H-NS Family Members MvaT and MvaU Regulate the Pseudomonas aeruginosa Type III Secretion System

Abstract

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen capable of causing severe disease in immunocompromised individuals. A major P. aeruginosa virulence factor is the type III secretion system (T3SS). The T3SS is used to translocate effector proteins into host cells, causing cytotoxicity. The T3SS is under the transcriptional control of the master regulator ExsA. ExsA is encoded in the exsCEBA operon and autoregulates transcription via the P _exsC promoter. There is also a Vfr-dependent promoter (P _exsA ) located in the intergenic region between exsB and exsA A previous chromatin immunoprecipitation (ChIP)-on-chip experiment identified strong binding signatures for MvaT and MvaU in the intergenic region containing the P _exsA promoter. MvaT and MvaU are DNA-binding histone-like nucleoid-structuring proteins that can repress gene expression. As predicted from the previous ChIP data, purified MvaT specifically bound to the P _exsA promoter region in electrophoretic mobility shift assays. Whereas disruption of mvaT or mvaU by either transposon insertion or clustered regularly interspaced short palindromic repeat interference (CRISPRi) derepressed P _exsA promoter activity and T3SS gene expression, overexpression of MvaT or MvaU inhibited P _exsA promoter activity. Disruption of mvaT, however, did not suppress the Vfr requirement for P _exsA promoter activity. Mutated MvaT/MvaU defective in transcriptional silencing exhibited dominant negative activity, resulting in a significant increase in P _exsA promoter activity. Because no effect of MvaT or MvaU on Vfr expression was detected, we propose a model in which the primary effect of MvaT/MvaU on T3SS gene expression is through direct silencing of the P _exsA promoter.IMPORTANCE Global regulatory systems play a prominent role in controlling the P. aeruginosa T3SS and include the Gac/RsmA, c-di-GMP, and Vfr-cAMP signaling pathways. Many of these pathways appear to directly or indirectly influence exsA transcription or translation. In this study, the histone-like proteins MvaT and MvaU are added to the growing list of global regulators that control the T3SS. MvaT and MvaU bind AT-rich regions in the genome and silence xenogeneic genes, including pathogenicity islands. The T3SS gene cluster has been horizontally transmitted among many Gram-negative pathogens. Control by MvaT/MvaU may reflect a residual effect that has persisted since the initial acquisition of the gene cluster, subsequently imposing a requirement for active regulatory mechanisms to override MvaT/MvaU-mediated silencing.

Keywords: ExsA; H-NS; MvaT; MvaU; Pseudomonas aeruginosa; type III secretion system.