HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/1/44 02-0031fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by NANTHAKUMAR, N. N. Articles by WALKER, W. A. Search for Related Content PubMed PubMed Citation Articles by NANTHAKUMAR, N. N. Articles by WALKER, W. A.

The role of indigenous microflora in the development of murine intestinal fucosyl- and sialyltransferases¹

N. NANDA NANTHAKUMAR^*, DINGWEI DAI^*,, DAVID S. NEWBURG and W. ALLAN WALKER^*2

^* Developmental Gastroenterology Laboratory, Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA;
Shanghai Institute for Pediatric Research, Xinhua Hospital and Shanghai Second Medical University, China; and
Program in Glycobiology, Shriver Center for Mental Retardation, Waltham, Massachusetts, USA

²Correspondence: Developmental Gastroenterology Laboratory, Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, 114 16th St. (114–3650), Charlestown, MA 02129, USA. E-mail: wwalker{at}partners.org

SPECIFIC AIMS

The ratio of sialic acid to fucose on rodent brush-border glycoconjugates reverses from a predominance of sialic acid to fucose during postnatal development; this inversion is attributed to a reciprocal developmental change in the level of fucosyl- and sialytransferase activities. Since enteric bacteria use intestinal brush-border glycoconjugates as their target host cell receptors, we hypothesized that initial bacterial colonization of the rodent gut may help regulate those specific glycosyltransferases responsible for synthesizing brush-border glycoconjugates. To investigate this hypothesis, glycosyltransferase enzymes activities were measured in mucosal microsomal fractions from different intestinal regions of maturing conventional (CONV), germ-free (GF), and ex germ-free (XGF) mice and compared with general enzyme markers of gut development (e.g., disaccharidases).

PRINCIPAL FINDINGS

1. Age-dependent and tissue-specific changes in fucosyl- and sialyltransferase and disaccharidase activities in the gut of conventional and germ-free mice
To determine regional differences during postnatal development of murine intestinal glycosyltransferases, enzyme activities were measured in the duodenum, jejunum, ileum, and colon. CONV mice expressed very little 1,2-fucosyltransferase (FT) activity during the first 2 wk, reaching an adult level by the fourth week, with an increasing gradient from the proximal (duodenum) to distal (colon) gut. However, FT activity in GF mice (Fig. 1 A) was unchanged in the gut throughout the 4 wk of study.

View larger version (46K): [in this window] [in a new window]   Figure 1. Specific activities of A) 1,2-fucosyltransferase (nmol·mg protein-1·h-1); B) 2,3/6-sialyltransferase (nmol·mg protein-1·h-1), C) sucrase (µmol·mg protein-1·h-1), and D) lactase (µmol·mg protein-1·h-1) were measured in the developing ileum of GF and CONV mice up to 6 wk of age. Each bar represents the mean ± SE of three samples. Samples from 1- and 2-wk-old suckling mice were pooled, 6–8 mice per sample; samples from older mice were pooled, 3 mice per sample. *P < 0.05 vs. CONV group; **P < 0.001 vs. CONV group.

Suckling CONV mice displayed an increasing gradient of 2,3/6-sialyltransferase (ST) activity along the proximal to distal axis with highest activity in the ileum (Fig. 1B ) and colon. In the small intestine, however, a developmental decline began in the ileum (Fig. 1B ) by the second week and extended to the proximal gut by wk 4 in CONV and GF mice. Though the significantly high level of ST activity in GF mice of all ages suggests a specific role for microbes, they do not regulate the developmental decline (Fig. 1B ). There were no changes with either age or microbial status in the colon.

Using sucrase and lactase activities as general markers of mature and immature intestine, respectively, the intrinsic program of postnatal development of the small intestine was examined (Fig. 1C, D ). Ontogenic changes in sucrase and lactase occurred during the third week in GF and CONV mice, reaching adult levels by the fourth week. The absence of luminal microbes had no effect on level of activity or the ontogeny of these two enzymes.

