Capsular Polysaccharide From Bacteroides fragilis Protects Against Ulcerative Colitis in an Undegraded Form.
ABSTRACT: The prominent human symbiont Bacteroides fragilis protects animals from intestinal diseases, such as ulcerative colitis, and its capsular polysaccharide plays a key role in reducing inflammation. B. fragilis strain ZY-312 was isolated from the feces of a healthy breast-fed infant, and the zwitterionic capsular polysaccharide zwitterionic polysaccharide, TP2, was extracted. In rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced enteritis, TP2 at an optimal dose of 2.5 mg/kg could significantly alleviate enteritis and reduced the degree of intestinal adhesions, the intestinal ulcer area, and the incidence of ulcers in rats. To understand the underlying mechanism, TP2 was labeled with Fluorescein isothiocyanate and orally administered at a dose of 2.5 mg/kg in rats. TP2 was mainly distributed in the cecum and colorectum, but it was not detected in the blood and other organs except that a compound with a molecular weight greater than that of TP2-FITC was found in liver tissue. During the absorption, distribution, metabolism, and excretion, TP2 was indigestible. These results were further confirmed by investigation in the simulated gastric, intestinal fluid, and colonic fluid with fecal microbiota in vitro, where TP2 remained unaltered at different time points. Furthermore, flora composition was analyzed in simulated colonic fluid with TP2 added and it was found that TP2 increased the abundance of Faecalibacterium, Enterococcus romboutsia, and Ruminococcaceae, whereas the abundance of the phylum Proteobacteria represented by Sutterella, Desulfovibrio, and Enterobacteriaceae was decreased. However, the amount of short-chain fatty acids in the simulated colonic fluid was not changed by intestinal flora post-TP2 addition. In conclusion, these findings confirmed that TP2, a capsular polysaccharide of B. fragilis, protects against ulcerative colitis in an undegraded form.
Project description:Bacteroides species are the most abundant Gram-negative bacteria of the human colonic microbiota. These endogenous organisms are unique in that they synthesize an extensive number of phase-variable surface polysaccharides. Pathogenic bacteria phase vary expression of surface molecules for immune evasion, but the importance of the synthesis of multiple phase-variable polysaccharides to these commensal bacteria is unknown. We previously showed that a Bacteroides fragilis mutant unable to synthesize 4 of the 8 capsular polysaccharides and unable to glycosylate proteins properly is rapidly outcompeted by the wild-type strain for colonization of the gnotobiotic mouse intestine. In the present study, we constructed mutants defective only in capsule polysaccharide synthesis to define better the importance of these surface molecules to intestinal colonization. We discovered a key enzymatic activity required for synthesis of 7 of the 8 capsular polysaccharides. Deletion of its gene resulted in the first B. fragilis mutant able to synthesize only one phase-variable polysaccharide, and further mutation resulted in a stable acapsular mutant. We show that the acapsular mutant is rapidly outcompeted, but synthesis of a single polysaccharide is sufficient for the organism to colonize the gnotobiotic intestine competitively. These data demonstrate that initial colonization of the gnotobiotic mouse intestine by B. fragilis requires that the organism synthesize only a single polysaccharide and suggest that the synthesis of multiple phase-variable polysaccharides is important for the bacteria's long-term maintenance in the normally complex and competitive ecosystem.
Project description:A genetic approach was used to assess the heterogeneity of the capsular polysaccharide C (PS C) biosynthesis locus of Bacteroides fragilis and to determine whether distinct loci contain genes whose products are likely to be involved in conferring charged groups that enable the B. fragilis capsular polysaccharides to induce abscesses. A collection of 50 B. fragilis strains was examined. PCR analysis demonstrated that the genes flanking the PS C biosynthesis region are conserved, whereas the genes within the loci are heterogeneous. Only cfiA(+) B. fragilis strains, which represent 3% of the clinical isolates of B. fragilis, displayed heterogeneity in the regions flanking the polysaccharide biosynthesis genes. Primers were designed in the conserved regions upstream and downstream of the PS C locus and were used to amplify the region from 45 of the 50 B. fragilis strains studied. Fourteen PS C genetic loci could be differentiated by a combination of PCR and extended PCR. These loci ranged in size from 14 to 26 kb. Hybridization analysis with genes from the PS C loci of strains 9343 and 638R revealed that the majority of strains contain homologs of wcgC (N-acetylmannosamine dehydrogenase), wcfF (putative dehydrogenase), and wcgP (putative aminotransferase). The data suggest that the synthesis of polysaccharides that have zwitterionic characteristics rendering them able to induce abscesses is common in B. fragilis.
