Role of Oligosaccharides and Glycoconjugates in Intestinal Host Defense

Abstract

For a long time, carbohydrates were believed to serve solely as a source of energy and as structural materials and not to have biologic significance. Today it is widely accepted that carbohydrates on cell surfaces play a key role in the cell-cell recognition process, and carbohydrate-binding proteins called lectins mediate many cellular interactions, including the adhesion of microorganisms to host cells and the control of leukocyte traffic and their recruitment to inflammatory sites (1-3). The importance of carbohydrates and their conjugates (glycoconjugates) in many biologic processes has provided new carbohydrate-based compounds for investigation. In this review, we focus mainly on microbial interaction with enterocyte surface carbohydrates. In particular, we examine the developmental changes in enterocyte surface carbohydrates and their mechanisms, in an attempt to understand the developmental regulation of microbial receptors on the apical surface of enterocytes. These developmental regulatory processes may help to explain an increased or decreased host sensitivity to specific infectious disease during early childhood. MICROBIAL ADHESION TO ENTEROCYTE SURFACE CARBOHYDRATES Once a microbe reaches the host's cellular surface, it must adhere to host cells to colonize them. This is particularly important in the gastrointestinal tract where mucosal surfaces are washed by fluids. In these locations, only microbes that can adhere to mucosal surfaces are able to stay at that site. Even in relatively stagnant areas such as the colon, Brownian motion can move a microbe that has made contact with a mucosal cell away from the surface of the cell. The adhesion of microbes to host epithelial surfaces, in addition to being a requisite first step in the colonization process, can also serve to induce the expression of virulence factors (4). The best understood mechanism of surface adherence is attachment by lectins. Host cell receptors for lectins are commonly carbohydrate residues of glycoconjugates (glycoproteins or glycolipids). Cell surface carbohydrates are expressed in a species-specific, tissue-specific, and developmentally regulated manner (5,6). Binding of lectins to the receptors is quite specific. This specificity is important, because the availability of suitable receptors is often determined by the age of the host and what body site is infected by the microbe. Therefore, the oligosaccharide repertoire on the host cell surface, whether in the form of glycoproteins or glycolipids, is a key genetic susceptibility factor in microbial infection and in toxin action; and it has been postulated that the control mechanisms by which the expression of cell surface carbohydrates are regulated may have a direct impact on host and age susceptibility to different microbial infections. It was found that a large number of bacterial, viral, and protozoan pathogens bind in vitro to carbohydrate structures of glycoconjugates present on the intestinal microvillus membrane (MVM) (Table 1) and that this binding can be readily inhibited by suitable mono- or oligosaccharides (2,7-10), which compete with the host-cell glycoproteins or glycolipids on the carbohydrate binding domain of the bacterial adhesin lectin. Some bacterial and viral surface lectins are specific for terminal (nonreducing) sugars, and may recognize internal structures as well (8). Glycolipids play a special role, because they are usually strictly membrane-bound and usually do not appear in secretions as potential inhibitors of membrane attachment, as do glycoproteins. A large number of bacteria have been shown to adhere to a carbohydrate sequence with lactose as a minimum requirement. The bacteria are both normal microflora and important pathogens, such as Bordetella pertussis, Vibrio cholerae, Shigella dysenteriae, and Neisseria gonorrhoeae. Among the carbohydrate sequences are lactosylceramide species with separate ceramide structures (R1-Galβ4Glc-R2) (11). Sialic acid residues (NeuAc-R2) may also be used by several viruses (influenza A, B, and C; polyoma virus; rotavirus; reovirus), bacteria (Mycoplasma, Escherichia coli CFA/1, E. coli S, N. gonorrhoeae) and bacterial toxins (cholera toxin [CT], E. coli heat-labile toxin [LT]) as binding sites. Streptococcus pneumoniae bind to buccal epithelial cells by a specific attachment to the carbohydrate sequence Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (12). The best characterized receptor is the acidic glycolipid ganglioside GM1 (Galβ1-3GalNAcβ1-4[NeuAcα2-3]Galβ1-4Glcβ1-1Cer) for CT and E. coli LT (11,13,14). Two neutral glycolipid receptors have also been identified. One is globotriaosylceramide (Gb3, Galα1-4Galβ1-4Glcβ1-1Cer) for Shiga toxin (15) and Shiga-like toxin from E. coli(16), and the other is neolactopentaosylceramide (nLc5Cer, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer) for Clostridium difficile toxin A (17). All three of these previous glycolipid receptors are synthesized from a common precursor, lactosylceramide (LacCer; Galβ1-4Glcβ1-1Cer) in mammalian tissues. Ganglioside GM1 is formed by the sequential addition of sialic acid, N-acetylgalactosamine, and galactose to LacCer, catalyzed by three enzymes: α2,3-sialyl-, β1,4-N-acetylgalactosaminyl-, and β1,3-galactosyl- transferases, respectively. Gb3 is formed by the addition of a galactose to LacCer, catalyzed by α1,4-galactosyltransferase. nLc5Cer is formed by the sequential addition of N-acetylglucosamine, β-linked galactose, α-linked galactose, catalyzed by β1,3-N-acetylglucosyl-,β1,4-galactosyl-, and α1,3-galactosyltransferases, respectively. As previous mentioned, the terminal carbohydrate sequence is critical to microbial adhesion and to bacterial toxins' binding during intraenterocyte events. Thus, the availability of specific glycosyltransferases in the enterocyte Golgi apparatus is critical to the cellular responsiveness to microbes and their toxins.TABLE 1: Microbial carbohydrate receptors on the intestinal microvillus membrane CONSEQUENCES OF ADHESION Although adhesion of the microbe to a mucosal surface is an important determinant of mucosal colonization, especially in determining its site and density, it is becoming increasingly clear that this is not the complete interaction. Several critical postadhesion events are necessary for microbes to establish themselves successfully on mucosal surfaces and to initiate infection (18,19). After adhesion to the eukaryotic cell, Yersinia species may increase their rate of transcription of virulence genes and inject effector proteins into the host cell cytoplasm (20,21). The PapG adhesin-mediated adherence to the host cell Galα(1-4)Gal-containing receptor may induce gene expression in uropathogenic E. coli, and this interaction may also stimulate cytokine responses mediated by a ceramide-signaling pathway (4,22). Thus, a specific lectin-carbohydrate interaction involving a ceramide-linked saccharide may mediate not only adherence to enhance ability to trap nutrients and to multiply, but also an improved response from the host. Figure 1 illustrates the importance of bacterial attachment to colonization and to either toxin penetration or tissue invasion. Also, many known glycoconjugate-lectin binding specificities of fungi and parasites are essential for colonization and virulence. The importance of binding to host cells has been well documented in the case of many bacterial toxins (18). A receptor requirement for penetration and replication of several viruses is well documented, and there is advanced information on the structures and mechanisms involved (2,23). The availability of glycoconjugates on the microvillus surface may therefore represent an important factor in the colonization and pathogenicity of microbes within the gastrointestinal tract.FIG. 1: Diagrammatic representation of sequential steps in pathogenesis of bacterial diarrhea. Bacteria must first adhere to the mucosal surface before surface colonization can occur. These steps are necessary for both toxin production resulting in toxigenic diarrhea or for tissue invasion resulting in inflammatory diarrhea.DEVELOPMENT OF ANTIADHESION THERAPY Because adhesion to host cells is the key first step in causing microbial infections, it should be possible to prevent such infections by blocking the adhesion. A number of strategies have been suggested, including mucosal immunity (to induce secretory immunoglobulin A [SIgA] antiadhesin antibodies), metabolic inhibitors of the expression of adhesions (e.g., sublethal concentration of antibiotics), dietary inhibitors (e.g., human milk), lectin drugs (consisting of lectin-like molecules that can block attachment by competitively occupying the receptor), and receptor analogues (oligosaccharides and glycoconjugates). The last one is the most attractive approach today for the synthesis of antiadhesive drugs (19). This was originally demonstrated in the late 1970s, when it was shown that methyl α-mannoside can protect mice against urinary tract infection with type 1 fimbriated E. coli; methyl α-glucoside, which is not recognized by the bacteria, was not effective (1). Subsequent studies with E. coli, both type 1 and P fimbriated, with type 1 fimbriated Klebsiella pneumoniae, and with other pathogenic micro-organisms have confirmed and extended the initial observations and have shown the drug potential of antiadhesive compounds (8,19,25,26). Globotetraose used in mice and Galα1-4GalβOMe used in monkeys prevented urinary tract infections with P-piliated E. coli(27,28). A striking example of the successful application of receptor analogues is that sialylated glycoproteins, administered orally, protected colostrum-deprived newborn calves from