Loss of LasR function leads to decreased repression of Pseudomonas aeruginosa PhoB activity at physiological phosphate concentrations

Abstract

The Pseudomonas aeruginosa LasR transcription factor plays a role in quorum sensing (QS) across phylogenetically distinct lineages. However, isolates with loss-of-function mutations in lasR (LasR- strains) are commonly found in diverse settings, including infections where they are associated with worse clinical outcomes. In LasR- strains, the LasR-regulated transcription factor RhlR can also be stimulated by the activity of the two-component system PhoR-PhoB in low-inorganic phosphate (Pi) conditions. Here, we demonstrate a novel link between LasR and PhoB in which the absence of LasR increases PhoB activity at physiological Pi concentrations and increases the Pi concentration necessary for PhoB inhibition. PhoB activity was also less sensitive to repression by Pi in mutants lacking different QS regulators (RhlR and PqsR) and in mutants lacking genes required for QS-induced phenazine production, suggesting that decreased phenazine production is one reason for increased PhoB activity in LasR- strains. In addition, the CbrA-CbrB two-component system, which can be more active in LasR- strains, was necessary for increased PhoB activity in LasR- strains, and loss of the CbrA-CbrB-controlled translational repressor Crc was sufficient to activate PhoB in LasR+ P. aeruginosa. Phenazines and CbrA-CbrB affected PhoB activity independently. The ∆lasR mutant also had PhoB-dependent growth advantages in the Pi-deplete medium and increased virulence-associated gene expression at physiological Pi, in part through reactivation of QS. This work suggests PhoR-PhoB activity may contribute to the fitness and virulence of LasR- P. aeruginosa and subsequent clinical outcomes.IMPORTANCELoss-of-function mutations in the gene encoding the Pseudomonas aeruginosa quorum sensing (QS) regulator LasR occur frequently and are associated with worse clinical outcomes. We have found that LasR- P. aeruginosa have elevated PhoB activity at physiological concentrations of inorganic phosphate (Pi). PhoB activity promotes Pi acquisition as well as the expression of QS and virulence-associated genes. Previous work has shown that PhoB induces RhlR, another QS regulator, in a LasR- mutant in low-Pi conditions. Here, we demonstrate a novel relationship wherein LasR represses PhoB activity through the production of phenazines and Crc-mediated translational repression. This work suggests PhoB activity may contribute to the increased virulence of LasR- P. aeruginosa.

Keywords: CbrB; Crc; LasR; PhoB; PhoR; Pseudomonas aeruginosa; RhlR; phenazines; phosphate scavenging; quorum sensing.

Fig 1

PhoR-PhoB activity is elevated in LasR– P. aeruginosa at physiological Pi concentrations. (A) P. aeruginosa wild type (WT) and ∆lasR, ∆phoB, and ∆pstB mutants were spotted onto MOPS-glucose agar gradient plates with a range of Pi concentrations from 0.1 to 1 mM and 60 µg/mL bromo-4-chloro-3-indolyl phosphate (BCIP) to indicate alkaline phosphatase (AP) activity. (B) P. aeruginosa strains on MOPS-glucose agar with BCIP and 0.7 mM Pi. (C) Colony biofilms on MOPS agar with 0.2% glucose and BCIP with either 0.2 or 10 mM Pi. (D) P. aeruginosa strains grown as described in B +/– 5 µM 3OC12HSL dissolved in DMSO. (E) P. aeruginosa clinical isolate (CI) LasR+/LasR– pairs from the sputum of two pwCF grown as described in B. For panels F–H, P. aeruginosa was grown as colony biofilms on MOPS-glucose agar with 0.7 mM Pi. (F) AP activity in WT and ∆lasR was quantified using a colorimetric p-nitrophenylphosphate (PNPP) substrate. Data from replicates collected on the same day have the same shape. Data were analyzed using an unpaired, two-tailed t-test (n = 12). (G) phoA transcripts in WT and ∆lasR were measured by qRT-PCR on different days and normalized to the housekeeping gene transcript ppiD. Data were analyzed using a paired, two-tailed t-test (n = 6). (H) Levels of PhoB-controlled transcripts and phoB itself were assessed using NanoString multiplex technology. Nine PhoB-regulated transcripts are shown as normalized counts. Data were analyzed using a two-way ANOVA; there are significant differences between WT-∆lasR, WT-∆phoB, and ∆lasR– ∆lasR∆phoB (P < 0.0001, n = 2–3). There are no significant differences between the ∆phoB-∆lasR∆phoB mutants (P = 0.46, n = 3). Statistical differences for each transcript are available in File S1. For all panels, asterisks denote significance (P < 0.05 = *). For panels A–E, similar results were obtained in three replicate experiments; a representative experiment is shown.

Fig 2

P. aeruginosa quorum sensing mutants have active PhoB at higher Pi than the wild type. (A) Colony biofilms of WT and the indicated quorum sensing (QS) mutants (∆lasR, ∆rhlR, and ∆pqsR) were grown on MOPS-glucose agar with 0.7 mM Pi and BCIP (top) or on agar without BCIP for analysis of AP activity using the colorimetric PNPP substrate. Data from replicates collected on the same day have the same shape. Data were analyzed by an ordinary one-way ANOVA with Tukey’s multiple comparisons tests (n = 4). (B) P. aeruginosa colony biofilms grown on MOPS-glucose agar with BCIP and 0.5–1.2 mM Pi. (C) Threshold Pi was determined as described in the Methods. Data were analyzed using a one-way ANOVA and Tukey’s multiple comparisons tests (n = 3). Smaller asterisks denote significance from the WT. For all panels, asterisks denote significance (P < 0.05 = *).

