Primary contributions to this chapter were made by J.D. Esko (University of California at San Diego).

MANY PATHOGENIC MICROORGANISMS exploit host cell-surface glycoconjugates as receptors for cell attachment, tissue colonization, and invasion. This chapter provides examples of proteins on the surface of microorganisms (adhesins or hemagglutinins) and their carbohydrate-binding partners on mammalian cell surfaces (receptors). Examples include viruses binding to sialic acids and glycosaminoglycans, and bacteria and toxins binding to glycosphingolipids.

Introduction ^(1–8)

Many microbial interactions with animal hosts involve attachment to epithelial cells lining the respiratory tract or the gastrointestinal tract, which are exposed to the environment. For infection to occur, bacteria, viruses, and parasites must pass through the glycocalyx that surrounds the cells, bind to cell surfaces or exposed ECM, and colonize the tissue. The first step involves adhesion mediated through specific proteins on the surface of the microorganism, called either bacterial adhesins or viral hemagglutinins, and ligands on the surface of the mammalian cells, called receptors. (Note that the term receptor in this case is equivalent to “ligand” for animal cell lectins.) During the last 15 years, many adhesins and hemagglutinins have been described, cloned, and characterized. In some cases, binding involves protein ligands, but many bind to complex carbohydrates found on glycoproteins, glycolipids, and proteoglycans. Binding can lead to cell invasion, tissue destruction, and systemic infection.

Many adhesins are lectins and may contain carbohydrate recognition domains that bind to the same carbohydrates as endogenous mammalian lectins (see Chapter 22). The adhesin present on influenza A virus, the hemagglutinin, is by far the best-studied system to date, in part because of its importance in public health. Wiley and his associates crystallized the viral hemagglutinin, determined its structure in 1981, and later solved the structure of cocrystals prepared with sialyllactose. Since then, the crystal structures for several other viral hemagglutinins and bacterial toxins have been determined as well. Like animal cell lectins, some microbial adhesins bind to terminal sugar residues, whereas others bind to internal sequences found in linear or branched oligosaccharide chains (see Chapter 29). The detailed maps of the carbohydrate-recognition domains have provided much insight into binding mechanisms and have led to the development of ligands with even greater affinity. These synthetic ligands form the basis for therapeutic agents to treat infection.

It should be kept in mind that colonization of tissues by microorganisms is not always pathogenic. For example, the normal flora of the gastrointestinal tract is determined by appropriate and desirable colonization by beneficial bacteria. Selective colonization of tissues by microorganisms defines the tropism of the infectious agent. To a large extent, tropism is determined by the composition and structure of the carbohydrate receptors expressed by cells in the target tissue.

Methods for Studying Microbial Binding and Adhesion ^(7,9–12)

To study microbial adhesion in vitro, adherence assays have been developed in which microorganisms are challenged to adhere to cultured cells, tissue sections, thin-layer chromatography plates, blots prepared from SDS-PAGE gels, or plastic surfaces coated with oligosaccharides or glycoconjugates. Many intact viruses cause hemagglutination if the red blood cells contain the cognate carbohydrate receptor.

In a typical overlay procedure, an immobilized glycoconjugate (separated by electrophoreses or chromatography) is incubated with a suspension of bacteria, virus, or toxin, and the extent of binding is determined by autoradiography, antibody staining, or direct visualization of the bound microorganism. These techniques have great sensitivity, especially when the putative receptors have been concentrated into bands (e.g., on a thin-layer plate; see Figure 28.1), which is thought to mimic the high density of ligands found on cell surfaces. To learn more about the ligand-receptor interaction, adherence or binding can be measured in the presence of a competitive ligand, such as another carbohydrate, or at different pH values and salt concentrations, or in the presence of divalent cations.

Figure 28.1

One way to find carbohydrate receptors is through an overlay technique. In the depicted thin-layer plate, labeled bacteria bind to a relatively minor glycolipid band.

In addition to direct binding experiments, one can examine how removal of a suspected carbohydrate receptor from target cells affects adhesion. For example, treatment of red blood cells with sialidase ablates hemagglutination by influenza virus. Resialylation with a specific sialyltransferase and CMP-sialic acid restores defined types of linkages (α2–3 or α2–6), thus allowing measurement of the linkage specificity of binding. The glycoconjugate composition can also be modified metabolically (see Chapters 6 and 40), by mutation of genes encoding the biosynthetic enzymes (see Chapters 31 and 32), or by transfection of cells with glycosyltransferases.

