The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity

Abstract

Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signalling molecule adenosine 3', 5'-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems.

FIG. 1. Biochemical characterization of P. aeruginosa CyaB

A) Coomassie Blue-stained SDS-polyacrylamide gel of purified recombinant CyaB_217–463. A 1.5 µg portion of protein was applied and molecular-mass standards (in kDa) are indicated. B) CyaB_217–463 specific activity (n=4; ± standard error of the mean (SEM)) in the presence of 200 µM Mn²⁺-ATP (68 nM protein, 40°C, pH 8.5) or 1 mM Mg²⁺-ATP (13 µM protein, 40°C, pH 8.5). C) Wild type and mutant (K274A or T351A) CyaB_217–463 specific activities (n=8; ± SEM) assayed with 250 µM Mn²⁺-ATP (0.7 µM protein, 40°C, pH 7.5). Percentage indicates the portion of wild type activity. All values were significantly different (p<0.0001) compared to the value for wild type CyaB.

FIG. 2. Activity of the lacP1-lacZ transcriptional reporter is cAMP-dependent and reflects the level of intracellular cAMP

A) Schematic diagram of the lacP1-lacZ promoter reporter. The lacP1 promoter region of the E. coli lacZ gene contains two LacI binding sites (gray rectangles) and a CRP binding site (black rectangle). The transcriptional start site (+1) and −35 and −10 regions are indicated. The region of the lacP1 promoter extending 177 bp upstream of the transcriptional start site was fused to the lacZ gene. B) Direct measurement of cAMP by enzyme immunoassay (left axis, black bars) and β-galactosidase activity (right axis, gray bars) in the wild type strain (wt), the AC mutants (cyaA, cyaB and cyaAB), or the vfr mutant (vfr); all strains contained the lacP1-lacZ reporter. For the β-galactosidase assay, the data represent three independent experiments with four replicates each and values are reported as the mean ± SEM; values for all strains are significantly different (p<0.0001) when compared pair wise to the values for all other strains shown. For the cAMP assay, the data represent three independent experiments, each with three replicates and values are reported as the mean ± SEM. The asterisk (*) indicates that the values for the indicated strains are significantly different (p≤0.019) compared to the value for the wild type strain. C) A defect in cAMP synthesis can be restored by exogenous cAMP. β-galactosidase activity in the wild type strain (wt), AC double mutant (cyaAB) or vfr mutant (vfr) grown in the presence (+) or absence (−) of 50 mM cAMP. All strains contained the lacP1-lacZ reporter. The data represent two independent experiments with three replicates each and values are reported as the mean ± SEM.

FIG. 3. Quantitation of cAMP reporter activity in Tn insertion mutants

β-galactosidase activity of the cyaA mutant (parent), the AC double mutant (cyaAB) and representative Tn insertion mutants in the PAKcyaA∷lacP1-lacZ background grown in broth culture to mid-logarithmic growth phase. The gene disrupted by Tn insertion is indicated for each strain. The pirA mutant is a clone from the Tn insertion library screen that had a blue colony phenotype on X-gal indicator plates. The data represent three independent experiments with four replicates each and values are reported as the mean ± SEM. All values (except pirA∷Tn) were significantly different (p<0.0001) compared to the value for the parent strain.

FIG. 4. Inactivation of TFP/Chp genes affects intracellular cAMP

A) Direct measurement of cAMP by enzyme immunoassay (left axis, black bars) and β-galactosidase activity (right axis, gray bars) in the cyaA mutant (parent), the double AC mutant (cyaAB), or the indicated non-polar deletion mutant in the PAKcyaA∷lacP1ΔlacI-lacZ background. For the β-galactosidase assay, the data represent three independent experiments with two replicates each and values are reported as the mean ± SEM; all values (except pilB) were significantly different (p≤0.0003) compared to the value for the parent strain. For the cAMP assay, the data represent three independent experiments with three replicates each and values are reported as the mean ± SEM; the asterisk (*) indicates that the values for the indicated strains are significantly different (p≤0.04) compared to the value for the parent strain. B) Complementation of the TFP/Chp mutants by expression of the corresponding gene in trans. Activity of the lacP1ΔlacI-lacZ reporter in the non-polar deletion mutant strains harboring either the empty pMMB67EH vector (V, black bars) or the pMMB-based plasmid (C, gray bars) encoding the corresponding complementing gene. The TFP/Chp genes were each expressed in the pMMB plasmid backbone listed in the experimental procedures. The cyaAB mutant (cyaAB) harboring either pMMB67EH (V) or pMMBV2-cyaA (C) serve as controls for complementation of lacP1ΔlacI-lacZ reporter activity. Strains were grown in broth culture containing IPTG at the concentration determined to be optimal for complementation of the mutant phenotype (see text) and assayed at mid-logarithmic growth phase. Relative reporter activity is reported as the β-galactosidase activity of an individual mutant strain divided by that of the cyaA mutant parent strain harboring pMMB67EH. The data represent a minimum of three independent experiments with two replicates each and values are reported at the mean ± SEM.

