Strain competition restricts colonization of an enteric pathogen and prevents colitis.
ABSTRACT: The microbiota is a major source of protection against intestinal pathogens; however, the specific bacteria and underlying mechanisms involved are not well understood. As a model of this interaction, we sought to determine whether colonization of the murine host with symbiotic non-toxigenic Bacteroides fragilis could limit acquisition of pathogenic enterotoxigenic B. fragilis We observed strain-specific competition with toxigenic B. fragilis, dependent upon type VI secretion, identifying an effector-immunity pair that confers pathogen exclusion. Resistance against host acquisition of a second non-toxigenic strain was also uncovered, revealing a broader function of type VI secretion systems in determining microbiota composition. The competitive exclusion of enterotoxigenic B. fragilis by a non-toxigenic strain limited toxin exposure and protected the host against intestinal inflammatory disease. Our studies demonstrate a novel role of type VI secretion systems in colonization resistance against a pathogen. This understanding of bacterial competition may be utilized to define a molecularly targeted probiotic strategy.
Project description:Enterotoxigenic Bacteroides fragilis is an enteric pathogen which is described as a causative agent of various intestinal infections and inflammatory diseases. Moreover, various research studies have reported it to be a leading factor in the development of colorectal cancer. As a part of the normal human microbiome, its treatment has become quite a challenge due to the alarming resistance against the available antibiotics. Although, this particular strain of B. fragilis shows susceptibility to few antibiotics, it is pertinent to devise an effective vaccine strategy for its elimination. There is no vaccine available against this pathogen up to date; therefore, we systematically ventured the outer membrane toxin producing proteins found exclusively in the toxigenic B. fragilis through the in-silico approaches to predict a multi-epitopic chimeric vaccine construct. The designed protein constitutes of epitopes which are predicted for linear B cells, Helper and T cells of outer membrane proteins expected to be putative vaccine candidates. The finalized proteins are only expressed in the enterotoxigenic B. fragilis, thus proving them to be exclusive. The 3D structure of the protein was first predicted followed by its refinement and validation via utilizing the bioinformatic approaches. Docking of the designed protein with the TLR2 receptor forecasted apt binding. Upon immune simulation, notable levels were observed in the expression of the immune cells.
Project description:Enterotoxigenic strains of Bacteroides fragilis produce an extracellular metalloprotease toxin (termed fragilysin) which is cytopathic to intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops. We report here that the fragilysin gene is contained within a small genetic element termed the fragilysin pathogenicity islet. The pathogenicity islet of B. fragilis VPI 13784 was defined as 6,033 bp in length and contained nearly perfect 12-bp direct repeats near its ends. Sequencing across the ends of the pathogenicity islet from two additional enterotoxigenic strains, along with PCR analysis of 20 additional enterotoxigenic strains, revealed that the islet is inserted at a specific site on the B. fragilis chromosome. The site of integration in three nontoxigenic strains contained a 17-bp GC-rich sequence which was not present in toxigenic strains and may represent a target sequence for chromosomal integration. In addition to the fragilysin gene, we identified an open reading frame encoding a predicted protein with a size and structural features similar to those of fragilysin. The deduced amino acid sequence was 28.5% identical and 56.3% similar to fragilysin and contained a nearly identical zinc-binding motif and methionine-turn region.
Project description:Bacteroides fragilis is the leading cause of anaerobic bacteremia and sepsis. Enterotoxigenic strains that produce B. fragilis toxin (BFT, fragilysin) contribute to colitis and intestinal malignancy, yet are also isolated in bloodstream infection. It is not known whether these strains harbor unique genetic determinants that confer virulence in extra-intestinal disease. We demonstrate that BFT contributes to sepsis in mice, and we identify a B. fragilis protease called fragipain (Fpn) that is required for the endogenous activation of BFT through the removal of its auto-inhibitory prodomain. Structural analysis of Fpn reveals a His-Cys catalytic dyad that is characteristic of C11-family cysteine proteases that are conserved in multiple pathogenic Bacteroides spp. and Clostridium spp. Fpn-deficient, enterotoxigenic B. fragilis has an attenuated ability to induce sepsis in mice; however, Fpn is dispensable in B. fragilis colitis, wherein host proteases mediate BFT activation. Our findings define a role for B. fragilis enterotoxin and its activating protease in the pathogenesis of bloodstream infection, which indicates a greater complexity of cellular targeting and activity of BFT than previously recognized. The expression of fpn by both toxigenic and nontoxigenic strains suggests that this protease may contribute to anaerobic sepsis in ways that extend beyond its role in toxin activation. It could thus potentially serve as a target for disease modification.
