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.
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.
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.
The oligosaccharide fraction of human milk is the third largest solid component, after fat and lactose. The several laboratories reporting quantitative data of this milk fraction are in general agreement that it represents more than 12 g/l in mature milk and approximately 22 g/l in colostrum (10). These oligosaccharides usually display lactose at the reducing end and fucose or sialic acid at the nonreducing terminus. Although the function of most of them is unknown, antiadherence effects by several of these molecules have been well documented. A prominent example is that a trisaccharide, Galβ1-4GlcNAcβ1-3Galβ, inhibits the adherence of S. pneumoniae to buccal epithelial cells (12). The bacteria that are inhibited from binding to enterocytes including Campylobacter jejuni, strains of E. coli and their heat-stable toxin, and enteopathogenic E. coli by fucosylated oligosaccharides (33-36). At physiologic concentrations, sialylated oligosaccharides strongly inhibit the binding of influenza A virus and S-fimbriated enteropathogenic E. coli to their respective host target cells (26). However, the concentrations of oligosaccharides naturally achieved at various points along the gastrointestinal tract is not clear. It has been estimated that the mean daily intake of sialylated oligosaccharides in breast-feeding infants reaches 170 mg/kg during the first 2 weeks of lactation, then gradually diminishes to 20 mg/kg beyond 10 weeks (37).
The human fat-globule membrane has extensive glycocalyx-like filaments consisting of glycosaminoglycan-containing proteoglycans, mucin, and mucin-associated proteins, including lactadherin and butyrophilin (38,39). Intact human milk fat globules and mucin inhibit, through sialic acid moieties, the binding of S-fimbriated E. coli to buccal epithelial cells (40). Human milk mucin also interferes with experimental rotavirus infection in mice (41). Rotavirus binds not only to the mucin but also to lactadherin. Lactadherin is a 46-kDa glycoprotein. This binding to lactadherin is also dependent on sialic acid (41). More recently, it was reported that lactadherin also protected infants against symptomatic rotavirus infection (39), which demonstrates that one of these human milk glycoconjugates capable of inhibiting pathogens in the laboratory settings also has clinical relevance in the human infant population.
Many glycoconjugate protective components of human milk are now known. A representative group of them is shown in Table 2. Examples include the ganglioside GM1, which binds to cholera toxin, labile toxin of E. coli(42), and labile toxin of C. jejuni(43), the neutral glycolipid globotriosyl ceramide (Gb3), which binds to Shiga toxin of Shigella and the Shiga-like toxins (verotoxin) of E. coli(44), and a small mannosylated glycoprotein that inhibits binding of some strains of E. coli to human intestinal epithelium (45). There are several types of glycosaminoglycans in human milk. Either a chondroitin-4 or a chondroitin-6 sulfate moiety inhibits the binding of gp 120, the outer membrane protein of AIDS virus to CD4, its host-cell receptor, the obligatory step in human immunodeficiency virus-1 infection (46). These inhibitory substances each contain a complex glycoconjugate moiety essential for activity.
Certain glycoproteins, particularly κ-caseins and some N-acetylglucosamine-containing oligosaccharides in human milk are shown to be "growth factors" for bifidobacteria and lactobacilli (48). These factors are prominent in human milk but not in nonprimate milks. Consequently, a breast-fed infant has a preferred intestinal microflora in which bifidobacteria predominate over potentially harmful bacteria (48). It is becoming increasingly apparent that indigenous intestinal microflora are of major importance in stimulating the development of the infant's mucosal defenses as well as in preventing colonization and invasion by pathogenic micro-organisms (49).
CHANGES IN CARBOHYDRATE RECEPTORS IN INTESTINAL MEMBRANE
The enterotoxigenic E. coli K99 is an organism responsible for outbreaks of life-threatening diarrhea in piglets, calves, and lambs. N-glycolyl neuraminic acid α2-3Gal, the receptor recognized by E. coli K99 adhesins, is abundantly expressed on glycoproteins and glycolipids of the gastrointestinal tract surface in these newborn animals but is largely replaced during postnatal development by N-acetyneuraminic acid α2-3Gal (50). Disappearance of the adhesion receptor during intestinal maturation correlates with decreased susceptibility to E. coli K99 enteritis. Humans produce only N-acetyneuraminic acid α2-3Gal from birth onward and therefore are not susceptible to E. coli K99 infection.
