Identification of a constitutively active variant of LuxO that affects production of HA/protease and biofilm development in a non-O1, non-O139 Vibrio cholerae O110

Pathogenesis of Vibrio cholerae depends on the concerted action of numerous virulence factors that includes a secreted hemagglutinin (HA) protease. Recent studies have evidenced that the expression of these virulence factors as well as the genes responsible for biofilm development is subject to control by quorum sensing in this organism. At low cell density, LuxO, the pivotal regulator of quorum-sensing circuit, has been shown to be phosphorylated at aspartate-47. Working in concert with sigma-54, LuxO-P activates the downstream repressor, which turned out to be four sRNAs [Lenz, D.H., Mok, K.C., Lilley, B.N., Kulkarni, R.V., Wingreen, N.S., Bassler, B.L., 2004. The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae. Cell 118, 69–82]. Subsequently, these sRNAs form complex with sRNA chaperone, Hfq. The Hfq-sRNA complex causes the destabilization of hapR mRNA transcript. HapR is a positive regulator of hapA that encodes HA/protease. At high cell density, dephosphorylation of LuxO impairs its function to activate the expression of sRNA, which in turn promotes HapR expression and causes protease production. It has been demonstrated that conversion of aspartate to glutamate (D47E) renders the LuxO molecule active without being phosphorylated. This variant of LuxO is referred as constitutively active LuxO or con-LuxO [Freeman, J.A., Bassler, B.L., 1999. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. Mol Microbiol 31, 665–677]. Other than D47E, mutation at L104Q also develops con-LuxO [Vance, R.E., Zhu, J., Mekalanos, J.J., 2003. A constitutively active variant of the quorum-sensing regulator LuxO affects protease production and biofilm formation in Vibrio cholerae. Infect. Immun. 71, 2571–2576]. The purpose of this study was to investigate the cause of protease negative phenotype of a non-O1, non-O139 strain of V. cholerae O110. In the process of exploring the nature of the phenotype, a constitutively active variant of LuxO molecule was characterized which represses protease production and enhances biofilm formation by this strain. Unlike luxU, disruption of luxO restored the protease production, which showed the constitutively active nature of LuxO protein in this strain.