Many glycoreceptors and microbial adhesins have been identified in the manner described above. As adhesin cDNAs become available through molecular cloning, refined binding assays can be carried out using various types of protein-carbohydrate assays (cf. Chapter 29). With the development of transgenic and gene-targeted mice with altered glycosylation (see Chapter 33), it should be possible to correlate the results of binding studies performed in vitro with microbial pathogenicity in vivo.

Microbial Adhesins and Cell Surface Glycoconjugate Receptors ^(8,13–15)

Microorganisms have evolved adhesins that interact with glycoproteins, proteoglycans, and glycolipids. On bacteria, many of the adhesins are protein subunits of pili (hairs), also known as fimbrae (threads). These structures typically have a diameter of 5–7 nm and can extend 100–200 nm in length, or about one-tenth the diameter of a bacterial cell. Thus, pili extend well beyond the glycocalyx formed from lipopolysaccharide and capsular polysaccharides (see Chapter 21), which can actually interfere with adhesin activity. The carbohydrate recognition domain of the adhesin is typically at the tip of the pilus.

Some adhesins are monomeric or oligomeric membrane proteins. Most bacteria (and perhaps other microorganisms) have multiple adhesins with different carbohydrate specificities, which help define the range of susceptible tissues (i.e., the microbe's ecological niche). Binding is generally of low affinity, but because the adhesins and the receptors often cluster in the plane of the membrane, the resulting strength of the interaction (avidity) can be quite strong. In fact, adhesion can require several hundred times the force of gravity to dissociate. Perhaps an appropriate analogy for adhesin-receptor binding is the interaction of the two faces of Velcro™ strips.

Adhesin-receptor interactions can result in signal transduction events critical for colonization and infection. Many microbes must fuse with the cell surface (e.g., herpes simplex virus) or with endosomal membranes after internalization (e.g., influenza). In some cases, the microbe survives and replicates within a phagolysosome (e.g., Chlamydia and Leishmania), which implies that the microbe can subvert normal processing pathways inside the cell. In other cases, binding results in a defense reaction (e.g., binding might cause epithelial cells to secrete interleukins, which results in a mucosal immune response). The relationship between microbes and the host can be quite complex. For example, colonization of germ-free mice with Bacteroides thetaiotaomicron, a normal resident microbe of the small intestine, induces an α1–2 fucosyltransferase in the mucosal epithelial cells. The bacteria bind to l-fucose residues and also use it as a carbon source. Rotaviruses, the major killer of children worldwide, can only bind to the intestinal epithelium of newborn infants during a period that appears to correlate with the expression of specific types and arrangements of sialic acids on glycoproteins. Thus, the intestine is operationally a functioning micro-ecosystem in which glycosylation plays an important part.

Adhesins That Bind to Glycolipids ^{(1,5,8,16–20)}

Many bacteria, bacterial toxins, and parasites bind to glycolipids, and a large number of adhesins target Galβ4Glc-containing oligosaccharides. Sometimes the adhesin binds to terminal Galβ4Glc in lactosylceramide, but the core oligosaccharide is often capped by other sugars (e.g., blood group antigens). Some bacteria secrete glycosidases that expose Galβ4Glc-Cer determinants, whereas others bind to the internal sequence Galα4Gal. Other binding specificities have been described as well (Table 28.1).

Table 28.1

Examples of interactions of bacterial adhesins with glycans.

The specificity of binding can explain the tissue tropism of the organism. The columnar epithelium that lines the large intestine expresses Galβ4Glc-Cer, whereas the cells lining the small intestine do not. Thus, Bacterioides, Clostridium, Escherichia coli, and Lactobacillus only colonize the large intestine under normal conditions. Several uropathogenic E. coli strains recognize Galα4Gal-containing glycolipids as either internal or terminal structures on bladder epithelia, consistent with the correlation of urinary tract infections with the P1 blood group phenotype (see Chapter 16). The presence or absence of these antigens may be a factor influencing the adherence of bacteria to the uroepithelial cells.

