Oligosaccharides in Human Milk and Bacterial Colonization

Field: Medicine (Health Sciences)

In the first year of life, a stable population of microflora is established in the human gut as a complex ecology that includes the intestinal mucosa and more than 400 interdependent species of bacteria. During this period, a child is vulnerable to enteric and other diseases, but breast-fed infants have a lower rate of morbidity and mortality than do artificially fed infants. The lower incidence of disease in infants while they are breast-feeding has been attributed to many causes: less exposure to contaminated water, better nutritional status, and psychological factors. Although these factors probably contribute, the most direct determinant of reduced disease incidence among the breast-fed seems to be factors in the milk itself. This protection is thought to result from two major kinds of components in human milk: those that provide a prebiotic effect that enhances colonization by Bifidobacterium bifidum, and others that inhibit the colonization and growth of pathogens during this vulnerable period. Three major classes of human milk components are known to inhibit pathogens: secretory antibodies; multifunctional agents, such as fatty acids and lactoferrin; and homologues of host cell surface glycoconjugates. Evidence is mounting that these homologues, consisting of glycoconjugates and oligosaccharides, have a major role. Glycoconjugates are molecules that have conjugated carbohydrate moieties (glycoproteins, glycolipids, glycosaminoglycans, and mucins); milk oligosaccharides have carbohydrate moieties conjugated to lactose. These milk carbohydrate moieties often share structural motifs with the cell surface glycoconjugates that pathogens adhere to when gaining entry to host target cells. Indeed, specific human milk oligosaccharides and glycoconjugates have been shown to block binding of specific pathogens to host cell glycoconjugate receptors. This article presents three examples: 1) A fucosylated oligosaccharide from human milk inhibits the pathogenicity of stable toxin of Escherichia coli in vivo, and the blocking effect has been demonstrated in vitro. 2) Fucosylated structures also inhibit binding by Campylobacter jejuni to host cell receptors and limit its colonization in vivo. 3) A glycoprotein, lactadherin, inhibits rotavirus in vitro and in vivo. High concentrations of lactadherin in human milk are associated with reduced incidence of symptoms of rotavirus infection in nursing infants. These findings, along with other examples of pathogen inhibition by milk glycoconjugates, suggest that this is a general mechanism of protection by human milk. Furthermore, human milk oligosaccharides survive transit through the intestine of the breast-feeding infant, implying that they are available to protect against gastroenteritis over the entire length of the intestinal tract. However, pronounced individual variation in the expression of oligosaccharides in human milk suggests heterogeneity in the ability of individual human milks to inhibit specific pathogens. Oral supplementation of vulnerable infants with glycoconjugates, oligosaccharides, or their homologues may be feasible. The milks of other species also contain homologous oligosaccharides and glycoconjugates, suggesting that this mechanism of protection in early life may be common to many mammals. The presence of a large spectrum of oligosaccharides and glycoconjugates in human milk may represent a major mechanism whereby breast-feeding protects infants from enteric and other pathogens during the period when the microflora of the gut are becoming established. BREAST-FEEDING PROTECTS INFANTS AGAINST DISEASES Evidence that breast-feeding protects infants against diseases during their vulnerable first year of life was first convincingly reported from the Massachusetts General Hospital in 1935 (1). In this study of 20,000 patients, breast-feeding was associated with a lower incidence of morbidity and mortality, especially of enteric disease, otitis media, and respiratory infection. Although subsequent studies yielded mixed results regarding the efficacy of breast-feeding against these diseases, more recent studies using relatively sophisticated experimental design and data analysis have yielded findings that mostly support the earlier report. A noteworthy example is a study performed in Lahore, Pakistan (2). The population was divided into rural poor, urban poor, and urban upper middle class; the efficacy of breast-feeding against enteric disease was determined by the same type of analysis used for testing the efficacy of drugs (Fig. 1). Breast-feeding was effective in preventing disease among all three populations, with the protection most pronounced in the