Fig 3

The loss of phenazines promotes PhoB activity. (A) P. aeruginosa WT and mutants ∆phz1, ∆phz2, and ∆phz (lacking phz1 and phz2 operons) grown on MOPS-glucose agar with 0.7 mM Pi and BCIP (top) or on a medium without BCIP for analysis of the AP activity using the colorimetric PNPP substrate. Data from replicates collected on the same day have the same shape (n = 7). (B) WT and the ∆phz, ∆lasR, and ∆lasR∆phz mutants grown as described in A. Data points represent the average of 2–4 colonies analyzed the same day. There were no significant differences between ∆lasR and ∆lasR∆phz (P = 0.92, n = 11). Data in A–B were analyzed using a one-way ANOVA and Tukey’s multiple comparisons tests. (C) P. aeruginosa colony biofilms grown on MOPS-glucose agar with BCIP and 0.5–1.2 mM Pi. The WT and ∆lasR mutant are also represented in Fig. 2C. Threshold Pi was determined as described in the Methods. Data were analyzed using a one-way ANOVA and Tukey’s multiple comparisons tests (n = 3). (D) P. aeruginosa was grown as described in C with either 200 µM PCA in DMSO or DMSO alone (n = 3). Data were analyzed using a matched two-way ANOVA with Sidak’s multiple comparisons tests. For all panels, asterisks denote significance (P < 0.05 = *).

Fig 4

The CbrA-CbrB-Crc pathway promotes PhoB activity in LasR– strains. (A) A proposed model of the relationship between the CbrA-CbrB and PhoR-PhoB two-component systems; crcZ, a small RNA; Crc, which acts as a translational repressor in complex with Hfq. For figures B–D, AP activity in indicated strains grown on MOPS-glucose agar with 0.7 mM Pi and BCIP (top) or on the medium without BCIP for analysis of AP activity using the colorimetric PNPP substrate. Data from replicates collected on the same day have the same shape. Data were analyzed using a one-way ANOVA and Tukey’s multiple comparisons tests. (B) AP activity in ∆lasR, ∆lasR∆cbrB, ∆lasR∆cbrB +cbrB, and ∆lasR∆cbrB∆crc mutants (n = 3). (C) AP activity in the WT, ∆crc mutant, and its complemented derivative (n = 3-6). (D) AP activity in ∆lasR, ∆crc, and ∆lasR∆crc (n = 8). (E) P. aeruginosa was grown on MOPS-glucose agar with BCIP and a gradient of Pi (0.5–1.2 mM). The WT and ∆lasR mutant are also represented in Fig. 2C. The Pi threshold was determined as described in the Methods. Data were analyzed using a one-way ANOVA and Tukey’s multiple comparisons tests (n = 3). For all panels, asterisks denote significance (P < 0.05 = *).

Fig 5

Loss of phenazines and loss of Crc have an additive effect on PhoB activity in LasR+ P. aeruginosa at 0.7 mM Pi. (A) P. aeruginosa colony biofilms grown on MOPS-glucose agar with 0.7 mM Pi for AP activity analysis using a colorimetric PNPP substrate. Shapes indicate data collected on the same day. Data were analyzed using a one-way ANOVA and Tukey’s multiple comparisons tests (n = 6). (B) P. aeruginosa colony biofilms grown on MOPS-glucose agar with BCIP and 0.5–1.2 mM Pi. Threshold Pi was determined as described in the Methods. Data were analyzed using a one-way ANOVA and Tukey’s multiple comparisons tests (n = 3). For all panels, asterisks denote significance (P < 0.05 = *).

Fig 6

PhoB mediates growth advantages and gene expression in ∆lasR P. aeruginosa. (A) P. aeruginosa WT and ∆lasR, ∆phoB, ∆lasR∆phoB, and ∆pstB mutants were grown in MOPS-glucose medium with no Pi. Data were analyzed using a two-way ANOVA with Tukey’s multiple comparisons tests comparing each mutant to the WT (n = 3). ∆lasR is significantly different after 4 hours (P < 0.02). ∆pstB is significantly different at 10 hours (P = 0.037). (B) NanoString analysis of PhoB-regulated transcripts shown as log₁₀ normalized counts (n = 2–3). Transcripts were significantly elevated in ∆lasR compared to the WT and ∆lasR∆phoB (P < 0.0001). There was no significant difference between the WT and ∆phoB mutant (P = 0.28). (C) NanoString analysis of QS-regulated transcripts shown as log₁₀ normalized counts (n = 2–3). There was no significant difference between the WT or ∆phoB mutant (P = 0.96). Transcripts were significantly elevated in ∆lasR compared to ∆lasR∆phoB (P < 0.0001). Complete NanoString data set with statistical analysis of each transcript is available in File S1.

Fig 7

Model of P. aeruginosa PhoB activity regulation at physiological Pi. LasR+ P. aeruginosa have increased repression of PhoB by Crc and phenazines at physiological Pi. LasR activity contributes to the expression of RhlR and QS activity. LasR– P. aeruginosa have increased PhoB activity, leading to the expression of RhlR and QS activity as well as phospholipases at physiological Pi.