between surface lectins on one cell and carbohydrates on the opposing cell. The second mechanism involves protein-protein interactions via the Arg-Gly-Asp (RGD) sequence. The final mechanism involves hydrophobic interactions between the two cell types.

Animal Cell Membranes as Substrata for Bacterial Adhesion

The purpose of this chapter is to review briefly the composition and organization of animal cell surface structures that may be potential receptors for adhesins of bacteria. The understanding of the specificity of animal cell—bacteria interactions requires a basic knowledge of the molecular structure of the animal cell surface, especially of those molecules that serve as receptors for ligands in general and for bacterial adhesins in particular. All animal cell membranes share common compositional and organizational features (Figure 3-1): (1) The major membrane lipids are arranged in a planar bilayer configuration that is predominantly in a “fluid” state under physiological conditions. The membrane lipids are commonly composed of glycerolphospholipids, sphingolipids, and sterols. (2) The bilayer membrane contains integral membrane constituents composed of both glycolipids and glycoproteins that are inserted or “intercalated” into the bilayer structure. (3) Other glycoproteins and proteins are bound to the surface of the plasma membrane by weak ionic interactions, hydrogen bonding, or the hydrophobic effect. These surface-associated glycoproteins and proteins bound to integral membrane structures are referred to as peripheral or extrinsic components. (4) In many animal cells there is a substantial layer of carbohydrate-containing materials of variable thicknesses outside the plasma membrane but in close or intimate association with the membrane. This layer is known as the cell coat or extracellular matrix. The distinction between membrane constituents as being integral, peripheral, or belonging to the cell coat is based on the method required to dissociate the constituent in question from the cell membrane. The integral constituents may be released only after disruption or perturbation of the phospholipid bilayer, usually by detergents (Lichtenberg et al., 1983). Nonintegral surface constituents are commonly released by washing the cells with buffers of different pH or ionic strength, or by using chelating agents, such as ethylenediaminetetraacetic acid (EDTA). There is no general method, however, to release selectively either peripheral or extracellular matrix constituents. As a result, the distinction between the two classes of membrane constituents is sometimes difficult to resolve and very often they are referred to as nonintegral membrane constituents. One of the key features of the membrane is its asymmetry. For nonglycosylated lipids the asymmetry is only partial, in that every phospholipid is present on both sides of the bilayer but in different amounts. In human erythrocytes, for example, lipids with positively charged head groups (e.g., phosphatidylethanolamine and phosphatidylserine) are predominant in the internal leaflet facing the cytoplasm (Marinetti and Crain, 1978). The asymmetry with respect to proteins, glycoproteins, and glycolipids is absolute: every molecule of a given membrane constituent has the same orientation across the lipid bilayer, with the carbohydrate moieties of the glycosylated compounds always exposed on the outer surface. For further information on the organization of the animal cell membrane, the reader is referred to reviews (Lodish et al., 1981; Lotan and Nicolson, 1981; Singer, 1981; Aplin and Hughes, 1982) and a book (Sim, 1982).