first 6, and especially in the first 3, months of life. This protection was most pronounced among the poor but was also clearly demonstrated in the upper middle class, whose socioeconomic status and level of hygiene is closer to that of many western societies. A prospective study in a Scottish population on the effect of breast-feeding on morbidity and mortality due to enteric diseases (3) showed that even in developed countries breast-feeding during the first 3 months of life can be demonstrated to confer protection against gastrointestinal illness. Curiously, in this study the benefits persisted beyond the period of breast-feeding itself. This phenomenon is explicable if breast-feeding contributes to establishing a healthy ecology of the gut, the ramifications of which extend beyond weaning.FIG. 1: Efficacy of breast-feeding in preventing enteric disease was high in both rural (village and mud hut) and urban (slum) poor, especially during the first 6 months of lactation, and the protection persisted for the 2-year duration of this study. Note that breast-feeding was also efficacious in preventing enteric disease, albeit to a lesser extent, in the upper middle class, a population whose living conditions are closer to those of developed countries. (Reprinted, with permission, from Kluwer Academic/Plenum Publishers. Hansen et al. Adv Exp Med Biol 1991;310:1-15).COLONIZATION BY MICROFLORA The adult human intestine represents a complex ecosystem of more than 400 species of bacteria, interdependent among themselves and with the human gut per se. The human intestinal microflora typically contains more cells than the entire number of cells in the human body. These intestinal bacteria have many functions, such as digesting food components that are indigestible by human intestinal enzymes, and can be a source of important factors such as vitamins. Moreover, a stable, healthy intestinal microflora is thought to contribute to overall health by excluding foreign, potentially harmful bacteria (4). The formation of a stable, interdependent ecosystem of intestinal microflora during the first year of life requires selective acquisition from among the many bacteria in the environment. Important environmental sources of bacterial exposure include the mother and other individuals with whom the infant has intimate contact (5). The colonization of the gut by new bacteria is influenced by the current resident bacteria. Moreover, colonization by any new strain of bacteria is influenced by intestinal epithelial cells, which in turn are affected by the bacteria that colonize the gut, as is discussed in greater detail in the review in this issue by Walker. Diet greatly influences the selection of intestinal microflora during this first year of life. For example, in the gut of the breast-fed infant, bifidobacteria predominate and enterobacteria are present (6), whereas in the artificially fed infant, bifidobacteria, bacteroides, clostridia, and streptococci are dominant (7). Bacteroides dominate the adult flora (8). Human milk, normally the principal food during this first year of life, contains factors that stimulate the growth of commensal bacteria. Prebiotics (9) enhance colonization by specific bacteria; for example, bifidus factor stimulates growth of Bifidobacterium bifidum (formerly called Lactobacillus bifidum) (10,11). Lactose affects colonization directly, by selecting for bacteria able to use this unique sugar, and indirectly, when fermented to acetate, making the gut acidic and stimulating the growth of some bacteria while inhibiting colonization by many pathogenic organisms. Human milk also contains three types of components that inhibit colonization by harmful bacteria. One is the secretory immunoglobulin A (sIgA), which is produced by the immune system of the mother against enteric pathogens to which she has a history of exposure (12). Another type is the multifunctional agents that serve as nutrients while protecting against a broad spectrum of pathogens. Examples are fatty acids that, when released from the triglycerides of milk in the stomach of the infant (and in expressed human milk that has been stored) destroy envelope viruses (13) and also act against other pathogens such as Giardia(14,15). Lactoferrin, the major protein in human milk, is active against a broad spectrum of pathogenic bacteria, and its digestion products include lactoferricin, a porin that is toxic to many strains of bacteria (16). The third class of milk components that are active against pathogens is molecules containing structural carbohydrates, the glycoconjugates and oligosaccharides. The oligosaccharides are complex carbohydrate structures that are generally attached to lactose (17). The glycoconjugates contain complex carbohydrate