Characterization of Vibrio VopS, an AMPylator of Rho GTPases

Vibrio parahaemolyticus is a gram-negative marine bacterium that causes gastroenteritis associated with the consumption of contaminated shellfish. The emergence of pandemic strains of V. parahaemolyticus has increased the need for characterization of the virulence factors of this pathogen. Sequencing of the genome of a clinical isolate revealed the presence of two type III secretion systems (T3SSs), one on each chromosome. The T3SS on chromosome one (T3SS1) has been shown to be responsible for cytotoxicity in HeLa cells, and it shares a high degree of homology to the T3SS of the Yersinia spp. Our studies have shown that infection of HeLa cells with a strain of V. parahaemolyticus capable of secreting only from T3SS1 indicated that T3SS1 mediates several events during infection including the rapid induction of autophagy, cell rounding, and finally lysis of the cell. Defining the T3SS1-mediated events of infection gives insight into virulence mechanisms of V. parahaemolyticus that have not been well characterized and provide a basis for the elucidation of the functions associated with T3SS1 effectors. One of the T3SS effectors, VopS, contains a Filamentation induced by cAMP (Fic) domain that we have shown is critical for the function of this effector. Our studies have found that VopS inhibits Rho GTPase signaling during infection by directly modifying Rho, Rac, and Cdc42, preventing their interaction with downstream effectors. These observations reveal a unique activity for VopS, which targets a pathway that is critical in the cellular response to V. parahaemolyticus infection. In addition, they provide insight into a novel post-translational modification that may expand our knowledge of eukaryotic cell signaling. Fic domains are found in proteins from several bacterial and eukaryotic species and are recognized by their conserved motif, HPFX(D/E)GNGR. The presence of Fic domains in higher eukaryotes suggested that this modification could be utilized in cell signaling. Our preliminary studies indicated that AMPylation is utilized by eukaryotes. We have shown that a Fic protein from humans, HYPE, possesses auto-AMPylation activity, confirming our hypothesis that these domains are involved in AMPylation. Ongoing and future studies seek to identify the substrates of HYPE activity and identify other components involved in this new layer of eukaryotic cell signaling.