A Study on Vibrio vulnificus: Screening and Identification of Anti-toxic Signaling Pathway

Abstract

Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The outcome from the infection is notorious for severe destruction of host tissues, thereby high mortality rate. In the present study, to seek out the way to deal with this severe pathogenesis caused by V. vulnificus, a cell-based functional screening was conducted. Prior to the screening, I first deduced the type of V. vulnificus-induced cell death from an experiment with a chemical necrosis inhibitor, necrostatin-1 and the occurrence of necrosis in the cells infected with vibrio was identified. I performed cell-based screening using cDNA library in mammalian expression vectors, including cDNA encoding 650 kinases, 100 phosphatases, and 340 ER-resident proteins. From the primary and secondary screening, p21-activated kinase 1 (PAK1) was isolated and proved to have the most profound activity for host cell survival against vibrio infection. Among PAK family members, PAK1 was the most effective to protect host cells infected with V. vulnificus, despite high similarity of their primary functions. The kinase activity of PAK1 is required for cell survival and PAK1 acts on the downstream of Rho GTPase, Cdc42 and Rac1, but not on that of RhoA. Upon an experiment with rtxA deletion mutant strain of V. vulnificus, a toxin with known as the most virulent factor, almost every clone isolated from the screening exerted their protective activity against the infection. Especially, signal pathways that function in cytoskeleton, unfolded protein responses and MAP kinase affected cell viability by V. vulnificus. Taken together, to overcome severity of the illness caused by an acute and intensive death from vibrio infection, many factors of virulence of V. vulnificus should be considered. Knowledge about existence of diverse possible ways of host defense mechanisms would be also helpful to cope with a disease cause by V. vulnificus.