structures that are attached to proteins, lipids, and other structures (e.g., glycoproteins, glycolipids, mucins, and glycosaminoglycans). The carbohydrate portion of these molecules is largely indigestible and passes through the intestinal tract of the infant mostly intact. These carbohydrate structures potentially act as decoys to which pathogens bind. It is this fraction of human milk that will be the focus of this review. HUMAN MILK OLIGOSACCHARIDES Oligosaccharides are, collectively, the third largest solid constituent in human milk, after lactose and fat. Human milk oligosaccharides consist of lactose at the reducing end, with a carbohydrate core that often contains a fucose or a sialic acid at the nonreducing end (17). When they were initially discovered and studied, these human milk oligosaccharides were thought to be the accidental consequence of the high levels of glycosyltransferases in mammary epithelial cells (required for formation of the abundant milk glycolipids and glycoproteins) in proximity to the high levels of lactose unique to these cells. However, the idea that these oligosaccharides may have a biologic purpose is supported by two lines of evidence in relatively recent investigations. First, it was discovered that the approximately 100 milk oligosaccharides that had been isolated and characterized represent only a very small portion of the total that remain to be characterized. The potential for many thousands of fucosylated oligosaccharides in human milk was suggested when the total human milk oligosaccharide fraction was analyzed by time-of-flight mass spectrometry: Evidence was found for the existence of oligosaccharides with up to 32 sugars and up to 15 fucoses (18). The huge potential for structural isomers suggests that there may be tens of thousands, or perhaps even hundreds of thousands, of potential human milk oligosaccharide structures. Such a large number of possible structures greatly increases the probability that some are biologically active. A second, independent line of investigation has identified several biologic functions attributable to the milk oligosaccharides. Oligosaccharides have been found in human milk that inhibit the adherence of Streptococcus pneumoniae to target cells in vitro (19). Human milk oligosaccharides also inhibit binding by enteropathogenic E. coli(20), binding and infection by invasive strains of C. jejuni(21), and the toxicity of stable toxin of E. coli(22). The findings from both lines of investigation, taken together, suggest that many more biologically active oligosaccharides remain to be discovered. MAMMALIAN CELL SURFACE GLYCOCONJUGATES ARE PATHOGEN RECEPTORS Mammalian cell membranes contain glycoproteins and glycolipids, and the membrane structure is such that the oligosaccharide moieties of these cell surface glycoconjugates protrude into the extracellular region (Fig. 2). These complex carbohydrate moieties participate in cell-cell interactions and can act as receptors for humoral mediators such as hormones. They can also act as receptors for antibodies, toxins, and pathogens. They are the usual sites where viruses and bacteria adhere to the host target cell to initiate the pathogenic process.FIG. 2: Cell surface interactions of glycoconjugates. Glycoconjugates (glycolipids, glycoproteins, and others) can participate in cell-cell interactions (top). These glycoconjugates can also bind to humoral mediators and can participate in transmembrane signaling (left). These same cell surface glycoconjugates can be used by pathogens (viruses, bacteria, and toxins) for recognizing, binding to, and gaining entry to host cells (right) as the essential first step in pathogenesis.The glycosyltransferases that make human milk oligosaccharides are similar to the glycosyltransferases that synthesize the oligosaccharide moieties of mammalian cell surface receptors. Thus, the milk oligosaccharides are likely to have some structural motifs similar to those found at the cell surface. These human milk oligosaccharides can therefore act as soluble homologues to the cell surface receptors that are the targets of specific pathogens. Serving as decoys, they bind to the pathogens and inhibit their ability to bind to the host cells. Because pathogen binding to the host is an essential first step in pathogenesis, specific milk oligosaccharides can protect against disease. Three examples will be discussed in which specific human milk components inhibit specific pathogens; two of the components are oligosaccharides, and one is a glycoprotein. Stable Toxin of Escherichia coli Infants born in areas where enterotoxigenic E. coli are endemic are at high risk for diarrhea caused by these organisms. E. coli produce both a labile toxin, which resembles cholera toxin