2. Intestinal glycosyltransferase activities in mature CONV, GF, and ex GF mice
FT and ST activities of XGF mice (GF mice colonized at 4 wk of age with intestinal microflora from age-matched CONV mice) approached levels found in CONV mice of the same age, but differed significantly from GF mice. FT activity throughout the gut (Fig. 2 A) was significantly higher in XGF and CONV than GF mice, which maintained the suckling phenotype. CONV and XGF mice both displayed an increasing gradient of FT activity from the proximal to distal intestine. ST activity was significantly lower, but indistinguishable between XGF and CONV mice, compared with GF mice except in the colon (Fig. 2B ). To confirm enzyme studies, expression of sialic- and fucose-containing glycoconjugates was analyzed by lectin immunofluorescence using tissues from CONV, GF, and XGF mice. The pattern of fucosyl and sialyl glycoconjugates paralleled the activity of their respective glycosyltransferases. Compared with CONV and XGF mice, fucosylated glycoconjugate expression was very low in the GF mice in all regions, with a specific reduction in goblet cells. This observation was most pronounced in the colon, suggesting that luminal bacteria may specifically regulate fucosylation of mucin in goblet cells whereas sialic acid-containing glycoconjugates were significantly enhanced in GF mice, again more prominent in the goblet cells but only in the small intestine. However, colonic expression of sialyl-glycoconjugates was unaffected by GF status, suggesting that microbes may down-regulate sialylated mucin in goblet cells but only in the small intestine.

View larger version (58K): [in this window] [in a new window]   Figure 2. Effects of indigenous microflora on A) 1,2-fucosyltransferase (nmol·mg protein-1·h-1); B) 2,3/6-sialyltransferase (nmol·mg protein-1·h-1), C) sucrase (µmol·mg protein-1·h-1), and D) lactase (µmol·mg protein-1·h-1) of conventional (CONV), germ-free (GF), and ex germ-free (XGF) mice. The specific activities of these enzymes were assayed in all 4 regions of the gut of 6-wk-old mice. Each region of gut tissue from 6 mice per sample was analyzed. Each bar represents the mean ± SE of three samples. *P < 0.05 vs. CONV and XGF group; **P < 0.01 vs. CONV and XGF group.

Both sucrase (Fig. 2C ) and lactase (Fig. 2D ) activities were unaffected by the GF status of the mature intestine while maintaining a bell-shaped proximal to distal gradient at 6 wk of age. In all regions, the activities of these two enzymes in GF mice were not significantly different from those in CONV or XGF mice. These observations suggest that luminal microflora may alter the expression of intestinal glycosyltransferases but does not affect other more generalized developmental parameters such as sucrase and lactase. Two weeks after the reintroduction of microflora, FT and ST in mice reached mature levels. These observations suggest that bacterial-epithelial cross-talk may contribute to the level of glycosylation found in conventional adult mice via developmental changes in these enzyme activities over the period of initial colonization.

CONCLUSIONS AND SIGNIFICANCE

Our results indicate that the changing luminal microflora during initial colonization affects the ontogeny of fucosyltransferase in a region-specific manner in the mouse gut. During postnatal development, FT activity increases markedly in both the small and large intestine, but is inversely related to ST activity only in the small intestine. These coordinated changes might be part of a developmental program designed to modify surface glycoconjugates by mechanisms that are not yet fully understood. Such modifications may mediate some of the striking structural and functional changes seen in the intestine during postnatal development and could influence surface colonization of resident bacteria.

In CONV animals, immature glycosyltransferase patterns (high ST and low FT activities) are relatively stable for the first 2 wk, when microflora of the mouse gut are thought to be relatively constant. On weaning, the microbial ecosystem undergoes changes that coincide with oncogenic changes in FT and ST activities. A developmental increase in FT is not seen in GF mice. A developmental decline of ST is unaffected by microflora, but the level of activity is significantly reduced by the presence of luminal bacteria. With the introduction of conventional microflora (i.e., XGF mice), the activities of developmental markers returned to the mature levels of age-matched CONV mice. These observations suggest that microflora are an important extrinsic factor that regulate specific aspects of the developing gut of the mouse (summarized in Fig. 3 ). However, the mechanism by which bacterial-epithelial cross-talk alters the expression of glycosyltransferases in the gut in a region-specific manner is not known. The novel finding of this study is that changes in gut physiology during postnatal maturation are not controlled exclusively by an intrinsic genetic program. These findings have significant implication for the function of brush-border glycoproteins and glycolipids since this is the site for intestinal adaptation and colonization.