The immature small intestine of the rat (51), pig (52), and human infant (53) exhibits an increased sensitivity and response to E. coli heat-stable enterotoxin (STa) due to a receptor-dependent mechanism. For example, the receptor number for STa in rat jejunum decreased from the neonatal level of 2.35 × 1012 to the adult level of 0.79 × 1012 receptors per milligram brush border membrane protein (51). The increased receptor number in the neonate is correlated with a parallel increase in the sensitivity of host responsiveness to 600-fold over that in the adult when a jejunal loop is exposed to various concentrations of STa. These results suggest that receptor events may contribute to developmental variations in host responsiveness to bacterial toxins causing diarrhea.
An age-dependent increase in the expression of the glycolipid receptor Gb3 for Shiga toxin has been noted in the rabbit intestine, and the Gb3 level was directly related to the secretory effects of the toxin in ligated rabbit ileal loops (54,55). In rats, Gb3 is the major microvillus membrane glycolipid on crypt cells in the adult intestine and is detectable only after weaning (56). So far, there are no data available about this glycolipid content in human intestine tissues. Thus, it remains to be confirmed that Gb3 in humans is developmentally regulated as it is in the rat and rabbit. If this is the case, it offers one possible explanation for the observation that human neonatal shigellosis is a rather uncommon event. On the other hand, the protection mechanism from C. difficile infection in neonates may vary from species to species. A decrease in the binding of C. difficile toxin A to membrane receptors in the neonatal intestine was found in rabbits (57), but not in hamsters (52). In addition to being protected by receptor underdevelopment, other mucosal mechanisms may also be needed to explain the neonatal resistance to C. difficile infection. In humans, C. difficile toxin A is frequently detected in infant stools in the absence of any clinical symptoms (58). However, it remains to be determined whether this phenomenon is related to a developmental variation in the expression of toxin receptor in human intestine.
CELL SURFACE CARBOHYDRATES
Developmental Regulation of Glycosyltransferase and Surface Carbohydrate Expression
As previously discussed, some microbes and bacterial toxins need 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. Known association between microbes and intestinal glycoconjugates are listed in Table 1. Thus, glycosylation of intestinal surface glycolipids and glycoproteins seem to play a central role in microbial receptor expression. Our hypothesis is that an increase in availability of certain glycoconjugates in the intestinal microvillus membrane (MVM) may account for an increased microbial attachment. For example, an immature glycosylation of the MVM may be an important determinant in microbial colonization and intestinal infection in infants, and some trophic factors may protect the gastrointestinal tract through regulation glycosylation.
To examine the accessibility of carbohydrates on the side chains of glycoproteins and glycolipids in the MVM, we compared the binding of radiolabeled lectins to membrane preparations from neonatal and adult intestine (59). Standard lectins were used and their specific carbohydrate moiety determined. When radiolabeled concanavalin A agglutinin, a lectin that binds to glucose and mannose residues, was exposed to newborn and adult MVM preparations, more lectin bound to the adult MVM, suggesting increased availability of mannose and glucose. In contrast, when wheat germ agglutinin lectin, which binds sialic acid and N-acetylglucosamine, was exposed to the MVM, more bound to the newborn preparations, suggesting a striking increase in these glycoconjugates. Dolichos biflorus agglutinin, a lectin-measuring N-acetylgalactosamine, bound avidly to adult MVM preparations and not to newborn MVM preparations, suggesting an absence of this sugar moiety on the newborn enterocyte surface. In like manner, when ulex europaeus agglutinin (UEA)-1, which is specific for fucose, was exposed to the MVM from newborn and adults, considerably greater quantities bound to adult preparations than to newborn preparations, suggesting an absence of fucose in the newborn MVM. To further examine glycosylation of glycoproteins and glycolipids in the newborn, we measured the incorporation of radiolabeled sugars into isolated MVM preparations from newborn and adult animals. In these studies a striking difference between the two membrane preparations was noted. Very little fucose was incorporated into the newborn MVM preparation compared with that in the adult preparation (60).
