An orphan chemotaxis sensor regulates virulence and antibiotic tolerance in the human pathogen <em>pseudomonas aeruginosa</em>

The synthesis of virulence factors by pathogenic bacteria is highly regulated and occurs in response to diverse environmental cues. An array of two component systems (TCSs) serves to link perception of different cues to specific changes in gene expression and/or bacterial behaviour. Those TCSs that regulate functions associated with virulence represent attractive targets for interference in anti-infective strategies for disease control. We have previously identified PA2572 as a putative response regulator required for full virulence of Pseudomonas aeruginosa, the opportunistic human pathogen, to Galleria mellonella (Wax moth) larvae. Here we have investigated the involvement of candidate sensors for signal transduction involving PA2572. Mutation of PA2573, encoding a probable methyl-accepting chemotaxis protein, gave rise to alterations in motility, virulence, and antibiotic resistance, functions which are also controlled by PA2572. Comparative transcriptome profiling of mutants revealed that PA2572 and PA2573 regulate expression of a common set of 49 genes that are involved in a range of biological functions including virulence and antibiotic resistance. Bacterial two-hybrid analysis indicated a REC-dependent interaction between PA2572 and PA2573 proteins. Finally expression of PA2572 in the PA2573 mutant background restored virulence to G. mellonella towards wild-type levels. The findings indicate a role for the orphan chemotaxis sensor PA2573 in the regulation of virulence and antibiotic tolerance in P. aeruginosa and indicate that these effects are exerted in part through signal transduction involving PA2572

Domain organizations of PA2571, PA2572 and PA2573.

<p>Domains were predicted by SMART with amino acid positions indicated. Domain abbreviations are as follows; HisKA (Histidine Kinase A phosphoacceptor domain), HATPase_c (Histidine kinase-like ATPase), REC (Receiver domain), HD (superfamily with predicted or known phosphohydrolase activity), HAMP (Histidine kinase, Adenylyl cyclase, Methyl binding protein, Phosphatase domain), and MA (Methyl-accepting chemotaxis-like domain). Vertical bars represent predicted transmembrane domains (SOUSI). Black lines below figures represent constructs cloned into either pBT or pTRG vectors to assess potential protein-protein interactions using the bacterial two-hybrid assay.</p

Production of virulence factors.

<p>Effects of mutations of <i>PA2572</i> and <i>PA2573</i> on production of pyoverdine (A) and pyocyanin (B) in <i>P. aeruginosa</i>. Complemented strains (indicated by pPA2572 and pPA2573) had wild-type levels of the factors. Phenotypic effects of mutations were complemented with reintroduction of genes into mutant strains. Error bars represent the mean ± standard deviation of three independent experiments in triplicate.</p

Venn diagrams representing unique and overlapping gene regulation in PA2572 and PA2573 mutants.

<p>Mutant strains were compared to the parental PAO1 strain during exponential growth phase (0.6–0.7 OD₆₀₀) in LB media. Genes were considered to have significant alteration in expression based on two-fold changes compared with wild-type. The numbers of genes regulated in an upward direction (A) and a downward direction (B) are shown.</p

Validation of transcriptome data using quantitative and semi-quantitative real-time PCR.

<p>Abbreviations and symbols: NS (Not significant, fold change was less than 2.0), RND (Resistance-Nodulation-Cell Division), + (represents increased gene expression in mutant strain based on increased abundance of PCR amplicon compared to PAO1), − (represents decreased gene expression in mutant strain based on decreased abundance of PCR amplicon compared to PAO1), NC (no apparent change in abundance was observed).</p

Motility of strains.

<p>Motility phenotypes of <i>P. aeruginosa</i> for swimming (0.3% agar) and swarming (0.5% Eiken agar) after 24 hours at 37°C. Mutant strains (indicated by PA2572 and PA2573) have reduced swimming and swarming, whereas complemented strains (indicated by pPA2572 and pPA2573) had wild-type levels of motility. Each plate is representative of the motility phenotype observed for strains in triplicate in three independent experiments.</p

Bacterial two-hybrid analysis investigating potential protein-protein interactions.

<p>(+) indicates a positive protein-protein interaction based on growth of colonies on Selective Screening Media (SSM) (M9+ His-dropout media containing 5 mM 3-AT). (−) indicates a negative interaction based on lack of growth on SSM. (-TM) represents a PA2573 construct without the N-terminal transmembrane domains. Results were observations from three independent experiments.</p