a lethal dose of enterotoxigenic E. coli K99 (29). With a rapidly increasing occurrence of antibiotic resistant strains, alternative therapies against infectious diseases are urgently needed. Carbohydrate analogues represent a viable alternative. Because their action does not require the blocking of any fundamental metabolic processes of micro-organisms, emergence of resistance is unlikely. The development of antiadhesion therapy targeted at the microbial lectins has been hampered by the great difficulty in large-scale synthesis of the required inhibitory saccharides. An alternative is glycomimetics, compounds that structurally mimic the inhibitory carbohydrates but that may be more readily obtainable. More detailed information about the specificity of microbial surface lectins and the elucidation of the structure of their receptors will certainly be of benefit in the design of such drugs. OLIGOSACCHARIDES AND GLYCOCONJUGATES IN HUMAN MILK AND THEIR PROTECTIVE EFFECTS The nonimmunoglobulin fraction of human milk also has protective effects against infections of the gastrointestinal, respiratory, and urinary tract during the first year of life. The protection has been attributed to the presence of oligosaccharides and glycoconjugates and their antiadhesive properties against microbes (10,18,30-32). There is a wide spectrum of oligosaccharides and glycoconjugates in human milk that interfere with microbial adhesion. In contrast to the other large class of antiadherent molecules in human milk, SIgA, these molecules are produced by antigen-independent mechanisms in mammary gland epithelial cells. Oligosaccharides The oligosaccharide fraction of human milk is the third largest solid component, after fat and The several of this milk fraction are in that it more in milk and in These oligosaccharides usually lactose at the and or sialic acid at the Although the of most of is effects by several of these molecules have been well A example is that a the adherence of pneumoniae to buccal epithelial cells (12). The bacteria that are inhibited from binding to including of E. coli and their and E. coli by oligosaccharides sialylated oligosaccharides the binding of A and E. coli to their host cells the of oligosaccharides at the gastrointestinal tract is not It has been that the of sialylated oligosaccharides in reaches during the first of to The human membrane has of and including and human milk fat and sialic acid the binding of E. coli to buccal epithelial cells milk also with infection in mice not only to the but also to is a This binding to is also on sialic acid More it was that also protected against infection which that one of these human milk glycoconjugates of pathogens in the also has in the human protective of human milk are A of is shown in the ganglioside which to toxin of E. and toxin of the neutral glycolipid ceramide which to Shiga toxin of Shigella and the Shiga-like toxins of E. and a that binding of of E. coli to human intestinal There are several of in human a or a the binding of the membrane of to its host-cell the step in human infection These inhibitory a essential for Oligosaccharides and glycoconjugates in human milk that adhesion or its toxin binding glycoproteins, particularly and oligosaccharides in human milk are shown to be for and These factors are in human milk but not in a has a intestinal microflora in which bacteria It is becoming increasingly that intestinal microflora are of importance in the development of the mucosal as well as in colonization and invasion by pathogenic micro-organisms IN IN The enterotoxigenic E. coli K99 is an for of diarrhea in and acid the receptor recognized by E. coli K99 is expressed on glycoproteins and glycolipids of the gastrointestinal tract surface in these newborn but is during development by acid of the adhesion receptor during intestinal with decreased susceptibility to E. coli K99 only acid from and therefore are not to E. coli K99 The of the and human an increased sensitivity and response to E. coli to a For the receptor number for in decreased from the of to the of receptors membrane The increased receptor number in the is with a increase in the sensitivity of host responsiveness to that in the when a is to of These that receptor events may to developmental in host responsiveness to bacterial toxins causing diarrhea. An increase in the expression of the glycolipid receptor Gb3 for Shiga toxin has been in the and the Gb3 was to the secretory effects of the toxin in In Gb3 is the microvillus membrane glycolipid on cells in the and is only after there are about this glycolipid in human tissues. Thus, it to be confirmed that Gb3 in is developmentally regulated as it is in the and this is the it one possible for the that human is a the other the protection mechanism from difficile infection in may from species to A in the binding of difficile toxin A to membrane