Mining the Pseudomonas aeruginosa genome for regulators of type III secretion system gene expression using FT-Tn-seq

The Pseudomonas aeruginosa type III secretion system (T3SS) plays a crucial role in disabling and/or weakening host immune responses. Previous transposon mutagenesis screens have been successful in pinpointing genes essential for T3SS gene expression, yet the mechanisms behind their regulatory control are still largely unclear. In order to gain a comprehensive understanding of the genes involved in controlling T3SS gene expression, researchers developed a fluorescently tagged transposon mutagenesis screen (FT-Tn-seq).

This approach led to the identification of more than 70 novel genes crucial for T3SS gene expression, with 31 of these genes validated through a secondary screening process.

Upon further investigation, it was discovered that the deletion of shaC, a gene encoding a sodium-proton antiporter subunit, resulted in a notable impairment of T3SS gene expression. This effect was due to the simultaneous influence on exsA transcription and ExsA translation. Additionally, the disruption of wspF, a cyclic-di-GMP phosphodiesterase, led to a significant reduction in T3SS expression in strain PAK, although not in PA103. This difference in effect was likely attributed to the elevated levels of c-di-GMP in strain PAK.

The study also uncovered a new small regulatory RNA (ivy), which negatively influences T3SS gene expression. The expression of ivy inhibits T3SS gene expression and requires the RNA-chaperone Hfq for its inhibitory activity. These findings not only provide valuable insights into the regulation of T3SS gene expression in P. aeruginosa but also pave the way for the development of targeted anti-virulence therapeutics that specifically interfere with the T3SS.