In addition to the organisms listed in Table 28.1, a variety of secreted bacterial toxins also bind to glycolipid determinants (Table 28.2). The best-studied example is the toxin from Vibrio cholera (cholera toxin), which consists of A and B subunits, in the ratio AB₅. Its crystal structure shows that the B subunits bind to G_M1 ganglioside receptors through carbohydrate-recognition domains located on the base of the subunits (Figure 28.2 and Figure 4.2). The A subunit (toxin) is loosely held above the plane of the B subunits, with a single α-helix penetrating through a central core created by the pentameric B subunits. Upon binding to membrane glycolipids through the B subunits, the A subunit is delivered to the interior of the cell by an unknown mechanism. The structures of related toxins from Shigella dysenteria, Bordetella pertussis, and E. coli have also been solved.

Table 28.2

Examples of glycosphingolipid receptors for bacterial toxins.

Figure 28.2

Crystal structure of cholera toxin B subunit pentamer with bound G_M1 pentasaccharide. (A) Bottom view; (B) side view. (Reprinted, with permission, from [17] Merritt et al. 1994 [© Cambridge University Press].)

Adhesins That Bind to Glycoproteins ^(5,21–34)

Compared to glycolipids, fewer interactions are known to occur between microorganisms and glycoproteins. This apparent preference for glycolipids may be related to the juxtaposition of glycolipid glycans to the membrane surface compared to the more distal location of glycoprotein glycans. Binding of a toxin or bacterium to a glycolipid might provide a higher likelihood of further interactions with the membrane (e.g., binding to another receptor or membrane intercalation). In fact, Shiga toxin will bind to Galα4Gal determinants on both glycolipids and glycoproteins, but only the binding to glycolipids results in cell death. Alternatively, the apparent preference for glycolipids may often reflect the better methodology available for analyzing glycolipids as receptors (e.g., overlay methods). In any case, several viruses and parasites have infection strategies based on binding to glycoproteins (Table 28.3). For example, Entamoeba histolytica expresses a 260-kD heterodimeric adhesin that binds to terminal Gal/GalNAc residues on glycoproteins and glycolipids. Binding may have a role in attachment, invasion, and cytolysis of intestinal epithelium, and it may function in binding bacteria as a food source.

Table 28.3

Examples of glycoprotein carbohydrate receptors for microorganisms.

By far, the best studied example of a glycoprotein adhesin is the influenza hemagglutinin, which binds to sialic-acid-containing glycans. Influenza A hemagglutinin associates into trimeric oligomers that enhance the overall binding to multivalent surfaces. The specificity of this interaction for A and B subtypes of influenza varies considerably, with human influenza viruses binding only to cells containing Siaα6Gal and other animal influenza viruses binding to Siaα3Gal linkages. This linkage preference is due to a single amino-acid change in the hemagglutinin. Influenza C, in contrast, binds preferentially to glycoproteins containing 9-O-acetylated sialic acids. Having the crystal structure available has made it possible to design better synthetic ligands. For example, the crystal structure revealed a hydrophobic pocket near the carbohydrate-binding site, which predicted that sialosides containing a hydrophobic aglycone would bind with greater affinity.

In addition to the hemagglutinin, influenza A and B virions express a sialidase (traditionally called neuraminidase) that cleaves sialic acid from glycoproteins. Its function may include prevention of viral aggregation by removal of sialic acid residues from virion envelope glycoproteins, dissociation of virions as they bud from the cell surface, or desialylation of soluble mucin from sites of infection in order to improve access to membrane-bound sialic acids. Interestingly, the sialidase specificity with respect to sialic acid linkage tends to evolve in parallel to the hemagglutinin, suggesting an important trophic function as well. In influenza C virus, a single glycoprotein contains both the hemagglutinin activity and the receptor destroying activity, which in this case is an esterase that cleaves the 9-O-acetyl group from acetylated sialic acid receptors. Powerful inhibitors have been designed based on the crystal structure of the sialidase from influenza A. Some of these inhibit enzyme activity at nanomolar concentrations and are in clinical studies to determine their utility as antiviral agents (see Chapter 41). Many other viruses (e.g., reovirus, rotavirus, Sendai, and polyomavirus) also appear to use sialic acids for infection.

