Identification of a novel RhlI/R-PrrH-LasI/Phzc/PhzD signalling cascade and its implication in <i>P. aeruginosa</i> virulence

Small regulatory RNAs (sRNAs) act as key regulators in many bacterial signalling cascades. However, in <i>P. aeruginosa</i>, the sRNAs involved in quorum sensing (QS) regulation and their function are still largely unknown. Here, we explored how the <i>prrH</i> locus sRNA influences <i>P. aeruginosa</i> virulence in the context of the QS regulatory network. First, gain- and loss-of-function studies showed that PrrH affects pyocyanin, elastase and rhamnolipid production; biofilm formation; and swimming and swarming motility and impaired the viability of <i>P. aeruginosa</i> in human whole blood. Next, our investigation disclosed that LasI and PhzC/D were directly repressed by PrrH. In addition, RhlI, the key member of the <i>rhl</i> QS system, diminished the expression of PrrH and enhanced the expression of downstream genes. Bioinformatics analysis found two binding sites of RhlR, the transcription factor of the <i>rhl</i> system, on the promoter region of <i>prrH</i>. Further β-galactosidase reporter and qPCR assays confirmed that PrrH was transcriptionally repressed by RhlR. Collectively, our data identified a novel RhlI/R-PrrH-LasI/PhzC/PhzD regulatory circuitry that may contribute to <i>P. aeruginosa</i> pathogenesis. Our findings indicate that PrrH is a quorum regulatory RNA (Qrr) in <i>P. aeruginosa</i> and provide new insight into PrrH’s function.