LasR Variant Cystic Fibrosis Isolates Reveal an Adaptable Quorum-Sensing Hierarchy in Pseudomonas aeruginosa

Abstract

Chronic Pseudomonas aeruginosa infections cause significant morbidity in patients with cystic fibrosis (CF). Over years to decades, P. aeruginosa adapts genetically as it establishes chronic lung infections. Nonsynonymous mutations in lasR, the quorum-sensing (QS) master regulator, are common in CF. In laboratory strains of P. aeruginosa, LasR activates transcription of dozens of genes, including that for another QS regulator, RhlR. Despite the frequency with which lasR coding variants have been reported to occur in P. aeruginosa CF isolates, little is known about their consequences for QS. We sequenced lasR from 2,583 P. aeruginosa CF isolates. The lasR sequences of 580 isolates (22%) coded for polypeptides that differed from the conserved LasR polypeptides of well-studied laboratory strains. This collection included 173 unique lasR coding variants, 116 of which were either missense or nonsense mutations. We studied 31 of these variants. About one-sixth of the variant LasR proteins were functional, including 3 with nonsense mutations, and in some LasR-null isolates, genes that are LasR dependent in laboratory strains were nonetheless expressed. Furthermore, about half of the LasR-null isolates retained RhlR activity. Therefore, in some CF isolates the QS hierarchy is altered such that RhlR quorum sensing is independent of LasR regulation. Our analysis challenges the view that QS-silent P. aeruginosa is selected during the course of a chronic CF lung infection. Rather, some lasR sequence variants retain functionality, and many employ an alternate QS strategy involving RhlR.

The Fitness of Pseudomonas aeruginosa Quorum Sensing Signal Cheats Is Influenced by the Diffusivity of the Environment

“…Our work opens up new avenues for exploring how, when, and why bacterial signaling evolves in different environments and why we find a variety of QS mutant genotypes and phenotypes in natural environments (8–14, 38, 39). P. aeruginosa clones carrying mutations in the lasI (3O-C 12 -HSL) and rhll (C 4 -HSL) QS signals have been isolated from chronically infected cystic fibrosis patients and mechanically ventilated hospital patients (8–11).…”

Three-Dimensional Microbiome and Metabolome Cartography of a Diseased Human Lung

“…The spatial variation in quinolones and rhamnolipids may stem from the variation in genetic mutations in quinolone biosynthetic pathways and physical separation of genetically different variants of Pseudomonas , and co-occurrence of other micro-organisms and exposure to different levels of antibiotics. Previously, different isolates cultured from one CF patient at a given time and between patients over time had accumulated different mutations and varied in genetic potential for production of quinolones (Feltner et al, 2016; Markussen et al, 2014; Smith et al, 2006; Wilder et al, 2009). Our maps showed that antibiotic penetration or presence of a specific local microbial community did not result in the spatial variation observed for quinolones and rhamnolipids and hence is most likely accounted for by the genetic variation in the Pseudomonas subpopulation and local environmental variation.…”

Quorum Quenching of Nitrobacter winogradskyi Suggests that Quorum Sensing Regulates Fluxes of Nitrogen Oxide(s) during Nitrification

“…While mutational analysis is the preferred method to study QS, there are many bacteria, including those of environmental importance, with no established genetic system and new techniques are needed to determine the purpose of QS in these organisms. Quorum quenching also represents a different methodology to study animal pathogens and social evolution (20, 22–24). Mutant construction in most bacteria takes several generations of selection that may introduce unintended changes, particularly during social-evolution experiments.…”

“…Mutant construction in most bacteria takes several generations of selection that may introduce unintended changes, particularly during social-evolution experiments. As previously suggested, quorum quenching through addition of a lactonase or chemical inhibitor is a quicker method to induce a QS-deficient phenotype without the need for several generations of selection and the possibility of pleiotropic effects (23, 24, 37). …”

“…AiiA, an AHL lactonase identified from Bacillus spp., is a well-characterized enzyme that specifically hydrolyzes the homoserine lactone (HSL) ring of AHLs, regardless of the chain length of the acyl group or other moiety (20, 21). So-called “quorum quenching” approaches have been implemented through both heterologous expression in a host of interest and addition of purified AHL lactonase (18, 20, 22–24). In this study, we used purified AiiA lactonase to identify QS-controlled gene expression and phenotypes in Nitrobacter winogradskyi , a well-characterized NOB that is currently not genetically tractable.…”