Project description:Polysaccharide A (PSA), an immunogenic capsular component of non-toxigenic Bacteroides fragilis (NTBF) strain NCTC 9343, is reported to promote mucosal immune development and suppress colitis. Contrastingly, enterotoxigenic Bacteroides fragilis (ETBF) is highly associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC), rapidly inducing IL-17-dependent murine colitis and tumorigenesis. In specific-pathogen-free (SPF) C57BL/6 wild-type (WT) and multiple intestinal neoplasia (MinApc716+/-) mice, we show that sequential treatment of the NTBF strain, 9343, followed by the ETBF strain, 86-5443-2-2 (86), diminished colitis and tumorigenesis. Mice treated simultaneously with 9343 and 86 exhibited both severe colitis and tumorigenesis. Abrogated disease severity in sequentially treated mice was attributed to 9343 strain dominance and decreased IL-17A, but 86 colonization prior to or simultaneous with 9343 mitigated the anti-inflammatory effect of 9343. Remarkably, 9343-mediated protection was independent of PSA, as sequentially treated mice receiving ?PSA 9343 exhibited similar protection. Further, SPF WT and Min mice colonized with PSA-competent or PSA-deficient 9343 exhibited similar IL-10, IL-17, and IFN-? responses. Treatment of 86-colonized mice with 9343 failed to disrupt 86 pathogenesis. Our findings demonstrate that 9343 colonization, independent of PSA, offers prophylaxis against colitis-inducing 86 but may not be a valid therapy once colitis is established.
Project description:Although gut microbiome composition is well defined, the mechanisms underlying community assembly remain poorly understood. Bacteroidales possess three genetic architectures (GA1-3) of the type VI secretion system (T6SS), an effector delivery pathway that mediates interbacterial competition. Here we define the distribution and role of GA1-3 in the human gut using metagenomic analysis. We find that adult microbiomes harbor limited effector and cognate immunity genes, suggesting selection for compatibility at the species (GA1 and GA2) and strain (GA3) levels. Bacteroides fragilis GA3 is known to mediate potent inter-strain competition, and we observe GA3 enrichment among strains colonizing infant microbiomes, suggesting competition early in life. Additionally, GA3 is associated with increased Bacteroides abundance, indicating that this system confers an advantage in Bacteroides-rich ecosystems. Collectively, these analyses uncover the prevalence of T6SS-dependent competition and reveal its potential role in shaping human gut microbial composition.
Project description:Background:Enterotoxigenic Bacteroides fragilis (ETBF) associated with the initiation and progression of colorectal cancer (CRC) has been alarmingly reported all over the world. In this study, simultaneous investigation of toxigenic and non-toxigenic patterns I, II and III and biofilm formation ability of Bacteroides fragilis isolated from patients with colorectal cancer was performed. Methods:Thirty-one patients diagnosed with CRC and thirty-one control subjects were recruited in this study. Specimens were cultured on BBE and BBA culture media. Classical phenotypic identification tests and PCR was performed to verify Bacteroides fragilis presence. Also, biofilm-forming ability and expression of bft gene were assessed under biofilm and planktonic forms. Results:A total of 68 B.fragilis was isolated from all colorectal tissue, of which 13 isolates (19.1%) (11 isolates from CRC and 2 from normal tissue) were positive for bft gene. The abundance patterns of I, II and III were as follow in descending order; pattern I?>?pattern III?>?pattern II in CRC subjects and pattern II?>?pattern III?>?pattern I in normal tissues. Also, pattern I showed higher biofilm formation ability compared to other patterns. Toxin expression was significantly reduced in biofilm form comparing with planktonic form. Conclusions:Based on our findings, there was a difference between the abundance of patterns I, II, and III and biofilm formation in isolates obtained from CRC and normal tissues. Biofilm formation ability and toxin encoding gene (bft) are two main virulence factors in B. fragilis pathogenicity which require more investigation to treat B. fragilis infections effectively.