Project description:The tumor-associated carbohydrate antigen/hapten Thomsen-nouveau (Tn; a-D-GalpNAc-ONH2) was conjugated to a zwitterionic capsular polysaccharide, PS A1, from commensal anaerobe Bacteroides fragilis ATCC 25285/NCTC 9343 for the development of an entirely carbohydrate cancer vaccine construct and probed for immunogenicity. This communication discloses that murine anti-Tn IgG3 antibodies both bind to and recognize human tumor cells that display the Tn hapten. Furthermore, the sera from immunization of mice with Tn-PS A1 contain cytokine interleukin 17 (IL-17A), which is known to possess anti-tumor function and represents a striking difference to an IL-2, and IL-6 profile obtained with anti-PS A1 sera.
Project description:Microbes occupy countless ecological niches in nature. Sometimes these environments may be on or within another organism, as is the case in both microbial infections and symbiosis of mammals. Unlike pathogens that establish opportunistic infections, hundreds of human commensal bacterial species establish a lifelong cohabitation with their hosts. Although many virulence factors of infectious bacteria have been described, the molecular mechanisms used during beneficial host-symbiont colonization remain almost entirely unknown. The novel identification of multiple surface polysaccharides in the important human symbiont Bacteroides fragilis raised the critical question of how these molecules contribute to commensalism. To understand the function of the bacterial capsule during symbiotic colonization of mammals, we generated B. fragilis strains deleted in the global regulator of polysaccharide expression and isolated mutants with defects in capsule expression. Surprisingly, attempts to completely eliminate capsule production are not tolerated by the microorganism, which displays growth deficits and subsequent reversion to express capsular polysaccharides. We identify an alternative pathway by which B. fragilis is able to reestablish capsule production and modulate expression of surface structures. Most importantly, mutants expressing single, defined surface polysaccharides are defective for intestinal colonization compared with bacteria expressing a complete polysaccharide repertoire. Restoring the expression of multiple capsular polysaccharides rescues the inability of mutants to compete for commensalism. These findings suggest a model whereby display of multiple capsular polysaccharides provides essential functions for bacterial colonization during host-symbiont mutualism.
Project description:Bacteroides is an abundant genus of bacteria of the human intestinal microbiota. Bacteroides species synthesize a large number of capsular polysaccharides (PS), a biological property not shared with closely related oral species, suggesting importance for intestinal survival. Bacteroides fragilis, for example, synthesizes eight capsular polysaccharides per strain, each of which phase varies via inversion of the promoters located upstream of seven of the eight polysaccharide biosynthesis operons. In a single cell, many of these polysaccharide loci promoters can be simultaneously oriented on for transcription of the downstream biosynthesis operons. Here, we demonstrate that despite the promoter orientations, concomitant transcription of multiple polysaccharide loci within a cell is inhibited. The proteins encoded by the second gene of each of these eight loci, collectively designated the UpxZ proteins, inhibit the synthesis of heterologous polysaccharides. These unique proteins interfere with the ability of UpxY proteins encoded by other polysaccharide loci to function in transcriptional antitermination of their respective operon. The eight UpxZs have different inhibitory spectra, thus establishing a hierarchical regulatory network for polysaccharide synthesis. Limitation of concurrent polysaccharide synthesis strongly suggests that these bacteria evolved this property as an evasion-type mechanism to avoid killing by polysaccharide-targeting factors in the ecosystem.
Project description:The mammalian gut represents a complex and diverse ecosystem, consisting of unique interactions between the host and microbial residents. Bacterial surfaces serve as an interface that promotes and responds to this dynamic exchange, a process essential to the biology of both symbionts. The human intestinal microorganism, Bacteroides fragilis, is able to extensively modulate its surface. Analysis of the B. fragilis genomic sequence, together with genetic conservation analyses, cross-species cloning experiments, and mutational studies, revealed that this organism utilizes an endogenous DNA inversion factor to globally modulate the expression of its surface structures. This DNA invertase is necessary for the inversion of at least 13 regions located throughout the genome, including the promoter regions for seven of the capsular polysaccharide biosynthesis loci, an accessory polysaccharide biosynthesis locus, and five other regions containing consensus promoter sequences. Bacterial DNA invertases of the serine site-specific recombinase family are typically encoded by imported elements such as phage and plasmids, and act locally on a single region of the imported element. In contrast, the conservation and unique global regulatory nature of the process in B. fragilis suggest an evolutionarily ancient mechanism for surface adaptation to the changing intestinal milieu during commensalism.
