Cholera, a waterborne disease caused by Vibrio cholerae of serogroups O1 and O139, is characterized by the passage of voluminous, rice-watery stools. In endemic areas in Asia and Africa, this disease continues to be a public health concern, with a prevalence of 5 million cases and 130,000 deaths per year. A major obstacle to the eradication of cholera is the persistence of V. cholerae in aquatic environments, which is facilitated by its capacity to form biofilms. Vibrios in biofilms are more resistant to environmentl stresses, including biocides and disinfectants. Further, Vibrios in a biofilm exhibit a hyperinfective phenotype that facilitates the rapid dissemination of the disease in outbreaks. Thus, a better understanding of the factors that regulate biofilm formation is important for the prevention and control of cholera. The genes responsible for the biosynthesis of the Vibrio polysaccharide (VPS) extracellular matrix, a major component of biofilms, are located in two operons, in which vpsA and vpsL are the first genes of operons I and II, respectively. These genes are regulated by quorum sensing, the second messenger cyclic diguanylate (c-di-GMP), and by the transcription factors AphA, VpsR and VpsT. We recently showed that the histone-like nucleoid structuring protein (H-NS) directly represses the expression of genes involved in the biosynthesis of the VPS matrix. A common theme in H-NS transcriptional silencing is the presence of anti-repressors that disrupt its interaction with DNA promoters. The objective of this application is to test the novel hypothesis that c-di-GMP enhances biofilm formation in V. cholerae by activating the expression/activity of H-NS anti-repressors acting at vps promoters. To this end, we will characterize the interaction between H-NS and the c-di-GMP-receptor protein VpsT in the regulation of the vpsA and vpsL promoters using a combination of genetic and molecular biology approaches (Aim 1).
In Aim 2, we will identify the transcription factor(s) capable of antagonizing H-NS repression at the vpsT promoter and use chromatin immunoprecipitation (ChIP) to demonstrate that elevated c-di-GMP intracellular content initiates an anti-repression cascade at the vpsT promoter that is subsequently transmitted to the downstream vpsA and vpsL promoters. Finally, in Aim 3, we will use confocal microscopy and a mouse competitive colonization assay to characterize the molecular architecture, composition and properties of the biofilm expressed in V. cholerae hns mutants.
Biofilm formation is involved in cholera transmission and in survival of pathogenic Vibrio cholerae in aquatic environments. With this research effort, we will determine the mechanism by which a bacterial regulator, known as the histone-like nucleoid structuring protein, modulates this process.
|Ayala, Julio C; Wang, Hongxia; Benitez, Jorge A et al. (2018) Molecular basis for the differential expression of the global regulator VieA in Vibrio cholerae biotypes directed by H-NS, LeuO and quorum sensing. Mol Microbiol 107:330-343|
|Ayala, Julio C; Silva, Anisia J; Benitez, Jorge A (2017) H-NS: an overarching regulator of the Vibrio cholerae life cycle. Res Microbiol 168:16-25|
|Wang, Hongxia; Ayala, Julio C; Benitez, Jorge A et al. (2015) RNA-seq analysis identifies new genes regulated by the histone-like nucleoid structuring protein (H-NS) affecting Vibrio cholerae virulence, stress response and chemotaxis. PLoS One 10:e0118295|
|Ayala, Julio C; Wang, Hongxia; Silva, Anisia J et al. (2015) Repression by H-NS of genes required for the biosynthesis of the Vibrio cholerae biofilm matrix is modulated by the second messenger cyclic diguanylic acid. Mol Microbiol 97:630-45|
|Ayala, Julio C; Wang, Hongxia; Benitez, Jorge A et al. (2015) RNA-Seq analysis and whole genome DNA-binding profile of the Vibrio cholerae histone-like nucleoid structuring protein (H-NS). Genom Data 5:147-150|
|Wang, Hongxia; Ayala, Julio C; Benitez, Jorge A et al. (2014) The LuxR-type regulator VpsT negatively controls the transcription of rpoS, encoding the general stress response regulator, in Vibrio cholerae biofilms. J Bacteriol 196:1020-30|