Vfr Directly Activates exsA Transcription To Regulate Expression of the Pseudomonas aeruginosa Type III Secretion System

Abstract

The Pseudomonas aeruginosa cyclic AMP (cAMP)-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single-cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in a vfr mutant, wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive-feedback loops, we tested the hypothesis that virulence factor regulator (Vfr) regulates the expression of exsA. ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (P exsA ) located immediately upstream of exsA. P exsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a P exsA promoter probe was demonstrated by electrophoretic mobility shift assays, and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and P exsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the P exsA promoter, which is internal to the previously characterized exsCEBA operon. IMPORTANCEVfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa. Regulation by Vfr allows for the coordinate production of related virulence functions, such as type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA. Pseudomonas aeruginosa is an environmental bacterium typically found in soil and water. The organism is also an important opportunistic pathogen of humans, especially in those with neutropenia, severe burns, and cystic fibrosis (1, 2). Both the physical and host environments expose P. aeruginosa to unique stresses that challenge survival. Reprogramming gene expression is critical for adaptation. The host signals to which the bacteria respond are not entirely clear but likely include contact with host cell surfaces or host-derived macromolecules, temperature, osmolarity, pH, iron limitation, and oxidative stress (3-5). Bacterial genes induced within mammalian hosts include those important for iron acquisition, carbon utilization, and ...

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

“…1). More recently, the same intergenic region was shown to contain an exsA-specific promoter (P exsA ) (30,32). We hypothesized that MvaT and MvaU control T3SS gene expression by modulating P exsA promoter activity.…”

“…ExsA is encoded in an operon beginning with exsC and autoregulates its transcription via the P exsC promoter (29). ExsA is also transcribed from another promoter, P exsA , which is located in the intergenic region between exsB and exsA (30). The P exsA promoter is under the transcriptional control of the global virulence regulator Vfr, a cAMP-binding protein (30).…”

“…ExsA is also transcribed from another promoter, P exsA , which is located in the intergenic region between exsB and exsA (30). The P exsA promoter is under the transcriptional control of the global virulence regulator Vfr, a cAMP-binding protein (30). Transcriptional regulators Fis (31) and VqsM (32) have also been reported to have positive effects on exsA transcription.…”

Targeting the Type Three Secretion System in Pseudomonas aeruginosa

“…Together with cAMP (produced by CyaB), Vfr (cAMP-dependent DNA-binding protein) increases exsA transcription from a newly identified promoter located immediately upstream of exsA [76]. GacSA-RsmYZ-RsmA: in the presence of environmental stimuli, the tripartite sensor histidine kinase GacS activates its cognate response regulator GacA by phosphorylation, which in turn induces the expression of regulatory small RNAs RsmY and RsmZ.…”

Comparison of the Structure, Regulation and Functions between Type Three and Type Six Secretion System in Gram-Negative Bacteria

“…T3SS gene expression have also been likened to quorum sensing. Recently, Marsden et al discovered that virulence factor regulator (Vfr) can regulate expression of the Pseudomonas aeruginosa type III secretion system, by directly activating exsA transcription [24]. Vfr is a cAMP-dependent DNA-binding protein.…”