Pseudomonas aeruginosa PA80 is a cystic fibrosis isolate deficient in RhlRI quorum sensing

Abstract

Pseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI’s) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.

Evolution of Quorum Sensing in Pseudomonas aeruginosa Can Occur via Loss of Function and Regulon Modulation

“…In contrast, we found evidence for QS regulon modulation in the Rhl mutants. These mutants arose at a much lower frequency than the Las mutants, similar to previous studies ( 54 , 55 ). Both the phenotypic profiles and gene expression analyses revealed that there is no complete loss of QS function, but rather a change in the expression of the QS-regulated trait profile.…”

Bacterial community structure and mucin drive Pseudomonas aeruginosa diversification and pathoadaptation

“…These results support the recent findings of Luján et al, who demonstrated that pqsR-regulated QS system is retained in the presence of competing taxa and mucin, likely due to the fitness benefits of retaining the redox agent pyocyanin, an effective anti-competitor toxin 18 . Mutations in QS transcriptional regulators pqsR and vqsM are commonly observed during in vitro evolution of P. aeruginosa [17][18][19]38,48,49 , but mutations in these genes are rarely observed in chronic adapted strains isolated from CF sputum 5,50 . Our results suggest that mutations within these QS transcriptional regulators are dependent upon the interaction between the community composition and the physical environment, whereby intense competition coupled with the presence of mucin provide strong selection for the retention of these QS transcriptional regulators.…”

Citrinin Is a Potential Quorum Sensing Inhibitor against Pseudomonas aeruginosa

“…However, in recent years, more and more studies have found that the rhl system occupies a more dominant position. The rhl system is directly related to the drug resistance of P. aeruginosa , and in the case of poor nutritional conditions, structural level changes, and can regulate the expression of related genes of the las system [ 27 ]. In addition, lasR deletion mutants are prevalent in patients with chronic P. aeruginosa infections, and these mutants have stronger rhl system activity than the wild strains [ 28 ].…”