View larger version (26K): [in this window] [in a new window]   Figure 3. Intrinsic (genetic programs and endogenous hormones) as well as extrinsic regulators (microflora) control the postnatal development of the mouse intestine. Luminal bacteria are important extrinsic regulators of fucosyltransferases in the gut. In the absence of microflora (germ-free), the level of these glycosyltransferases is significantly affected and thus maintain an immature pattern of surface glycosylation. Unlike intrinsic regulators, the introduction of microbes initiates the developmental induction of fucosyltransferase and the level of sialyltransferase via an unknown mechanism.

Previous studies have investigated interactions between a single genetically manipulated bacterial component of indigenous microflora and the small intestinal epithelium of GF mice. Expression of fuc1,2Gal-specific glycoconjugates in the ileum was developmentally controlled but absent in GF mice. Innoculation of adult GF mice with a complete microflora or fucose using autochthonous bacteria, Bacteroides thetaiotaomicron, could reinitiate and establish the production of this specific glycoconjugate. Our studies shows that the immature pattern of sialyl/fucosyl ratio on brush-border glyconjugates is maintained through development in GF mice and the reintroduction of luminal microflora reverses this immature pattern to a mature pattern present in CONV mice via altering the level of expression of respective glycosyltransferases in a region-specific manner throughout the gut. However, it is not known whether a specific bacterium can induce changes in multiple glycosyltransferase activities by the same mechanism or requires introduction of commensal flora collectively.

A basic understanding of host-microbial interactions in the mammalian gut could lead to a better appreciation of normal development and of disease processes in the developing gastrointestinal tract, ultimately providing new strategies for prevention and treatment of infectious diseases in infants. For example, probiotics have been used to restore the normal microflora and treat infections such as Clostridium difficile pseudomembranous colitis, which result from an imbalance in resident microflora when patients are given broad-spectrum antibiotics. It is reasonable to hypothesize that probiotics protect the gastrointestinal tract by stimulating glycosyltransferase expression, thereby changing the glycosylation pattern of the intestinal mucosa and influencing bacterial adherence.

We conclude that glycosyltransferase activities in the intestinal mucosa of mice are under region-specific developmental regulation and that acquisition of indigenous microflora is an important regulatory factor in the process.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0031fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0031fje

This article has been cited by other articles:

				D. Meng, D. S. Newburg, C. Young, A. Baker, S. L. Tonkonogy, R. B. Sartor, W. A. Walker, and N. N. Nanthakumar Bacterial symbionts induce a FUT2-dependent fucosylated niche on colonic epithelium via ERK and JNK signaling Am J Physiol Gastrointest Liver Physiol, October 1, 2007; 293(4): G780 - G787. [Abstract] [Full Text] [PDF]

				N. N. Nanthakumar, D. Dai, D. Meng, N. Chaudry, D. S. Newburg, and W. A. Walker Regulation of intestinal ontogeny: effect of glucocorticoids and luminal microbes on galactosyltransferase and trehalase induction in mice Glycobiology, March 1, 2005; 15(3): 221 - 232. [Abstract] [Full Text] [PDF]

				C. Iordache, L. Drozdowski, M. T. Clandinin, G. Wild, Z. Todd, and A. B. R. Thomson Treatment of suckling rats with GLP-2 plus dexamethasone increases the ileal uptake of fatty acids in later life Am J Physiol Gastrointest Liver Physiol, January 1, 2005; 288(1): G54 - G59. [Abstract] [Full Text] [PDF]

				N. N. Nanthakumar, C. Young, J. S. Ko, D. Meng, J. Chen, T. Buie, and W. A. Walker Glucocorticoid responsiveness in developing human intestine: possible role in prevention of necrotizing enterocolitis Am J Physiol Gastrointest Liver Physiol, January 1, 2005; 288(1): G85 - G92. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/1/44 02-0031fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by NANTHAKUMAR, N. N. Articles by WALKER, W. A. Search for Related Content PubMed PubMed Citation Articles by NANTHAKUMAR, N. N. Articles by WALKER, W. A.