Because our hypothesis was that immature glycosylation accounted for the difference in available sugars, we examined certain glycosyltransferase activities in the developing intestine. When α2,6-sialyltransferase and sialic acid incorporation were measured as a function of age, we noted a striking increase of both during the newborn period and a rapid decline at the time of weaning. In contrast, α1,2/1,3-fucosyltransferase activity and fucose incorporation were measured as a function of age, the activity in newborns was at very low levels and increased at weaning (61). During postnatal development, β1,3-galactosyltransferase and β1,4-galactosyltransferase activities were increased by 17- and 24-fold, respectively, in rat small intestine (62). We have further studied the expression of these glycosyltransferases in different regions of mouse intestine during postnatal development. The results showed that α2,6-sialyltransferase activity was increased in the immature intestine of suckling mice, primarily in the ileum and to a lesser extent in the jejunum, but not in the duodenum and colon; α1,2-fucosyltransferase activity was decreased in the ileum and colon, but not in the duodenum and the jejunum compared with adults in the first 3 weeks of life, but it rapidly increased at 4 weeks; and β1,4-galactosyltransferase activity increased throughout the intestine (from duodenum to colon) during postnatal development (63). These results suggest that activities of intestinal glycosyltransferases are under region-specific and developmental regulation.
Table 3 shows examples of several glycosyltransferase activities in the rat intestine that are under developmental regulation. A reciprocal relationship exists between a decrease in sialyltransferase activity and an increase in fucosyltransferase activity (61) that appears to be well correlated with a shift from sialylation to fucosylation of MVM membrane glycoproteins and glycolipids in rat small intestine after weaning (59,64). Specifically, the increased sialylation in the neonatal intestine is reflected by an increased level of gangliosides mainly in the form of GM3 but not GM1. On the other hand, it is tempting to associate the postnatal increase of fucosyltransferase activities with the expression of carbohydrate antigens I, X, and Y by suggesting that the former may control the synthesis of fucoglycoproteins and fucoglycolipids and thereby enhance C. difficile toxin A receptor expression.
The specific Gb3 galactosyltransferase, α1,4-galactosyltransferase, activity increased with age, with a sharp increase seen at 18 days of age, whereas α-galactosidase activity followed an inverse pattern. Thus, regulation of both synthetic and degradative pathways for Gb3 appears to explain the observed changes in Gb3 levels with age (65). The activities of two other glycosyltransferases, galactosyltransferase (62) and N-acetylgalactosaminyltransferase (66) are noted to increase with age. The increased activities of these two enzymes may enhance the expression of glycoproteins and glycolipids containing galactose and N-acetylgalactosamine in the mature intestine, but their role in effecting the expression of carbohydrate sequences on microbial receptors remains to be determined.
Hormonal Effect on Glycosylation Processes in Intestine During Development
Alterations in the carbohydrate structures of glycoproteins and glycolipids can be attributed to a variable level of glycosyltransferase activity during development. However, the glycoconjugate patterns formed through activity of glycosyltransferase in tissues may depend on a number of internal and external factors. These include the luminal stimuli such as phorbol ester (62) and sodium butyrate (70), nutritional factors such as vitamin A deficiency, nutrient composition differences of the diet (71), hormonal stimuli, and bacterial colonization. These factors may affect the developmental program that modulates the control of the gene expression of glycosyltransferases, most likely at the transcriptional level (72-74). On the other hand, other regulatory mechanisms may be affected by substrate availability or by phosphorylation-dephosphorylation regulation of enzyme activities responsible for specific carbohydrate sequences (75). Thus, the presence or absence of intestinal chemical stimuli, microflora, and nutrients may help to modulate regulatory mediators that control the neonatal expression of microbial receptors during development.
