The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

Abstract

The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the wellcharacterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo. Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS. IMPORTANCEAlthough members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box helicases are now thought to promote translation by enhancing ribosomal recruitment. We identify here an RNA helicase that plays a critical role in promoting ExsA synthesis, the central regulator of the Pseudomonas aeruginosa type III secretion system, and provide additional evidence that DEAD box helicases directly stimulate translation of target genes. The finding that DeaD stimulates exsA translation adds to a growing list of transcriptional and posttranscriptional regulatory mechanisms that control type III gene expression. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that expresses a large array of virulence factors to cause both acute and chronic infections (1). P. aeruginosa infections generally initiate as acute infections that can range from self-limiting folliculitis to life-threatening ventilator-associated pneumonia (1). P. aeruginosa utilizes a type III secretion system (T3SS) to promote host cell cytotoxicity and avoid phagocytosis (2, 3). The T3SS is a multiprotein complex that spans the cell envelope and functions by directly ...

Post-transcriptional regulation of type III secretion in plant and animal pathogens

“…DEAD-Box RNA helicases, so named for a conserved Asp-Glu-Ala-Asp catalytic motif, are ATP-dependent RNA binding proteins that hydrolyze ATP to ADP and partially unfold RNA substrates [44]. P. aeruginosa has 7 putative DEAD box helicases, one of which (DeaD) is essential for T3SS gene expression [45]. A mutant lacking deaD has a specific decrease in ExsA synthesis.…”

“…A mutant lacking deaD has a specific decrease in ExsA synthesis. Although some DEAD-Box RNA helicases function as a component of the RNA degradosome, the half-life of exsA transcript is unaffected in a deaD mutant [45]. A role in translation seems more likely as purified DeaD stimulates ExsA translation in vitro and DeaD activity is sensitive to mutations in the DEAD motif that presumably disrupt catalytic activity (Fig.…”

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

“…Several distinct roles have now been described for the exsBexsA intergenic region. The region contains Vfr-and VqsM-dependent promoters (34) and contributes to posttranscriptional regulation of ExsA expression by the DeaD RNA-dependent helicase (35) and the small RNA binding protein RsmA (36). The nucleotide substitutions used to disrupt the Vfr binding site, therefore, may have also altered exsCEBA mRNA stability or posttranscriptional effects on ExsA synthesis or may have impaired the inherent activity of the P exsA promoter (i.e., Vfr independent).…”

Targeting the Type Three Secretion System in Pseudomonas aeruginosa

“…The DeaD RNA helicase increases ExsA expression at the post-transcriptional level, possibly by relieving an inhibitory structure within the exsA mRNA that normally prevents its translation [77]. Salicylidene acylhydrazides have been screened as inhibitors of T3SS transcription in different bacterial species, such as Chlamydia trachomatis or Yersinia pseudotuberculosis [49].…”