Characterization of cyclic-GMP-cAMP regulation in Vibrio cholerae

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ABSTRACT Vibrio cholerae is a pathogen capable of causing worldwide pandemics including seven in the scientific record. The seventh pandemic El Tor biotype that displaced the classical biotype responsible for the first six pandemics differ genetically by many single nucleotide polymorphisms and the acquisition of two large genetic islands called Vibrio seventh pandemic islands I and II (VSP-I and VSP-II). The VSP-I island encodes eleven genes, including dncV which encodes a dinucleotide cyclase enzyme that produces the cyclic- GMP-AMP (cGAMP) molecule. Virulence studies in the infant mice model of infection revealed that dncV is the only gene in VSP-I, that when mutated, has a competitive disadvantage compared to the parental strain. This provides strong evidence that DncV and cGAMP is important for virulence. How cGAMP regulated downstream pathways to enhance virulence remained unresolved. Recently, a study revealed that another gene in VSP-I called capV, that is immediately adjacent to dncV, encodes a phospholipase that is activated by binding to cGAMP. These results suggest that cGAMP acts on the cell by binding protein receptors in a manner similar in concept of other cyclic dinucleotides. While the discover of CapV explained the effect of DncV and cGAMP on lipid metabolism in V. cholerae, the effect of cGAMP on virulence, MSHA expression and chemotaxis expression are unexplained. This proposal hypothesizes that additional protein receptor(s) bind directly to cGAMP to mediate downstream regulation of MSHA, chemotaxis and pathogenesis. We will test our hypothesis by using biochemical and genetic approaches including: Aim 1. Identifying interacting proteins of cGAMP and Aim 2. Characterizing cGAMP down regulation of the MSHA operons. Together, results from the completion of this aim will reveal how cGAMP signaling occurs in V. cholerae and shed light on the intersection between cGAMP and cyclic-di-GMP (c-di-GMP), another cyclic dinucleotide molecule also used by V. cholerae. In addition, revealing the network of signaling molecules in V. cholerae will allow better understand of pathogens can adapt when they acquire cGAMP signaling via horizontal gene transfer.