Circulating hormones are some of the most important internal factors that regulate the glycosyltransferase expression in the gut. Among them glucocorticoids, thyroxine, and insulin have been demonstrated to play an important role in the development of the intestine in every organism studied so far. The circulating levels of these three hormones are markedly increased near the weaning period (76-78). We have previously reported that cortisone injection precociously induces decreased α2,3- and α2,6-sialyltransferase activities and sialylation (61) and an increase in the glycosidic-bound fucose in MVM as well as in α1,2/1,3-fucosyl-, β1,3-and β1,4-galactosyltransferase activities in 2-week-old suckling rats (61,62). The progressive loss of the sialic acids of the MVM glycoproteins is one major biochemical change that occurs in the rat small intestine during the transition from suckling to weaning, and this process is speeded up by an injection of glucocorticoids into the suckling animals. Hamr et al. (79) reported that in the ileum of suckling rats, α2,6-sialytransferase mRNA level was approximately four times higher than in the jejunum, whereas the membrane-bound enzyme activity was less than two times higher. In comparison with controls, hydrocortisone treatment significantly decreased the level of this transcript and of the corresponding enzyme activity in both segments of the small intestine of suckling rats. Additionally, the antiglucocorticoid mifepristone (RU-38.486) suppressed the effect of hydrocortisone. The expression of α2,6-sialyltransferase mRNA in the small intestine of weaned (25-day-old) rats was several times lower than that in suckling rats and was unresponsive to hydrocortisone as well as to mifepristone. These results indicate that the glucocorticoid-induced transcriptional down-regulation of α2,6-sialyltransferase expression in the small intestine of suckling rats is mediated through the glucocorticoid receptor pathway and support the notion that alterations in sialylation of MVM glycoconjugates are the result of a lower expression of α2,6-sialyltransferase.
A precocious enhancement of fucosyltransferase activity is also obtained after the treatment of suckling rats with hydrocortisone, but the same treatment administered to weaned rats is ineffective. Moreover, the precocious rise of suckling rat fucosyltransferase activity, induced by hydrocortisone, is antagonized by mifepristone, whereas the normal increase in fucosyltransferase activity cannot be prevented by this antagonist (80). This result suggests that fucosyltransferase response to hydrocortisone or cortisone in suckling rat intestine is due to an interaction with glucocorticoid receptors and disappears at the weaning period. It also suggests that the normal developmental increase in fucosyltransferase activity is independent of glucocorticoids (71,80).
Thyroxine administration to suckling rats raises fucose content and 125I-UEA-1 binding in MVM, but does not alter sialic acid content or 3H-fucose incorporation (81) and microsomal fucosyltransferase activity (80). These observations suggest an important role of thyroxine that should be studied further. Insulin in pharmacologic doses is known to stimulate intestinal differentiation and maturation in suckling mice (82). Jaswal et al. (83) showed that nutritionally deprived pups had low fucosylation level in MVM as a consequence of a delayed intestinal maturation induced by undernutrition. Insulin treatment of these pups induced adult-type fucosylation, whereas cortisone treatment was ineffective. However, the precise mechanism of insulin action needs further study.
Effect of Microflora on Glycosylation Processes in Intestine During Development
The human intestine contains a complex and dynamic society of nonpathogenic bacteria. At birth, colonization of the previously germ-free human gut begins. Normally, the first microbes to be established are derived from the mother during delivery and subsequently from other external environments. It is estimated that there are 1014 cells associated with the human body and that 90% of these are micro-organisms, most of which reside in the colon (84). The human colon contains at least 1011 organisms per milliliter of luminal contents. These organisms that normally live in the intestine are collectively referred to as the intestinal microflora, which contains more than 400 bacterial species. The intestinal microflora is an active metabolic entity that provides essential products to the host such as vitamins K and B12. The microflora also provides colonization resistance-a first line of defense against invasion by exogenous pathogenic organisms or indigenous opportunists (49,85). Comparisons between mice that have been conventionally raised with a normal microbiota and those that have been maintained in a germ-free state have shown that maturation of the intestine is also affected by interactions with its resident microbiota (86). Without a microflora the rate of epithelial cell renewal is reduced in the small intestine, the cecum becomes massively enlarged, and the composition and spatial organization of the diffuse gut-associated lymphoid tissue (GALT) are altered (87).