The interaction of Plasmodium falciparum (malaria) merozoites with red blood cells also depends on sialic acids present on the host cell. In this organism, attachment is mediated by a specific sialic-acid-binding adhesin on merozoites called EBA-175. The adhesin binds to sialic acids present on the major erythrocyte membrane protein, glycophorin, and prefers Neu5Ac rather than 9-O-acetyl Neu5Ac or Neu5Gc. Soluble Neu5Ac and Neu5Acα6Gal-containing oligosaccharides do not competitively inhibit the binding of EBA-175 to erythrocytes, but Neu5Acα3Gal-containing oligosaccharides are effective inhibitors, indicating that the adhesin is sensitive to the underlying oligosaccharide structure. Binding to erythrocytes leads to invasion and eventual production of additional merozoites. Other organisms expressing sialic acid adhesins also can bind to erythrocytes (e.g., influenza), but these interactions cannot lead to productive infections in these nonnucleated cells. Thus, in these circumstances, the erythrocyte might be considered a clearance mechanism for these agents (see Chapter 3). Plasmodium-infected erythrocytes also express glycosaminoglycan-binding proteins that are thought to facilitate adherence of the infected cells to tissues. As described below, in certain types of malaria, another developmental form of the parasite, the circumsporozoite, selectively invades hepatocytes by way of a heparan-sulfate-binding adhesin called the circumsporozoite protein.

Adhesins That Bind to Glycosaminoglycans ^(7,35–36)

Many bacteria, parasites, and viruses use proteoglycans as adhesion receptors (Table 28.4). Most microorganisms bind to heparan sulfate rather than chondroitin sulfate, possibly due to its greater prevalence on cell surfaces (see Chapter 29). Unlike adhesins that interact with glycolipids and glycoproteins, the GAG-binding adhesins presumably pick out binding sites within the polysaccharide chains as opposed to binding to terminal sugars. To date, the precise structure of the carbohydrate recognition domain of a microbial glycosaminoglycan adhesin has not yet been determined. Dengue flavivirus, the causative agent of dengue hemorrhagic fever, binds to heparan sulfate. Modeling the primary sequence of the viral envelope protein on the crystal structure of a related virion envelope protein suggests that the heparan-sulfate-binding site may lie along a groove in the protein lined by positively charged amino acids (Figure 28.3). Thus, the GAG-binding adhesins may have a more open structure, consistent with the binding sites of other heparin-binding proteins (see Chapter 29).

Table 28.4

Examples of microorganisms that bind proteoglycan receptors on eukaryotic cells.

Figure 28.3

Putative structure of the heparin-binding site in dengue virus envelope protein. Note the alignment of positively charged amino acids along an opening on the face of the protein. (Reprinted, with permission, from [35] Chen et al. 1997.)

The different tissue tropism of glycosaminoglycan-binding microbes may reflect variation in the fine structure of the heparan sulfate chains. Herpes simplex virus glycoproteins gpB and gpC bind to heparin and have different requirements for sulfate groups along the chains (Table 28.5). The differential expression of sequences rich in 2-O-sulfated uronic acids on various cells therefore could partly explain the different tissue tropism of HSV-1 and HSV-2 subtypes. P. falciparum sporozoites (malaria) also bind to heparin and heparan sulfate in a tissue-specific manner, with preferred binding to the basolateral surface of hepatocytes and the basement membrane of kidney tubules. The circumsporozoite protein that covers the sporozoite cell surface mediates binding. The carboxyl terminus of the protein contains positively charged residues. Clustering of the circumsporozoite protein on the surface of the organism may generate a high concentration of positively charged residues that facilitate binding. How this would achieve selective binding to hepatocyte proteoglycans is unclear.

Table 28.5

Microbial heparin-binding proteins.

