Peter J. Intile, Grant J. Balzer, Matthew C. Wolfgang, Timothy L. Yahr

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 well-characterized 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. Here we report that PA2840, renamed DeaD, stimulates ExsA synthesis at the post-transcriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. Additionally, 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. Although 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. Here, we identify 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 post-transcriptional regulatory mechanisms that control type III gene expression.