Vibrio cholerae replicates to high cell density in the human small intestine, leading to the diarrheal disease cholera. During infection, V. cholerae senses and responds to environmental signals that govern cellular responses. Spatial localization of V. cholerae within the intestine affects nutrient availability and metabolic pathways required for replicative success. Metabolic processes used by V. cholerae to reach such high cell densities are not fully known. We sought to better define the metabolic traits that contribute to high levels of V. cholerae during infection. By disrupting the pyruvate dehydrogenase (PDH) complex and pyruvate formate-lyase (PFL), we could differentiate aerobic and anaerobic metabolic pathway involvement in V. cholerae proliferation. We demonstrate that oxidative metabolism is a key contributor to the replicative success of V. cholerae in vivo using an infant mouse model in which PDH mutants were attenuated 100-fold relative to the wild type for colonization. Additionally, metabolism of host substrates, including mucin, was determined to support V. cholerae growth in vitro as a sole carbon source, primarily under aerobic growth conditions. Mucin likely contributes to population expansion during human infection as it is a ubiquitous source of carbohydrates. These data highlight oxidative metabolism as important in the intestinal environment and warrant further investigation of how oxygen and other host substrates shape the intestinal landscape that ultimately influences bacterial disease. We conclude from our results that oxidative metabolism of host substrates is a key driver of V. cholerae proliferation during infection, leading to the substantial bacterial burden exhibited in cholera patients. IMPORTANCE Vibrio cholerae remains a challenge in the developing world and incidence of the disease it causes, cholera, is anticipated to increase with rising global temperatures and with emergent, highly infectious strains. At present, the underlying metabolic processes that support V. cholerae growth during infection are less well understood than specific virulence traits, such as production of a toxin or pilus. In this study, we determined that oxidative metabolism of host substrates such as mucin contribute significantly to V. cholerae population expansion in vivo. Identifying metabolic pathways critical for growth can provide avenues for controlling V. cholerae infection and the knowledge may be translatable to other pathogens of the gastrointestinal tract.
Vibrio cholerae respires both aerobically and anaerobically and, while oxygen may be available to it during infection, other terminal electron acceptors are proposed for population expansion during infection. Unlike gastrointestinal pathogens that stimulate significant inflammation leading to elevated levels of oxygen or alternative terminal electron acceptors, V. cholerae infections are not understood to induce a notable inflammatory response. To ascertain the respiration requirements of V. cholerae during infection, we used Multiplex Genome Editing by Natural Transformation (MuGENT) to create V. cholerae strains lacking aerobic or anaerobic respiration. V. cholerae strains lacking aerobic respiration were attenuated in infant mice 105-fold relative to wild type, while strains lacking anaerobic respiration had no colonization defect, contrary to earlier work suggesting a role for anaerobic respiration during infection. Using several approaches, including one we developed for this work termed Comparative Multiplex PCR Amplicon Sequencing (CoMPAS), we determined that the bd-I and cbb3 oxidases are essential for small intestinal colonization of V. cholerae in the infant mouse. The bd-I oxidase was also determined as the primary oxidase during growth outside the host, making V. cholerae the only example of a Gram-negative bacterial pathogen in which a bd-type oxidase is the primary oxidase for energy acquisition inside and outside of a host.
A major challenge faced by bacteria is infection by bacteriophage (phage). Abortive infection is one strategy for combating phage in which an infected cell kills itself to limit phage replication, thus protecting neighboring kin. One class of abortive infection systems is the cyclic oligonucleotide based anti-phage signaling system (CBASS) which relies on two core enzymatic activities; an oligo-nucleotide cyclase that is activated following phage infection and a cyclic-oligo-nucleotide sensitive effector whose activity kills the infected cell. However, the mechanisms behind the deployment and activation of these lethal CBASS systems prior-to and following infection have largely remained a mystery. While exploring unique genomic features of the current pandemic Vibrio cholerae biotype El Tor for clues underlying its pandemic success we found its CBASS was spuriously activated by the folate biosynthesis inhibitor sulfamethoxazole, but only after the population had reached a high-cell density. This population density dependent activity revealed that transcription of both the oligo-nucleotide cyclase, dncV, and the CBASS phospholipase effector, capV, is enhanced at high-cell density by quorum sensing. Together, these results demonstrate that the V. cholerae CBASS is deployed when the environment is densely populated and activated in response to a perturbation in folate biosynthesis.
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