NON-RIBOSOMAL PEPTIDE-BASED QUORUM SENSING SIGNAL IN PSEUDOMONAS AERUGINOSA

Abstract

The quorum sensing (QS) network in Pseudomonas aeruginosa plays a key role in the coordination of virulence factors production and contributes to its pathogenesis. Since the las system is at the top of the QS hierarchy, knocking it out would be akin to paralyzing the intercellular communication network and disarming the pathogen. However, P. aeruginosa clinical strains isolated from chronically infected cystic fibrosis patients were often found to be bearing a defective las system, yet remain profligate producers of virulence factors such as pyocyanin and elastase. Further, under phosphate limitation conditions, a las¿independent activation of the rhl QS system and pyocyanin production could be observed. This lead us to question whether there exists an alternative intercellular communication circuit that functions independent of LasIR, and whose effects are only apparent under non-standard, stressful environmental conditions, the very same conditions that P. aeruginosa is subjected to during infections. Therefore by random transposon mutagenesis, we screened for mutants with alterations in production of pyocyanin under low phosphate condition, and isolated a mutant disrupted at PA2305, a putative non-ribosomal peptide synthase gene. Subsequent in-frame deletion of PA2305 and in trans complementation of the latter confirmed that PA2305 positively affects the production of virulence factors pyocyanin and elastase, PQS and the rhl quorum sensing systems, and contributes to the full virulence of the bacteria towards nematodes C. elegans and zebrafish D. rerio. We have purified the putative enzyme product of the PA2305 NRPS cluster and named it Qrp (quinolone regulating peptide). When exogenously added, only 10 nM of Qrp is required to restore PQS production levels and its dependent phenotypes back to twice that of wild type levels. Further, we discovered that the ferric-binding siderophore pyochelin functions as a downstream Qrp-dependent signaling intermediate and forms a tripartite relay between Qrp and PQS, with a similar minimum effective concentration of 10 nM. Lastly, we also assayed the applicability of Qrp as a las-independent activator of quorum sensing and QS-dependent phenotypes in non-standard conditions. During phosphate depletion, the ¿lasI¿2305 double mutant shows an overall downregulation in virulence genes expression and decreases in pyocyanin, elastase and PQS production compared to the lasI mutant strain, signifying its positive effects on quorum sensing systems downstream of las. The dramatic upregulation of PA2305 expression observed in P. aeruginosa clinical isolates further implies that PA2305/ Qrp could represent a formidable force in driving forward the expression of quorum sensing-regulated virulence factors in the context where quorum sensors become defunct.