in that it binds to ganglioside in the host cell surface, and a stable toxin, which binds to the extracellular domain of guanylate cyclase in intestinal mucosal cells, initiating a different cascade of pathogenic events resulting in secretory diarrhea. Human milk significantly reduced mortality rates in suckling mice given stable toxin of E. coli(22). The protective activity was found to reside solely in the neutral oligosaccharide fraction of pooled human milk and was active at its native concentration. When this neutral oligosaccharide fraction was passed over a Ulex europaeus lectin-affinity column, the activity was found exclusively in the fucosylated oligosaccharide fraction that bound to Ulex(23). When the active fucosylated fraction was separated into its component peaks on an amino column by high-performance liquid chromatography (HPLC), only one of the minor peaks consistently inhibited the diarrhea caused by stable toxin in vivo. When this peak was separated into its components by reversed-phase HPLC, the activity was found exclusively in one of its minor peaks. This peak was isolated to homogeneity and, at 2.5 times its nominal concentration in human milk (to take into account losses in its purification), this fraction inhibited stable toxin in vivo as effectively as the milk from which it was isolated (Table 1). This fraction is present in human milk at a concentration of approximately 40 parts per billion. Its high level of activity at such a low concentration suggests high degrees of specificity and avidity for its receptor.TABLE 1: Total and fucosylated milk oligosaccharides inhibit 125I-ST binding to T84 membranes An in vitro system commonly used to study stable toxin was used to determine the mechanism by which the fucosyloligosaccharide fraction may protect against diarrhea induced by stable toxin. T84 cells, an immortal line descended from intestinal mucosa, were grown under conditions that induce the expression of many characteristics of intestinal mucosal epithelial cells: tight junctions, polarity, expression of microvilli on the mucosal surface, and expression of guanylate cyclase among these microvilli. Guanylate cyclase is a transmembrane protein with an extracellular domain that binds to stable toxin, activating the intracellular domain, and thus increasing the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP; Fig. 3). Elevated levels of cGMP result in increased efflux of chloride ions from the cell, accompanied by an efflux of sodium ion and water. This movement of salts and water to the lumen of the gut is the secretory diarrhea induced by stable toxin. The inhibitory fucosyloligosaccharide binds to a site on the extracellular domain of the guanylate cyclase of T84 cells, blocking binding by stable toxin (24). This seems to be the mechanism whereby this oligosaccharide prevents the stable toxin from inducing secretory diarrhea.FIG. 3: The mechanism whereby a milk oligosaccharide inhibits the ability of stable toxin to cause secretory diarrhea. When stable toxin binds to the extracellular domain of guanylate cyclase, the intracellular enzymes are activated, causing an increase in intracellular cyclic guanosine monophosphate (cGMP) and loss of chloride from the cell. The efflux of chloride along with its counter ion, sodium, and water produce secretory diarrhea. By preventing stable toxin from binding to its extracellular receptor, homeostasis is preserved and secretory diarrhea avoided, despite the presence of stable toxin.Campylobacter Campylobacter jejuni, a common pathogen of birds, also infects humans. In areas where domestic fowl live in proximity to in contact with the or their are at high risk for Campylobacter diarrhea early in life. Campylobacter infection is often in cells in vitro. Human milk inhibits the ability of C. jejuni to cells. This activity in the oligosaccharide fraction of human milk and in the fucosylated oligosaccharides that bind to a europaeus column the concentrations found in human milk, these oligosaccharides inhibit the of pathogenic invasive strains of C. jejuni to cells, but the of A was used to the inhibition of C. jejuni binding in vitro into an inhibition of infection in vivo. of mice were given or high in the or the presence of the active human milk oligosaccharide that, at both the presence of the oligosaccharide fraction in several less colonization than in the Thus, milk fucosylated oligosaccharides only inhibited binding in vitro but infection in Human milk oligosaccharides inhibit the ability of Campylobacter to colonize the of both bacteria per and bacteria per the ability of Campylobacter to colonize the gut is inhibited by the presence of human milk oligosaccharides at one the concentration found in human findings provide