FIG. 5. CyaB protein levels do not account for altered intracellular cAMP in the TFP/Chp mutants

CyaB immunoblot of whole cell lysates from the cyaA parent strain (cyaA), the double AC mutant (cyaAB) and the indicated mutants in the PAKcyaA∷lacP1ΔlacI-lacZ background. Samples were normalized based on total protein and blots were probed with affinity-purified CyaB-specific rabbit antiserum.

FIG. 6. The Chp system regulates cAMP at the level of CyaB activity

β-galactosidase activity of the wild type strain (wt), the double AC mutant (cyaAB), and the cyaAB, pilG (cyaABpilG) and cyaAB, pilH (cyaABpilH) triple mutants. All strains contained the lacP1ΔlacI-lacZ reporter and carried either the pMMB67EH empty vector or pMMBV2-cyaA (cyaA) or pMMBV2-cyaB (cyaB). Bacteria were grown in broth culture in the presence (+) or absence (−) of 50 mM cAMP. The data represent two independent experiments with four replicates each and values are reported as the mean ± SEM. The asterisk (*) indicates that the values for the indicated strains are significantly different (p<0.0001) when compared pair wise to the value for the cyaAB mutant carrying pMMBV2-cyaB. B) CyaB immunoblot of whole-cell lysates from the above strains. Samples were normalized based on bacterial number and blots were probed with affinity-purified CyaB-specific rabbit antiserum.

FIG. 7. The role of the TFP/Chp genes in TFP production and twitching motility

A) Coomassie Blue-stained SDS-polyacrylamide gel showing the amount of pilin in purified surface pilus fractions from the cyaA mutant (cyaA), a non-piliated pilA mutant (pilA), the double AC mutant (cyaAB) and the indicated mutants in the PAKcyaA∷lacP1ΔlacI-lacZ background. Samples for pilus preparations were normalized based on bacterial number. Pilus preps were performed three times and a representative gel is shown. B) Immunoblot of whole-cell lysates from the above strains probed with a pilin-specific monoclonal antibody. C) Twitching motility zones for the indicated strains. The data represent at least three independent experiments with a minimum of three replicates each and values are reported as the mean ± SEM.

FIG. 8. The Chp system regulates TFP production but not twitching motility via control of intracellular cAMP

A) Coomassie Blue-stained SDS-polyacrylamide gel showing the amount of pilin in purified pilus fractions from the wild type (wt) strain, the double AC mutant (cyaAB), the pilG and pilH mutants in a cyaA background (pilG and pilH, respectively) or the cyaAB, pilG (cyaABpilG) and cyaAB, pilH (cyaABpilH) triple mutants. All strains contained the lacP1ΔlacI-lacZ reporter. Bacteria were grown on LB agar plates in the presence (+) or absence (−) of 20 mM cAMP and were normalized based on bacterial number. B) Immunoblot of whole-cell lysates from the above strains probed with a pilin-specific monoclonal antibody. C) Twitching motility zones for the indicated strains grown in the presence (+) or absence (−) of 20 mM cAMP. The data represent two independent experiments with three replicates each and values are reported as the mean ± SEM. The asterisk (*) indicates that the values for the indicated strains are significantly different (p<0.001) compared to the value for the cyaAB mutant grown in the presence of cAMP.

FIG. 9. Model for coupling environmental sensing to virulence factor expression via the P. aeruginosa Chp chemosensory system

1) In response to unknown signal(s), the Chp system controls cAMP synthesis by modulating activity of the AC enzyme CyaB, which is predicted to be integral inner membrane protein. The cAMP-dependent transcription factor Vfr mediates transcriptional activation of multiple virulence systems, including 2) T3S, QS, flagellar biosynthesis, multiple toxins and degradative enzymes and 3) the structural and regulatory factors responsible for TFP biogenesis. 4) The Chp system also exerts a regulatory effect on TFP function (twitching motility) that is cAMP-independent.