Characterization of a new mechanism of cell wall synthesis regulation in the cholera pathogen

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Peptidoglycan (PG) is an essential component of the bacterial cell wall, a structure that bacteria rely on to maintain cell shape and integrity. PG synthetic pathways have been extensively studied, and numerous enzymes that contribute to it have been identified. However, as many of these proteins appear to have overlapping activities, the specific roles of most factors have not been precisely determined. Moreover, there is little knowledge of how PG synthesis is spatio-temporally regulated. In Vibrio cholerae, the etiologic agent of cholera, two homologous proteins, PBP1a and PBP1b play key roles in PG synthesis. Our analyses of mutants suggest that PBP1a is the dominant factor, as cells lacking PBP1a (mrcA mutants) produce less PG, are highly susceptible to osmotic challenge, and fail to grow or maintain their rod shape in the presence of non-canonical D-amino acids, which are released in stationary phase. In contrast, cells lacking PBP1b lack a dramatic phenotype. Additionally, we have found that mrcA mutants are largely phenocopied by strains lacking either lpoA or vc1887. In E. coli, the role of LpoA is to activate PBP1a; however, no function has been identified for VC1887, a periplasmic protein that lacks recognizable motifs or homologs outside of the Vibrionaceae. Here, I present experiments to better characterize the process of PG synthesis in V. cholerae. In particular, I will assess the roles of PBP1a, PBP1b and VC1887, which is a novel contributor to PG production. Finally, I will conduct a high throughput screen to identify specific inhibitors of PBP1a/VC1887/LpoA, in order to identify agents that specifically inhibit the growth of Vibrios. In aggregate, these studies will yield new fundamental knowledge regarding the control of PG synthesis in an important pathogen and may yield new agents to treat cholera.