Vibrio cholerae is an enteric pathogen of the human small intestine that proliferates to high cell density during human infection. Although not typically classified as a virulence factor, metabolism is a cornerstone for fitness in the host environment. In this work, I explore the essential role of aerobic metabolism, including oxidative respiration, for successful colonization of V. cholerae in the infant mouse model. Oxidative respiration is the most efficient energy generating metabolic pathway in living organisms and supports the rapid proliferation of V. cholerae in the small intestinal environment. Despite knowledge that oxygen diffuses from the host epithelium into the gut lumen, the role of oxygen in supporting colonization and proliferation of V. cholerae had not been explored prior to the work presented here in Chapters 2 and 3.In Chapter 2, by targeting the pyruvate dehydrogenase (PDH) complex, an enzyme required to convert pyruvate to acetyl-CoA under aerobic conditions, I show that aerobic metabolism through the PDH complex is required for population expansion in the infant mouse. As the gut was predominantly considered anaerobic and exists in a state of low oxygen tension, I also examined the contribution of anaerobic metabolism to infant mouse colonization. By targeting cognate pyruvate formate-lyase (PFL) that similarly converts pyruvate to acetyl-CoA, but only under anaerobic conditions, I determined that anaerobic respiration is dispensable for colonization. In Chapter 3, I directly test the importance of aerobic and anaerobic respiration by targeting the complete set of terminal oxidases and terminal reductases encoded by V. cholerae. Using a modified Multiplex Genome Editing by Natural Transformation (MuGENT) approach, I generated strains denoted Aero7 and Ana4. Aero7 is a functionally strict anaerobe derivative of V. cholerae, lacking all four terminal oxidases (cbb3, bd-I, bd-II, and bd-III), whereas Ana4 lacked functionality in each of the four terminal reductase complexes (fumarate, trimethylamine-N-oxide, nitrate, and biotin sulfoxide reductases). Disruption in the oxidase complexes in strain Aero7 severely attenuated V. cholerae colonization in the infant mouse, however, no attenuation was observed for Ana4. These data supported our findings in Chapter 2 that aerobic, but not anaerobic metabolism was critical for V. cholerae growth in the infant mouse. Furthermore, I determined that the bd-I oxidase, and to a lesser extent the cbb3 oxidase, support oxidative respiration during infection with bd-II and bd-III oxidases being dispensable for colonization.In summation, aerobic metabolism through the PDH complex and the terminal reduction of oxygen by the bd-I oxidase are essential to V. cholerae colonization of the infant mouse. Through this work, I uncovered a role for oxidative metabolism for V. cholerae colonization. These findings expand our knowledge of V. cholerae biology and pathogenicity in the gastrointestinal tract and implicate oxygen as a critical electron acceptor that shapes the progression of enteric infections.
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