receptors in the was found in but not in In addition to being protected by receptor other mucosal mechanisms may also be to explain the resistance to difficile In difficile toxin A is in in the of any it to be determined whether this is to a developmental in the expression of toxin receptor in human SURFACE CARBOHYDRATES of and Carbohydrate As microbes and bacterial toxins a specific cell surface carbohydrate structure for attachment to occur. Figure 1 illustrates the importance of bacterial attachment to colonization and to either toxin action or tissue invasion. microbes and intestinal glycoconjugates are in Thus, of intestinal surface glycolipids and glycoproteins to play a role in microbial receptor is that an increase in availability of glycoconjugates in the intestinal microvillus membrane (MVM) may for an increased microbial For an of the may be an important determinant in microbial colonization and intestinal infection in and factors may protect the gastrointestinal tract regulation examine the of carbohydrates on the of glycoproteins and glycolipids in the we the binding of lectins to membrane from and lectins were used and their specific carbohydrate A a lectin that to and was to newborn and more lectin to the increased availability of and In when which sialic acid and N-acetylglucosamine, was to the more to the newborn a striking increase in these a N-acetylgalactosamine, to and not to newborn an of this on the newborn enterocyte In when which is specific for was to the from newborn and to to newborn an of in the newborn examine of glycoproteins and glycolipids in the we the of into from newborn and In these studies a striking the membrane was was into the newborn with that in the Because was that for the in sugars, we in the and sialic acid were as a of we a striking increase of both during the newborn and a at the of In and were as a of the in was at and increased at and were increased by and in have the expression of these glycosyltransferases in different of during The that was increased in the of in the and to a in the but not in the and was decreased in the and colon, but not in the and the with in the first of but it rapidly increased at and increased the to during development These that of intestinal glycosyltransferases are and developmental of several in the that are developmental A a in and an increase in that to be well with a from to of membrane glycoproteins and glycolipids in after the increased in the is by an increased of mainly in the form of but not the other it is to the increase of with the expression of carbohydrate and by that the may control the synthesis of and and enhance difficile toxin A receptor changes in the of glycosyltransferases in The specific Gb3 increased with with a increase at of an Thus, regulation of both and for Gb3 to explain the changes in Gb3 with age The of other and are to increase with The increased of these may enhance the expression of glycoproteins and glycolipids galactose and in the but their role in the expression of carbohydrate sequences on microbial receptors to be on in in the carbohydrate structures of glycoproteins and glycolipids can be attributed to a of during the formed of in may on a number of internal and These the such as and factors such as A of the and bacterial These factors may the developmental that the control of the gene expression of most at the the other other regulatory mechanisms may be by availability or by regulation of for specific carbohydrate sequences Thus, the presence or of intestinal and nutrients may help to regulatory that control the expression of microbial receptors during are of the most important internal factors that the expression in the Among and have been demonstrated to play an important role in the development of the in The of these three are increased the have that decreased and and and an increase in the in as well as in in The of the sialic of the glycoproteins is one that in the during the from to and this is by an of into the that in the of was in the the membrane-bound was In with decreased the of this and of the in both of the of the the of The expression of in the of was several that in and was to as well as to These that the of expression in the of is mediated the receptor pathway and the that in of glycoconjugates are the of a expression of A of is also after the of with but the administered to is the of by is by the normal increase in be prevented by this This that response to or in is to an interaction with receptors and at the It also that the normal developmental increase in is of to and binding in but does not sialic acid or and These observations an important role of that should be in is known to stimulate intestinal and in mice that in as a of a intestinal by of these was the mechanism of action of on in The human a and of colonization of the human the first microbes to be are from the during and from