Mining the Pseudomonas aeruginosa genome for regulators of type III secretion system gene expression using FT-Tn-seq
Abstract:
The Pseudomonas aeruginosa type III secretion system (T3SS) is an important virulence determinant used to mute and/or impair host immune defenses. Traditional transposon mutagenesis screens have been instrumental in identifying genes required for T3SS gene expression but our understanding of how those genes orchestrate regulatory control remains incomplete. A comprehensive inventory of the genes involved in the control of T3SS gene expression would help define regulatory mechanisms and provide additional targets for anti-virulence therapeutics. Here, we designed a fluorescently tagged transposon mutagenesis screen (FT-Tn-seq) and identified over 70 new genes important for T3SS gene expression. A role for 31 of those genes was validated using a secondary screen. Several candidate genes were selected for further studies. We demonstrate that deletion of shaC, which encodes a sodium-proton antiporter subunit, results in a significant defect in T3SS gene expression. That defect results from a combined effect on exsA transcription and ExsA translation. Deletion of wspF, a cyclic-di-GMP phosphodiesterase, results in a significant reduction of T3SS expression in strain PAK, but not in PA103. The effect on the T3SS is likely due to elevated levels of c-di-GMP in PAK. Finally, we identified a new sRNA (ivy) that participates in the control of the T3SS. Expression of ivy inhibits T3SS gene expression and requires the RNA-chaperone Hfq for inhibitory activity. Findings from this study will open new research avenues to better understand regulation of T3SS gene expression in P. aeruginosa and develop T3SS-specific therapeutic interventions.
