Vibrio cholerae is the etiologic agent of cholera, a severe diarrheal disease that represents a significant burden on global health and productivity. The high morbidity of cholera demands effective prophylactic strategies, but oral cholera vaccines exhibit variable immunogenicity and efficacy in human populations. One variable factor in human populations is the gut microbiome, the resident community of microorganisms in the gut, which in cholera-endemic areas is strongly and repeatedly modulated by malnutrition, cholera, and non-cholera diarrhea. However, the role of microbiome variations in immune responses against V. cholerae and oral cholera vaccination is not well understood. We conducted fecal transplants from healthy human donors and model communities of either human gut microbes resembling those of healthy individuals or those of individuals recovering from diarrhea or malnutrition in various mouse models. We show microbiome-specific effects on host antibody responses against V. cholerae, and that dysbiotic human gut microbiomes, commonly present in areas where malnutrition and diarrhea are common, suppresses the immune response against V. cholerae through the action of CD4+ lymphocytes. Our findings suggest that the composition of the gut microbiome at time of infection or vaccination may be pivotal for providing robust mucosal immunity, suggesting a target for the improvement of prophylactic and therapeutic strategies for cholera.

Vibrio cholerae causes the diarrheal disease cholera that affects millions of people across the globe and causes over 100,000 deaths yearly. V. cholerae must out-compete the commensal gut microbial community (the gut microbiome) to establish itself in the gut and cause disease. The major functions of normal gut microbiota include nutrient metabolism, nutrient absorption, drug metabolism, and contribute to gut immunomodulation. V. cholerae is a gram-negative bacteria and a major human GI pathogen, despite improvements in treatment and understanding of the mechanisms of pathogenesis, cholera remains a significant global health burden.We describe here that during infection with V. cholerae, individuals with different gut microbiome response differently. We used suckling and adult germfree mice models to introduce different gut communities and examine gut microbiome structure during V. cholerae infection. Next, we showed that the community which includes strains from healthy human is able to decrease cholera infection. Then we identified Vibrio-antagonistic microbes across many different microbiome contexts by setting up combinatorial experiment. Second, we explored how healthy community causes colonization resistance. We showed that this is mediated through the bile salt hydrolase enzyme activity which degrades the bile salt taurocholate that activates the expression of virulence genes. In this dissertation, we used the transplantation of complete human fecal microbiomes and defined communities in both germfree adult mice and suckling antibiotic treated mice models to demonstrate that the variation in gut microbiome can result in different V. cholerae colonization outcomes.

V. cholerae is a Gram-negative enteric pathogen that can cause severe watery diarrhea in human, which affects millions of people in developing countries each year. The gut microbiota that is recognized as a factor in driving susceptibility to the infection, has evolved a variety of strategies to restrict pathogen growth, including nutrient competition, stimulating host immunity, regulation of virulence gene expression, and direct killing. Diet plays a dominant role in shaping the gastrointestinal environment and the function of the gut microbiota. It also regulates the infection of pathogens as well as the interactions between pathogens and gut microbiota. Here, we explored the interspecies competition by direct killing between the gut commensal Escherichia coli and V. cholerae. We identified the genotoxicity of E. coli-produced colibactin toward V. cholerae and other gut microbial commensals. We also demonstrated that dietary proteins regulate V. cholerae colonization and competition against the colibactin-producing E. coli via type VI secretion system. In addition, we showed that gut microbial community regulates host hydrogen peroxide production and V. cholerae resistance to oxidative stress. Combining next generation sequencing analysis with mouse model and traditional molecular techniques provides more comprehensive understanding of host-microbiota-pathogen interactions, which offers new strategies in the design of prophylactic and therapeutic measures to improve global health.

Vibrio cholerae is a Gram-negative bacterium responsible for cholera, a highly contagious diarrheal disease posing a significant global public health threat, particularly in developing countries. To successfully colonize and cause disease in the host, V. cholerae must travel through the stomach's harsh conditions and withstand fluctuations in oxygen levels, pH, and encounter bile and antimicrobial peptides in the intestines. Bile acids, prominent components of bile, play pivotal roles in regulating V. cholerae's virulence gene expression and influencing disease progression. Bacterial bile salt hydrolases (BSH) facilitate bile acid modification by cleaving amino acids from bile acid molecules, thereby altering the composition of bile acid pool.In this study, we identified a novel Vibrio Bile Salt Hydrolase (VBH) capable of deconjugating taurodeoxycholic acid (TDCA) to deoxycholic acid (DCA) and converting taurochenodeoxycholic acid (TCDCA) to chenodeoxycholic acid (CDCA). Our findings demonstrate that VBH activity influences V. cholerae growth and colonization dynamics, as well as interactions with other gut microbes such as Blautia obeum through bile acid processing. Additionally, we uncovered a novel regulatory pathway where autoinducer-2 (AI-2) modulates secondary bile acid metabolism in Clostridium scindens, highlighting bile acids' broader impact on gut commensals. Furthermore, we explored how V. cholerae's disease outcomes are shaped by interactions with gut commensals, their ability to degrade mucin, and induce mucin production. Lastly, we propose the application of single-cell RNA sequencing (scRANseq) techniques to deepen our understanding of host-microbe interactions involving V. cholerae. These insights promise to inform novel therapeutic strategies combating V. cholerae infections and other enteric diseases.

Quorum sensing (QS) is a bacterial communication process that allows bacteria to alter gene expression based on environmental signals. Autoinducer-2 (AI-2) is one of these signaling molecules that regulates the virulence of the gastrointestinal pathogen Vibrio cholerae, which causes the diarrheal disease cholera that affects millions of people worldwide. Research on healthy and malnourished humans has shown that the structure of gut microbiome is different and differentially able to resist V. cholerae colonization, pathogen infection, and host immune response and treatment. However, the effect of AI-2 produced by different bacteria in susceptible and resistant microbiomes on their community and on V. cholerae is not well understood. To this end I constructed Escherichia coli expressing luxS gene from different microbes and combined them with different model communities of human gut microbes resembling health individuals or individuals recovering from diarrhea and tested in mouse models. We show microbiota-associated biofilm regulation in V. cholerae providing resistance against host intestinal environmental stress and AI-2 signal mediated microbiome structural and gene expression change. Our findings suggest that the composition of the gut microbiome is affected by AI-2 signal, and at the time of infection or treatment, the composition is crucial to host resistance against infection. This potentially provides a direction for the prevention and/or improved resistance to pathogen infection as well as immunotherapy.

V. cholerae is a diarrheal disease that affects millions in developing areas annually that lack adequate preventative measures. One possible therapeutic is the use of the gut microbiome, the resident microbial community of the gastrointestinal tract. This community is highly diverse, but how microbial diversity confers resistance or

susceptibility to intestinal pathogens is poorly understood. Using species identified through previous work and transplantation of human microbiomes into several animal models of infection, we show that these species shape the chemical environment of the gut by degrading small molecules used by V. cholerae to activate the expression of virulence genes, leading to reduced pathogen colonization. The absence of these functions and species permits increased infection loads on a personal microbiome-specific basis. These findings suggest new targets for individualized preventative strategies of V. cholerae infection through modulating the structure and function of the gut microbiome.