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.

Importance: Chronic Pseudomonas aeruginosa infections, such as those in patients with the genetic disease cystic fibrosis, are notable in that mutants with defects in the quorum-sensing transcription factor LasR frequently arise. In laboratory strains of P. aeruginosa, quorum sensing activates transcription of dozens of genes, many of which encode virulence factors, such as secreted proteases and hydrogen cyanide synthases. In well-studied laboratory strains, LasR-null mutants have a quorum-sensing-deficient phenotype. Therefore, the presence of LasR variants in chronic infections has been interpreted to indicate that quorum-sensing-regulated products are not important for those infections. We report that some P. aeruginosa LasR variant clinical isolates are not LasR-null mutants, and others have uncoupled a second quorum-sensing system, the RhlR system, from LasR regulation. In these uncoupled isolates, RhlR independently activates at least some quorum-sensing-dependent genes. Our findings suggest that quorum sensing plays a role in chronic P. aeruginosa infections, despite the emergence of LasR coding variants.

FIG 1

Amino acid changes, functions, and phenotypes of LasR variants. (A) Location of single-nucleotide substitutions in the EPIC isolates mapped to the LasR amino acid sequence. Each unique amino acid substitution or early termination in the collection is shown in this schematic of the polypeptide. The signal and DNA-binding domains of LasR are highlighted. *, a mutation resulting in a stop codon. Where multiple letters are present, there was more than one unique substitution within the collection at the same residue (e.g., “PR” at residue 23). (B) Activity of LasR in an E. coli expression system. We cloned lasR from the isolates and expressed the cloned gene under control of an arabinose-inducible promoter in E. coli, which also contained a LasR-responsive lacZ reporter. The E. coli reporters were grown in the presence of 2 µM 3OC₁₂-HSL. Data are the percentage β-galactosidase activity compared to lasR cloned from strain PAO1 (red bar). (C) Activity of LasR in clinical isolates. We electroporated clinical isolates of P. aeruginosa PAO1 (red) or a PAO1 ΔlasR mutant (blue) with a plasmid containing a LasR-responsive GFP reporter, and we measured fluorescence in the presence (dark bars) or absence (light bars) of 3OC₁₂-HSL after 18 h of growth. (D and E) Concentrations of 3OC₁₂-HSL (D) and C₄-HSL (E) after 18 h of P. aeruginosa growth. Strain PAO1 produced 2 µM 3OC₁₂-HSL and 9.5 µM C₄-HSL. Data are displayed as a percentage of the amount of AHL produced by PAO1 (red bar). (F) Pyocyanin production after 18 h in King’s A medium. PAO1 again was used as the reference (red bar) and produced 5.5 µg/ml pyocyanin. (G) Combined production of PQS and HHQ by the various isolates after 18 h in LB broth, normalized to production by PAO1. Error bars represent mean ± ranges for results of three individual experiments.

FIG 2

AHLs and RhlR mediate QS in some LasR-null clinical isolates. (A and B) AHL dependence of elastase and pyocyanin production. LasR-null mutants were grown in broth with (light bars) or without (dark bars) 20 µg/ml AiiA lactonase. In all cases, AiiA lactonase-grown cultures did not have measurable signals after 18 h. We measured production of elastase (A) and pyocyanin (B) from these cultures after 18 h of growth. PAO1 (red) and an AHL QS-deficient strain, mutant strain PAO1 ΔlasR ΔrhlR ΔqscR (ΔRRR) (blue), are shown for comparison. Elastase activity is shown in arbitrary units. (C) RhlR activity in LasR-null isolates. We electroporated AHL-responsive isolates with an RhlR-responsive rhlA-gfp reporter and measured fluorescence at 18 h. Fluorescence per cell is reported for clinical isolates (dark bars) with PAO1 (red), PAO1 ΔlasR, and PAO1 ΔRRR (blue) shown for comparison. Isolate E183, which demonstrated wild-type function, is also included. Growth with AiiA lactonase (light bars) abrogated the fluorescence in most cases. (D) Temporal profile of RhlR activation of the rhlA-gfp reporter. Strains E42 (closed circles), E131 (closed triangles), and E165 (open circles) are shown. PAO1 (closed squares) is shown in red, and PAO1 ΔlasR (inverted triangles) is shown in blue. In all panels, error bars show standard errors for results from four independent measurements. *, a statistically significant decrease (P < 0.05) in production of elastase (A), pyocyanin (B), or GFP (C) in the presence of AiiA, compared to the buffer control (two-tailed t test).