Project description:Commensal non-toxigenic Bacteroides fragilis confers powerful health benefits to the host, and has recently been identified as a promising probiotic candidate. We previously isolated B. fragilis strain ZY-312 and identified it as a novel strain based on 16S rRNA sequencing and morphological analyses. We also determined that ZY-312 displayed desirable probiotic properties, including tolerance to simulated digestive fluid, adherence, and in vitro safety. In this study, we aim to investigate whether ZY-312 meets the safety criteria required for probiotic bacteria through comprehensive and systematic evaluation. Consequently, the fatty acid profile, metabolite production, and biochemical activity of strain ZY-312 were found to closely resemble descriptions of B. fragilis in Bergey's manual. Taxonomic identification of strain ZY-312 based on whole genome sequencing indicated that ZY-312 and ATCC 25285 showed 99.99% similarity. The 33 putative virulence-associated factors identified in ZY-312 mainly encoded structural proteins and proteins with physiological activity, while the lack of bft indicated that ZY-312 was non-toxigenic. In vivo safety was proven in both normal and immune-deficient mice. The 11 identified antibiotic resistance genes were located on the chromosome rather than on a plasmid, ruling out the risk of plasmid-mediated transfer of antibiotic resistance. In vitro, ZY-312 showed resistance to cefepime, kanamycin, and streptomycin. Finally, and notably, ZY-312 exhibited high genetic stability after 100 passages in vitro. This study supplements the foundation work on the safety evaluation of ZY-312, and contributes to the development of the first probiotic representative from the dominant Bacteroidetes phylum.
Project description:To further understand the epidemiology of enterotoxigenic Bacteroides fragilis (ETBF), 89 extraintestinal B. fragilis strains from Seoul, Korea, were examined for secretion of B. fragilis toxin (BFT) by the HT29/C1 biologic assay and for the B. fragilis toxin gene (bft) by colony blot hybridization and PCR. Complete agreement between the three techniques was found. Overall, 34 B. fragilis strains (38%) were identified as ETBF. Eleven of the 34 ETBF strains (32%) expressed a new isoform of BFT (Korea-BFT). This new isoform is more related to BFT-2 than to BFT-1. Like BFT-1 and BFT-2, Korea-BFT cleaves E-cadherin, the zonula adherens protein.
Project description:Hcp (hemolysin-coregulated protein) is considered a vital component of the functional T6SS (Type VI Secretion System), which is a newly discovered secretion system. Our laboratory has previously sequenced the whole genome of porcine extraintestinal pathogenic E. coli (ExPEC) strain PCN033, and identified an integrated T6SS encoding three different hcp family genes. In this study, we first identified a functional T6SS in porcine ExPEC strain PCN033, and demonstrated that the Hcp family proteins were involved in bacterial competition and the interactions with other cells. Interestingly, the three Hcp proteins had different functions. Hcp2 functioned predominantly in bacterial competition; all three proteins were involved in the colonization of mice; and Hcp1 and Hcp3 were predominantly contributed to bacterial-eukaryotic cell interactions. We showed an active T6SS in porcine ExPEC strain PCN033, and the Hcp family proteins had different functions in their interaction with other bacteria or host cells.
Project description:The only recognized virulence factor of enterotoxigenic Bacteroides fragilis (ETBF) that accompanies bloodstream infections is the zinc-dependent non-lethal metalloprotease B. fragilis toxin (BFT). The isolated toxin stimulates intestinal secretion, resulting in epithelial damage and necrosis. Numerous publications have focused on the interrelation of BFT with intestinal inflammation and colorectal neoplasia, but nothing is known about the mechanism of its secretion and delivery to host cells. However, recent studies of gram-negative bacteria have shown that outer membrane vesicles (OMVs) could be an essential mechanism for the spread of a large number of virulence factors. Here, we show for the first time that BFT is not a freely secreted protease but is associated with OMVs. Our findings indicate that only outer surface-exposed BFT causes epithelial cell contact disruption. According to our in silico models confirmed by Trp quenching assay and NMR, BFT has special interactions with outer membrane components such as phospholipids and is secreted during vesicle formation. Moreover, the strong cooperation of BFT with polysaccharides is similar to the behavior of lectins. Understanding the molecular mechanisms of BFT secretion provides new perspectives for investigating intestinal inflammation pathogenesis and its prevention.