other It is that there are cells with the human body and that of these are micro-organisms, most of which in the The human at of These that in the are to as the intestinal which more bacterial The intestinal microflora is an metabolic that essential to the host such as and The microflora also colonization first of against invasion by pathogenic or mice that have been with a normal and that have been in a have shown that of the is also by with its a microflora the rate of epithelial cell is in the the and the and of the tissue are the interaction and microbes that the microbial be in a special The ability to mice any a critical for and this mice can be by one or more species of bacteria, becoming found that colonization of mice with a of micro-organisms from mice to the of in the increased expression of the neutral and in with a of intestinal bacteria, these changes in the host and also of the found in mice with a complete microflora a has been to the a bacterial of the normal microflora and the intestinal demonstrated that developmental in the of binding of a to the the and In both of these structures are not in the before and a the to and A of production and In cellular production of these structures is and in production to enterocytes. These that the of production in of its microflora but that the microflora is required to complete the of an normal to mice and a of can be at any after of mice also in of a host gene in the a of the microflora can these is a and a of the normal intestinal microflora in both mice and mice with this the changes in expression of glycoconjugates that are in with a complete microflora This to be a specific response of the intestinal to bacterial changes are when mice are with several other bacterial including and These observations a specificity of and microbes that to be There is a direct the number of viable cells in the and the of of glycoconjugates A minimum of bacteria are required to a bacteria the of the with is in the of bacteria to induce in the to are to do in the the to a of The this host response and microbial is with several possible a direct binding interaction bacterial cells and host intestinal that must a critical before can of a bacterial factor that a host changes in the metabolic properties of the bacteria that their ability to host metabolic direct binding interaction and the intestinal has been These that the of on bacterial is the of of a microbial or changes in bacterial or a of the A of in the mammalian will new strategies for the and of infectious diseases in microbial have been used to infections, such as that when are and the normal microflora during antibiotic therapy microbial receptor expression in the is by which bacterial colonization are not well understood but at three mechanisms of action have been 1) production of specific for adhesion and of mucosal responses In the may also be used to prevent infections. AND The attachment of microbes to carbohydrate on the host cell surface is essential for successful colonization and to membrane proteins and is an important host determinant in microbial colonization of the The for are studies and have shown that glycosyltransferases and microbial receptors are developmental genetic and and factors and bacterial may the of these and the expression of microbial Therefore, the developmental control of microbial receptors in the gastrointestinal tract may in to the host sensitivity to intestinal infection in and factors may also protect the gastrointestinal tract In the it may also be possible to microbial attachment by blocking with oligosaccharides or glycoconjugates specific for the lectins. The of microbial receptors in intestinal epithelial cells the importance of intestinal surface carbohydrate expression in interaction. With improved for receptor binding and the the availability of several human intestinal of human of human intestinal epithelial the cell and cell and of we may membrane receptors and the receptor sequences in the may glycoconjugates from human intestinal structurally and may the binding of microbial to epithelial surfaces with glycoproteins or Subsequent studies on the intestinal expression and developmental regulation of glycosyltransferases can be a has been used to in which the the was expressed in the gastrointestinal tract by with a human In the the potential by with for specific will be used to examine the role of oligosaccharides and glycoconjugates in cellular and the interaction. In the the of and cell biologic in intestinal cell and in of human or of human and will help to the developmental regulation of intestinal microbial receptors and events. the of microbial receptors and their effector responses in the the developmental and on receptor and the effector response and the biologic in host new may be in the and of with infectious intestinal diseases of This was by and from the of was in by