Project description:Leveraging systems biology approaches, we illustrate how metabolically distinct species of Clostridia protect against or worsen Clostridioides difficile infection in mice by modulating the pathogen's colonization, growth, and virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Paraclostridium bifermentans survive infection with reduced disease severity, while mice colonized with the butyrate-producer, Clostridium sardiniense, succumb more rapidly. Systematic in vivo analyses revealed how each commensal alters the gut-nutrient environment to modulate the pathogen's metabolism, gene regulatory networks, and toxin production. Oral administration of P. bifermentans rescues conventional, clindamycin-treated mice from lethal C. difficile infection in a manner similar to that of monocolonized animals, thereby supporting the therapeutic potential of this commensal species. Our findings lay the foundation for mechanistically informed therapies to counter C. difficile disease using systems biology approaches to define host-commensal-pathogen interactions in vivo.
Project description:We illustrate how metabolically distinct species of Clostridia can protect against or worsen Clostridioides difficile infection, modulating the pathogen's colonization, growth, and virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Paraclostridium bifermentans survived infection while mice colonized with the butyrate-producer, Clostridium sardiniense, more rapidly succumbed. Systematic in vivo analyses revealed how each commensal altered the gut nutrient environment, modulating the pathogen's metabolism, regulatory networks, and toxin production. Oral administration of P. bifermentans rescued conventional mice from lethal C. difficile infection via mechanisms identified in specifically colonized mice. Our findings lay the foundation for mechanistically informed therapies to counter C. difficile disease using systems biologic approaches to define host-commensal-pathogen interactions in vivo.
Project description:Prior antibiotic treatment is a risk factor for Clostridioides difficile infection (CDI); the commensal gut microbiota plays a key role in determining host susceptibility to the disease. Previous studies demonstrate that the pre-colonization of mice with a commensal fungus, Candida albicans, protects against a lethal challenge with C. difficile spores. The results reported here demonstrate that the cecum contents of antibiotic-treated mice with C. albicans colonization contained different levels of several lipid species, including non-esterified, unsaturated long-chain fatty acids compared to non-C. albicans-colonized mice. Mice fed olive oil for one week and challenged with C. difficile spores showed enhanced survival compared to PBS-fed mice. The amount of olive oil administered was not sufficient to cause weight gain or to result in significant changes to the bacterial microbiota, in contrast to the effects of a high-fat diet. Furthermore, the direct exposure of C. difficile bacteria in laboratory culture to the unsaturated fatty acid oleic acid, the major fatty acid found in olive oil, reduced the transcription of genes encoding the toxins and reduced the survival of bacteria in the post-exponential phase. Therefore, the effects of C. albicans on the metabolite milieu contributed to the attenuation of C. difficile virulence.
Project description:Clostridioides difficile colonizes the intestines of susceptible individuals and releases toxins that mediate disease. To replicate and expand in the intestines, C. difficile ferments proline, and this activity is influenced by the availability of proline and trace nutrients. C. difficile must also compete with the commensal microbiota for these limited nutrients. The specific microbes present in the intestines that may shape the ability of C. difficile to benefit from proline fermentation are unknown. In this study we developed a panel of commensal Clostridia to test the hypothesis that the microbiota influences C. difficile growth through proline fermentation. The experimental panel of Clostridia was composed of murine and human isolates that ranged in their capacity to ferment proline in different media. Competition between wild type C. difficile and a mutant strain unable to ferment proline (prdB:CT) in the presence of these Clostridia revealed that bacteria closely related to Paraclostridium benzoelyticum and Paeniclostridium spp. decreased the benefit to C. difficile provided by proline fermentation. Conversely, Clostridium xylanolyticum drove C. difficile towards an increased reliance on proline fermentation for growth. Overall, the ability of C. difficile to benefit from proline fermentation is contextual and in part dependent on the microbiota.
Project description:Analysis of Clostridium difficile (Cd) from the cecal contents of germ-free mice or Bacteroides thetaiotaomicron (Bt)-monocolonized mice on a standard, polysaccharide rich diet or polysaccharide deficient diet 5 days after infection. Results identify genes that are involved in the Cd response to diet, in vivo colonization and in interactions with Bt. In vitro transcriptional profiles of Clostridium difficile obtained from cecal contents of germ-free or Bt-monocolonized mice on a standard, polysaccharide rich or polysaccharide deficient diet. 4 samples/group. 2 control genomic DNA samples for Clostridium difficile and 2 reference genomic DNA samples for Bacteroides thetaiotaomicron Please note that 4 control samples (genomic DNA) were used to determine whether the genomic DNA correctly bound to the probes and thus, were not included in data processing (i.e no processed/normalized data).