In many cases, the proteoglycans may be part of a coreceptor system in which the microorganisms make initial contact with a cell surface proteoglycan, and later with another receptor. For example, HSV binds to heparan sulfate on the cell surface, but infection requires additional nonproteoglycan receptors. The coreceptor role of proteoglycan is reminiscent of the formation of ternary complexes required for antithrombin inhibition of thrombin and basic FGF signaling (see Chapters 29 and 34). Although it is clear that cell surface proteoglycans act as adhesion receptors, their role in invasion and pathogenesis is unclear. The HSV glycoprotein gpB binds heparan sulfate and promotes adherence, as well as virus-cell fusion and syncytium formation. The mechanism by which heparan sulfate facilitates membrane fusion is unknown, but perhaps it acts like a template facilitating the association of fusogenic membrane proteins (cf. Chapter 29).

Microbial Carbohydrate Ligands for Animal Cell Lectins ^(3,37–44)

Some microorganisms mimic carbohydrate receptors on mammalian cell surfaces. For example, Chlamydia has a complex mode of adhesion, in which heparan sulfate is thought to act as a bridge, binding both host-cell protein receptors and Chlamydia receptors. A minimal decasaccharide is needed, and on the basis of competition studies with chemically modified heparin, the binding sequences for host and microbial receptors may differ. Interestingly, Chlamydia produces its own sulfated heparin-like molecule, which may provide the opportunity to infect cells with low levels of endogenous heparan sulfate or with heparan sulfate that lacks appropriate binding sequences. Leishmania appears to utilize heparan sulfate in a similar way. This is an open area of research since the heparin-binding proteins have not yet been described in any detail, nor has the biosynthesis of a heparan-sulfate-like chain been studied to any extent in these microbes.

Trypanosoma cruzi has developed an interesting strategy of molecular camouflage in which a parasite-encoded trans-sialidase transfers sialic acid from serum glycoproteins in the host to membrane proteins on its own surface. Although the primary function of this reaction is most likely to cover surface glycans as a way of preventing host immune reactivity, the sialylated glycans might be recognized by sialic-acid-binding lectins on cells (see Chapters 22–27). The trans-sialidase may also act as an adhesin. Neisseria gonorrhoeae uses low levels of tissue CMP-sialic acid to cover itself with sialic acid residues, making it resistant to complement. Schistosomes, a parasitic filarial worm, contain the Lewis X antigen that is also found on human leukocytes. Since Lewis X is recognized by selectins, the presence of these carbohydrates may provide a mechanism for attachment or transcellular migration. (However, these glycans also generate a massive anti-Lewis X antibody response in the host.) In a similar way, the capsules surrounding bacteria, lipopolysaccharide, and yeast cell walls contain oligosaccharide sequences that may be recognized by mammalian cell lectins. For example, yeast mannans are recognized by both soluble and macrophage mannose-binding protein, which has an important role during the preimmune phase in infants. The structure and biology of these types of carbohydrates and carbohydrate-binding proteins are discussed in Chapters 19, 21, and 36.

Future Directions ^(5,45–48)

The interactions described above suggest a correlation between adhesin-receptor interactions and microbial pathogenesis. Examination of virulent and nonvirulent isolates has revealed a dependence on carbohydrate interactions in some cases, but much additional work is needed in this area. The use of mouse models with genetic defects in specific steps in glycosylation (see Chapters 32 and 33) will undoubtedly prove to be useful in this regard. In the end, however, it is the ability to interfere with these processes that will establish whether a causal relationship exists and whether the interaction is a suitable target for drug intervention.

Adhesin molecules represent potential targets for generating antibodies for vaccination. However, the presence of multiple adhesins on cells may frustrate this strategy for controlling infection. Another idea suggested by in vitro binding studies is to administer oligosaccharides known to interact with an adhesin and to measure microbial distribution, tissue colonization, and host survival in a suitable animal model. The ability of exogenous heparin and related polysaccharides to inhibit viral replication suggests that this approach might lead to polysaccharide-based antiviral pharmaceutical agents. Multivalent ligands should prove even more potent, but their use may be limited to the respiratory and gastrointestinal tracts because of difficulties in their delivery.

As more crystal structures become available, the ability to custom design small-molecule inhibitors that fit into the carbohydrate recognition domains and the active sites of adhesins should improve. Already, the structure of the influenza hemagglutinin and neuraminidase has suggested numerous ways to modify sialic acid to better fit the active sites. Some of these compounds are already being tested in human trials (see Chapters 40 and 41).

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