Understanding the interaction between enterocytes and microbes requires that the intestine's microbial ecology be controlled in a special manner. The ability to raise mice under germ-free conditions (i.e., without any resident micro-organisms) provides a critical technology for manipulating and simplifying this ecosystem. Germ-free mice can be colonized by one or more species of bacteria, thus becoming gnotobiotes. Umesaki et al. (88,89) found that colonization of germ-free BALB/c mice with a slurry of fecal micro-organisms obtained from conventional mice leads to the induction of guanosine diphosphate (GDP)-fucose asialo-GM1 α1,2-fucosyltransferase activity in the epithelium, increased expression of the neutral glycolipid, fucosyl asialo-GM1, and corresponding decrease in asialo-GM1. Colonization with a subset of indigenous intestinal microbes, segmented filamentous bacteria, recapitulates these biochemical changes in the host and also restores some features of the diffuse GALT found in mice with a complete conventional microflora (87).
Recently, a simplified model system has been created to study the interactions between a single genetically manipulatable bacterial component of the normal microflora and the mouse small intestinal epithelium (69,90). Bry et al. (69) demonstrated that developmental variations in the pattern of binding of UEA-1, a fucα1,2Gal-specific lectin, to the ileum between the developing germ-free and conventional NMRI inbred mice. In both groups of mice, these fucosylated structures are not detectable in the enterocytes before postnatal day 17, and a few scattered ileal villus enterocytes acquire the capacity to express fucα1,2Gal epitopes between postnatal days 17 and 21. A divergent pattern of fucosylated glycoconjugate production then appears between postnatal days 21 and 28. In germ-free mice, cellular production of these fucα1,2Gal structures is extinguished and remains suppressed thereafter; whereas, in conventional mice, production increases dramatically, generalizing to involve all ileal villus enterocytes. These findings indicate that the initiation of fucosylated glycoconjugate production in small intestine occurs independently of its microflora but that the microflora is required to complete the fucosylation program.
Inoculation of an intact normal flora to adult germ-free NMRI mice reinitiates and maintains a "conventional" pattern of fucα1,2Gal glycoconjugate production. Reinitiation can be engineered at any time after postnatal day 28 (69). Conventionalization of germ-free mice also results in transcriptional activation of a host GDP-L-fucose:β-D-galactoside 2-α-L-fucosyltransferase gene in the ileum (69). Remarkably, a single member of the autochthonous microflora can recapitulate these changes. Bacteroides thetaiotaomicron is a gram-negative anaerobe and a prominent component of the normal intestinal microflora in both mice and humans (91). Adult germ-free NMRI mice monocontaminated with this organism show the same changes in expression of fucosylated glycoconjugates that are seen in animals inoculated with a complete conventional microflora (69). This appears to be a specific response of the intestinal epithelium to bacterial colonization. By contrast, no changes are observed when mice are colonized with several other autochthonous bacterial species, including Peptostreptococcus micros(92) and Bifidobacterium infantis(93). These observations suggest a specificity of cross-talk between enterocytes and microbes that remains to be elucidated.
There is a direct correlation between the number of viable B. thetaiotaomicron cells in the ileum and the extent of induction of UEA-1-reactive fucosylated glycoconjugates (69). A minimum of 104 bacteria per milliliter are required to elicit a "patchy" fucose phenotype; 107 bacteria per milliliter fully recapitulates the "conventional" pattern. Colonization of the duodenum with B. thetaiotaomicron is less efficient than in the ileum. Nonetheless, levels of bacteria sufficient to induce fucosylation in the ileum (106 to 107 colony-forming units/ml) are unable to do so in the duodenum, demonstrating the organism's capacity to reproduce a region-specific conventional pattern of fucosylated glycoconjugate production. The correlation between this host response and microbial density is consistent with several possible mechanisms: a direct binding interaction between bacterial cells and host intestinal epithelium that must reach a critical threshold before induction can occur, secretion of a soluble bacterial factor that causes a concentration-dependent host response, and/or density-dependent changes in the metabolic properties of the bacteria that modulate their ability to influence host epithelia metabolic pathways (90). No direct binding interaction between B. thetaiotaomicron and the intestinal epithelium has been observed (69). These results suggest that the dependence of induction on bacterial density is the result of secretion of a soluble microbial factor, or density-dependent changes in bacterial metabolism-signal transduction, or a combination of the two (90).