Project description:Analysis of Clostridium difficile (Cd) from the cecal contents of germ-free mice or Bacteroides thetaiotaomicron (Bt)-monocolonized mice on a standard, polysaccharide rich diet or polysaccharide deficient diet 5 days after infection. Results identify genes that are involved in the Cd response to diet, in vivo colonization and in interactions with Bt.
Project description:Clostridioides difficile infection (CDI) imposes a substantial burden on the health care system in the United States. Understanding the biological basis for the spectrum of C. difficile-related disease manifestations is imperative to improving treatment and prevention of CDI. Here, we investigate the correlates of asymptomatic C. difficile colonization using a multi-omics approach. We compared the fecal microbiome and metabolome profiles of patients with CDI versus asymptomatically colonized patients, integrating clinical and pathogen factors into our analysis. We found that CDI patients were more likely to be colonized by strains with the binary toxin (CDT) locus or strains of ribotype 027, which are often hypervirulent. We find that microbiomes of asymptomatically colonized patients are significantly enriched for species in the class Clostridia relative to those of symptomatic patients. Relative to CDI microbiomes, asymptomatically colonized patient microbiomes were enriched with sucrose degradation pathways encoded by commensal Clostridia, in addition to glycoside hydrolases putatively involved in starch and sucrose degradation. Fecal metabolomics corroborates the carbohydrate degradation signature: we identify carbohydrate compounds enriched in asymptomatically colonized patients relative to CDI patients. Further, we reveal that across C. difficile isolates, the carbohydrates sucrose, rhamnose, and lactulose do not serve as robust growth substrates in vitro, consistent with their enriched detection in our metagenomic and metabolite profiling of asymptomatically colonized individuals. We conclude that pathogen genetic variation may be strongly related to disease outcome. More interestingly, we hypothesize that in asymptomatically colonized individuals, carbohydrate metabolism by other commensal Clostridia may prevent CDI by inhibiting C. difficile proliferation. These insights into C. difficile colonization and putative commensal competition suggest novel avenues to develop probiotic or prebiotic therapeutics against CDI.
Project description:Clostridium difficile is a leading cause of nosocomial infections, causing disease that ranges from mild diarrhea to potentially fatal colitis. A variety of surface proteins, including flagella, enable C. difficile colonization of the intestine. Once in the intestine, toxigenic C. difficile secretes two glucosylating toxins, TcdA and TcdB, which elicit inflammation and diarrheal disease symptoms. Regulation of colonization factors and TcdA and TcdB is an intense area of research in C. difficile biology. A recent publication from our group describes a novel regulatory mechanism that mediates the ON/OFF expression of co-regulated virulence factors of C. difficile, flagella and toxins. Herein, we review key findings from our work, present new data, and speculate the functional consequence of the ON/OFF expression of these virulence factors during host infection.
Project description:Recent studies describe in detail the shifts in composition of human-associated polymicrobial communities from health to disease. However, the specific processes that drive the colonization and overgrowth of pathogens within these communities remain incompletely understood. We used in vitro culture systems and a disease-relevant mouse model to show that population size, which determines the availability of an endogenous diffusible small molecule, limits the growth, colonization, and in vivo virulence of the human oral pathogen Porphyromonas gingivalis. This bacterial pathogen overcomes the requirement for an endogenous cue by utilizing a cell-density dependent, growth-promoting, soluble molecule provided by the symbiotic early colonizer Veillonella parvula, but not produced by other commensals tested. Our work shows that exchange of cell-density-dependent diffusible cues between specific early and late colonizing species in a polymicrobial community drives microbial successions, pathogen colonization and disease development, representing a target process for manipulation of the microbiome towards the healthy state.
Project description:Clostridioides difficile is the most common cause of nosocomial antibiotic-associated diarrhea, and is responsible for a spectrum of diseases characterized by high levels of recurrence, morbidity, and mortality. Treatment is complex, since antibiotics constitute both the main treatment and the major risk factor for infection. Worryingly, resistance to multiple antibiotics is becoming increasingly widespread, leading to the classification of this pathogen as an urgent threat to global health. As a consummate opportunist, C. difficile is well equipped for promoting disease, owing to its arsenal of virulence factors: transmission of this anaerobe is highly efficient due to the formation of robust endospores, and an array of adhesins promote gut colonization. C. difficile produces multiple toxins acting upon gut epithelia, resulting in manifestations typical of diarrheal disease, and severe inflammation in a subset of patients. This review focuses on such virulence factors, as well as the importance of antimicrobial resistance and genome plasticity in enabling pathogenesis and persistence of this important pathogen.