two examples of human milk oligosaccharides that inhibit human pathogens. The of these human milk glycoconjugates to the expression of disease in a breast-feeding human population has been for Campylobacter or stable E. However, this issue was in a third example of a human milk glycoconjugate that inhibits a specific lactadherin inhibition of and is the most common cause of especially in the developed where infection by bacteria and other is less The ability of rotavirus to cells in vitro is inhibited by human milk. of this inhibitory activity in the protein fraction and has been isolated to a The inhibition is general for all major strains of human rotavirus and is of sialic acid from this its inhibitory suggesting that a sialic acid attached to a specific carbohydrate of this is for the inhibitory The lactadherin binds to blocking its ability to bind to its host target cell This ability to inhibit rotavirus infection and its diarrhea has also been demonstrated in vivo in a The the lactadherin levels in milk and presence of diarrhea in their infants was determined in a population of nursing of the infants were to of milk were for lactadherin and the infants were for symptoms of diarrhea. the infants were with those with diarrhea were milk with significantly lower levels of lactadherin than those were was levels in milk and diarrhea among These data suggest that the expression of lactadherin in the milk among nursing and that the level of lactadherin expressed is significantly to the presence of diarrhea in breast-fed concentrations in milk are associated with less disease in breast-fed infants. In breast-feeding infants with those infants whose milk was high in lactadherin were less likely to symptoms of rotavirus than were those infants whose milk low levels of This that lactadherin, which binds to rotavirus and inhibits its ability to cells in vitro and to cause diarrhea in also inhibits the of diarrhea in seems likely that large of lactadherin bind to most of the receptors for host cell surface glycoconjugates, blocking the first step in However, when only a of lactadherin is as in produce low levels in their milk, or when lactadherin is as in infants are artificially the has receptors that are able to bind to intestinal mucosal cells and initiate GLYCOCONJUGATES It is noteworthy that in the study of lactadherin and rotavirus the lactadherin and disease was whereas the and disease was This that protection by glycoconjugates of human milk may be at as important as protection by Although in human milk were discovered early and are as protective agents they to represent only one mechanism whereby human milk protects infants. The glycoconjugates and oligosaccharides confer protection at the concentrations found in human milk, probably the large number of possible structures of these milk specificity and potential ability to protect host cells from a of pathogens. It is to that human milk for the protection of infants. The immune of infants are microflora is and is and other of against pathogens are Thus, human milk may be important to the of infants from disease and may therefore have protection MILK OLIGOSACCHARIDES The ability of individuals to glycoconjugates and oligosaccharides in human milk as in other and such as have been to oligosaccharide expression in human milk, but oligosaccharide expression is to variation among individual The two and are shown in The in is by a different than the in to all human milk oligosaccharides, these contain lactose at the reducing can be in the and shown in These also have lactose at the reducing end, but the fucose at the nonreducing end is attached by an or the is by one type of whereas the are by at one different Because the expression of is known to be glycoconjugates in human milk and are as This variation of fucosylated oligosaccharides in of human milk has to be of the two in human milk. In these fucose is attached to lactose by an or an of these is by a different The structures of three of human milk. These structures several common to They contain lactose at the reducing end (right) and contain fucose at the nonreducing end of the in an an or an The is by whose is independent from that synthesize the or of oligosaccharides among milk from different was by an that oligosaccharides of sugars or as peaks. This for more than by of human milk oligosaccharides variation was found among expression of glycoconjugates in their In the the are but in some the evidence of any the and This suggests expression of protective oligosaccharides among individual on their of The expression of oligosaccharides over the of was the milk from a given The overall concentration of oligosaccharides over the of whose milk oligosaccharide a of to produce such milk for the first 6 