Screen Regulators Of Virulence Gene TcpP And Functional Analysis Of LysR Proteins In Vibrio Cholerae

Vibrio cholerae is both an inhabitant in natural aquatic environments and the pathogens of the human diarrheal disease cholera. Major virulence factors in Vibrio cholerae are ctxAB gene which encodes cholera toxin CT, and pathogenicity island TCP which encodes TCP pili. These pili are Vibrio cholerae intestinal colonization factors, which are also CTXφreceptors. ToxR is a regulator of virulence genes. Genes encoding CT and TCP are regulated by the ToxR.In this study. Vibrio cholerae LZV608 was used and randomLy inserted by mariner transposon to establish mutant library. With luciferase gene as a reporter gene, five mutant strains were selected which affected expression of virulence gene tcpP in Vibrio cholerae. Through Arbitrary PCR amplification. DNA sequencing and BLAST analysis, we determined those transposon inserted genes are aphA, aphB, aroK which encodes a metabolic pathways related kinase, a gene that encodes hypothetical protein of unknown function, and a gene VC1405 which encodes a Methyl-accepting chemotaxis protein.In this experiment, in-frame deletion of VC1405 and complemental expression of VC1405 were constructed. We found that the expression of tcpP was reduced after deletion VC1405, showing reduced light value of reporter gene, while the light value restores in complemental expression strain. Previous reports suggested aphA and aphB have mutual coordinating effects on tcp operator and positively regulate tcpPH expression. By analysis strains with luciferase as reporter gene, we argued that the process of this regulation is not achieved through transcriptional factors aphB and aphA.LysR-type transcriptional regulators (LTTRs) are thoroughly studied as transcriptional regulators. LTTRs have wide range of regulatory functions in bacterial cells, which involved in bacterial metabolism, cell division, quorum sensing, virulence, motility, nitrogen fixation and oxidation reactions. There have approximately 40 types of LysR proteins in Vibrio cholerae. Some of these proteins'functions have been studied in depth. In this study, VC1561. one of these LysR family proteins whose function has unknown, was studied with gene knockout and complementary approaches, we prove VC1561 is not a necessary gene for Vibrio cholerae growth. By infant mice intestinal colonization assay, we proved VC1561 is not related to colonization capability of Vibrio cholerae in infant mice intestine. According to western bolt assay, VC1561 is also not related to cholera toxin production. With luciferase as reporter gene, we find VC1561 can activate the expression of its downstream gene VC1562. and inhibit the expression itself.