Project description:In response to phosphate limitation, bacteria employ the Pho regulon, a specific regulatory network for phosphate acquisition. The two-component signal transduction system of PhoRB plays a crucial role in the induction of Pho regulon genes, leading to the adaptation to phosphate starvation. Herein, we identified the PhoRB system in Bacteroides fragilis, a commensal gut bacterium, and evaluated its role in gut colonization and survival in peritoneal abscesses. BF1575 and BF1576 encoded PhoR (sensor histidine kinase) and PhoB (response regulator) in the sequenced B. fragilis strain YCH46, respectively. Transcriptome analysis revealed that deletion of phoB affected the expression of 585 genes (more than 4-fold change) in B. fragilis, which included genes for stress response (chaperons and heat shock proteins), virulence (capsular polysaccharide biosynthesis) and phosphate metabolism. Deletion of phoB reduced the ability of the bacterium to persist in peritoneal abscesses induced by an intra-abdominal challenge of B. fragilis. Furthermore, PhoB was necessary for survival of this anaerobe in peritoneal abscesses but not for in vitro growth in rich media or in intestinal colonization. These results indicate that PhoB plays an important role in the survival of B. fragilis under stressful extraintestinal conditions.
Project description:A major clinical manifestation of infection with Bacteroides fragilis is the formation of intra-abdominal abscesses, which are induced by the capsular polysaccharides of this organism. Transposon mutagenesis was used to locate genes involved in the synthesis of capsular polysaccharides. A 24,454-bp region was sequenced and found to contain a 15,379-bp locus (designated wcf) with 16 open reading frames (ORFs) encoding products similar to those encoded by genes of other bacterial polysaccharide biosynthesis loci. Four genes encode products that are similar to enzymes involved in nucleotide sugar biosynthesis. Seven genes encode products that are similar to sugar transferases. Two gene products are similar to O-acetyltransferases, and two products are probably involved in polysaccharide transport and polymerization. The product of one ORF, WcfH, is similar to a set of deacetylases of the NodB family. Deletion mutants demonstrated that the wcf locus is necessary for the synthesis of polysaccharide B, one of the two capsular polysaccharides of B. fragilis 9343. The virulence of the polysaccharide B-deficient mutant was comparable to that of the wild type in terms of its ability to induce abscesses in a rat model of intra-abdominal infection.
Project description:Mammals harbour a complex gut microbiome, comprising bacteria that confer immunological, metabolic and neurological benefits. Despite advances in sequence-based microbial profiling and myriad studies defining microbiome composition during health and disease, little is known about the molecular processes used by symbiotic bacteria to stably colonize the gastrointestinal tract. We sought to define how mammals assemble and maintain the Bacteroides, one of the most numerically prominent genera of the human microbiome. Here we find that, whereas the gut normally contains hundreds of bacterial species, germ-free mice mono-associated with a single Bacteroides species are resistant to colonization by the same, but not different, species. To identify bacterial mechanisms for species-specific saturable colonization, we devised an in vivo genetic screen and discovered a unique class of polysaccharide utilization loci that is conserved among intestinal Bacteroides. We named this genetic locus the commensal colonization factors (ccf). Deletion of the ccf genes in the model symbiont, Bacteroides fragilis, results in colonization defects in mice and reduced horizontal transmission. The ccf genes of B. fragilis are upregulated during gut colonization, preferentially at the colonic surface. When we visualize microbial biogeography within the colon, B. fragilis penetrates the colonic mucus and resides deep within crypt channels, whereas ccf mutants are defective in crypt association. Notably, the CCF system is required for B. fragilis colonization following microbiome disruption with Citrobacter rodentium infection or antibiotic treatment, suggesting that the niche within colonic crypts represents a reservoir for bacteria to maintain long-term colonization. These findings reveal that intestinal Bacteroides have evolved species-specific physical interactions with the host that mediate stable and resilient gut colonization, and the CCF system represents a novel molecular mechanism for symbiosis.
Project description:Bacteroides fragilis, though only a minor component of the human intestinal commensal flora, is the anaerobe most frequently isolated from intra-abdominal abscesses. B. fragilis 9343 expresses at least three capsular polysaccharides-polysaccharide A (PS A), PS B, and PS C. Purified PS A and PS B have been tested in animal models and are both able to induce the formation of intra-abdominal abscesses. Mutants unable to synthesize PS B or PS C still facilitate abscess formation at levels comparable to those of wild-type 9343. To determine the contribution of PS A to abscess formation in the context of the intact organism, the PS A biosynthesis region was cloned, sequenced, and deleted from 9343 to produce a PS A-negative mutant. Animal experiments demonstrate that the abscess-inducing capability of 9343 is severely attenuated when the organism cannot synthesize PS A, despite continued synthesis of the other capsular polysaccharides. The PS A of 9343 contains an unusual free amino sugar that is essential for abscess formation by this polymer. PCR analysis of the PS A biosynthesis loci of 50 B. fragilis isolates indicates that regions flanking each side of this locus are conserved in all strains. The downstream conserved region includes two terminal PS A biosynthesis genes that homology-based analyses predict are involved in the synthesis and transfer of the free amino sugar of PS A. Conservation of these genes suggests that this sugar is present in the PS A of all serotypes and may explain the abscessogenic nature of B. fragilis.