months of those whose milk had produced milk that was in these oligosaccharides during the first 6 months of However, 6 months and year of lactation, the milks from both classes of to contain both types of oligosaccharides, suggesting some in the mechanism of oligosaccharide in These are with the that human milk seems to its protection against disease during the first 6 months of These data also suggest a ability to protect against specific pathogens by breast-feeding whose expression of This phenomenon is a of current MILK OLIGOSACCHARIDES The oligosaccharides of human milk represent a major of by the One a mechanism for their digestion that this be the other if the of these oligosaccharides is protection from disease, they be to remain as they through the to protect even of the the of the milk oligosaccharides after by breast-fed the oligosaccharide of the and of the breast-fed infants were with those of the milk from their The of oligosaccharides in the and of the breast-fed infants resembles that in their milk, suggesting that their is human milk. The and of breast-fed infants were also to the and of infants. Oligosaccharides in the and of artificially fed infants have a different from milk and from the and of breast-fed infants. The oligosaccharides in of artificially fed infants probably from of intestinal epithelial cells and from bacteria. oligosaccharides in these infants may from of cells, from the of intestinal oligosaccharides by their into the The of oligosaccharides in the and of breast-fed infants are than those in infants (Table 2) that they may any oligosaccharide The concentration of milk oligosaccharides in the of breast-fed infants as than in the milk they are the other milk components are and as they through the gut, whereas the oligosaccharides remain and increase in 2: concentrations in milk, and of breast-fed and infants An of and of oligosaccharides, on for the of milk and for and suggests that the of oligosaccharides by breast-fed infants is in the whereas a small is and in the It is to that the small of oligosaccharides in the some protection against tract The increased concentration of oligosaccharides at the end of the that they are present the intestine in concentrations to protect against pathogens. These results also the that the of the oligosaccharides in milk is protective than HUMAN MILK GLYCOCONJUGATES INFANTS The three examples of protective oligosaccharides and glycoconjugates in human milk that have been discussed are of many classes of glycoconjugates found in human milk, oligosaccharides, glycoproteins, mucins, glycosaminoglycans, and glycolipids (Table 3). These all have in common a complex carbohydrate as of their structure and the ability to inhibit binding of pathogen to its host cell surface The of glycoconjugates in human milk that protect nursing infants from specific pathogens is evidence that the glycoconjugates in the may represent a major mechanism of human milk in protecting the infant from 3: Human milk inhibitory glycoconjugates The of human milk in includes three of in to nutrients and These three include the produced by the mother against of pathogens to which she has a history of exposure and into the milk. A line of is the multifunctional agents, which may serve as nutrients but also have that can inhibit a broad of pathogens. Glycoconjugates the third line of functions of human milk components These of are probably all important components of a For example, the glycoconjugates inhibit specific whereas the multifunctional agents to have a these of human milk be as a line of during the period that the exposure to a pathogen and the of secretory against it in milk. the other glycoconjugates be the line of against enteric pathogens. The data human milk lactadherin levels and to symptoms of rotavirus support this serve a essential protection from disease if the of the mother the of a to produce a specific milk component that inhibits a specific It is also possible that the of these three of protection for different pathogens. This of agents in human milk that inhibit common diseases in infants may a in in the study of human milk. than of human milk as a food with some antibodies, perhaps it is more to of it as a of biologically active protective agents that also nutritional Thus, milk may protect the infant from harmful pathogens during the first year of life, the immune system is and a stable microflora has been while an that stimulates the growth of commensal bacteria. These provide for health of breast-feeding in both and developed countries. Furthermore, the of the milk oligosaccharides in and during intestinal transit suggest that supplementation of the native oligosaccharides or their homologues may be feasible. by from the of and Human of