A basic understanding of host-microbial interactions in the mammalian gut will ultimately yield new strategies for the prevention and treatment of some infectious diseases in humans. Live microbial supplements, probiotics, have been used to treat certain infections, such as C. difficile-induced pseudomembranous colitis that occurs when patients are given broad-spectrum antibiotics (94), and restore the normal microflora during antibiotic therapy (9). Whether probiotics affect microbial receptor expression in the intestine is unknown. Mechanisms by which probiotics affect bacterial colonization are not well understood yet, but at least three mechanisms of action have been observed: 1) production of antimicrobial substances (bacteriocins), 2) specific competition for adhesion receptors, and 3) stimulation of mucosal immune responses (9). In the future, probiotics may also be used to prevent opportunistic infections.
SUMMARY AND CONCLUSIONS
The attachment of microbes to carbohydrate moieties on the host cell surface is considered essential for successful colonization and infection. Adding sugars to MVM membrane proteins and lipids (glycosylation) is an important host determinant in microbial colonization of the intestine. The enzymes responsible for glycosylation are glycosyltransferases. Our studies and others have shown that glycosyltransferases and microbial receptors are under developmental regulation. Intrinsic genetic control, hormones (glucocorticoids, insulin and thyroxine), and external factors (diet and bacterial colonization) may affect the ontogeny of these enzymes and the expression of microbial receptors. Therefore, the developmental control of microbial receptors in the gastrointestinal tract may in part contribute to the altered host sensitivity to intestinal infection in infancy. Probiotics and some trophic factors may also protect the gastrointestinal tract through differential glycosylation. In the future, it may also be possible to inhibit microbial attachment by blocking with oligosaccharides or glycoconjugates specific for the appropriate lectins.
The molecular nature of microbial receptors in intestinal epithelial cells underscores the importance of intestinal surface carbohydrate expression in host-microbe interaction. With improved techniques for characterizing receptor binding and the receptor's structure-i.e., the availability of several human intestinal models (organ culture of human fetus intestine, primary culture of human fetus intestinal epithelial cells, the H-4 cell line, and Caco-2 cell line) and of carbohydrate-specific monoclonal antibodies, we may identify additional membrane receptors and the receptor sugar sequences in the near future. We may isolate glycoconjugates from human intestinal tissue, then identify them structurally using mass spectrometry and nuclear magnetic resonance spectroscopy. We may test the binding of microbial ligands to epithelial surfaces with glycoproteins or glycolipids. Subsequent studies on the intestinal expression and developmental regulation of individual glycosyltransferases can then be pursued. Recently, a transgenic mouse model has been used to study Helicobacter pylori infection, in which the receptor, the primate-specific Lewisb(95), was expressed in the mouse gastrointestinal tract by transfection with a human α-1,3/4-fucosyltransferase (96). In the future, the potential transgenic animal models by transfection with constructs for specific glycosyltransferase(s) will be used to examine the role of oligosaccharides and glycoconjugates in regulating cellular differentiation and the host-microbe interaction. In the same manner, the use of molecular and cell biologic techniques in intestinal cell lines and in primary cultures of human enterocytes or organ culture of human fetal, neonatal, and adult intestine will help to determine the relationship between developmental regulation of intestinal microbial receptors and postreceptor-effector events. By understanding the molecular nature of microbial receptors and their effector responses in the intestine, the developmental programming and environmental influence on receptor expression, and the effector response and the biologic significance in neonatal host defenses, new approaches may soon be available in the prevention and treatment of infants with infectious intestinal diseases of various origins.
Acknowledgments: This work was supported by grants HD12437, HD31852, PO1-DK33506, HD13021, and P30-DK40561 from the National Institutes of Health (Bethesda, Maryland, U.S.A.); Dr. Dai was supported in part by Nestlé's Nutrition Research Fellowship (Nestec Ltd., Vevey, Switzerland).
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