A novel sensor kinase-response regulator hybrid regulates type III secretion and is required for virulence in Pseudomonas aeruginosa

Abstract

The type III secretion system (TTSS) of Pseudomonas aeruginosa is induced by contact with eukaryotic cells and by growth in low-calcium media. We have identified a protein, RtsM, that is necessary for expression of the TTSS genes in P. aeruginosa. RtsM possesses both histidine kinase and response regulator domains common to two-component signalling proteins, as well as a large predicted periplasmic domain and seven transmembrane domains. Deletion of rtsM resulted in a defect in production and secretion of the type III effectors. Northern blot analysis revealed that mRNAs encoding the effectors ExoT and ExoU are absent in the DeltartsM strain under TTSS-inducing conditions. Using transcriptional fusions, we demonstrated that RtsM is required for transcription of the operons encoding the TTSS effectors and apparatus in response to calcium limitation or to host cell contact. The operon encoding the TTSS regulator ExsA does not respond to calcium limitation, but the basal transcription rate of this operon was lower in deltartsM than in the wild-type parent, PA103. The defect in TTSS effector production and secretion of deltartsM could be complemented by overexpressing ExsA or Vfr, two transcriptional activators involved in TTSS regulation. DeltartsM was markedly less virulent than PA103 in a murine model of acute pneumonia, demonstrating that RtsM is required in vivo. We propose that RtsM is a sensor protein at the start of a signalling cascade that induces expression of the TTSS in response to environmental signals.

Fig. 1

Regulation of TTSS expression in P. aeruginosa. The expression of the operons encoding the P.aeruginosa TTSS (which encode the TTSS apparatus, regulators and effectors) is under the control of the known regulators ExsA, ExsD, ExsC, CyaB and Vfr. ExsA is an AraC-like transcriptional activator that directly binds to a conserved sequence upstream of the genes it regulates, while ExsD is an antiactivator that binds to and sequesters ExsA. When bacteria are grown in low-calcium media, ExsC binds to and sequesters ExsD, which releases active ExsA. A second input into the system is provided by CyaB, an adenylate cyclase that is induced under low-calcium conditions. Increased cAMP levels produced by CyaB in turn activate the cAMP binding transcription factor, Vfr; both proteins are required for TTSS gene transcription. The CyaB–Vfr pathway may work upstream of or in parallel to ExsA.

Fig. 2

Cytotoxicity toward HeLa cells as measured by LDH release. HeLa cells were infected with bacteria at an MOI of 10–15 in triplicate and sampled at 2 hpi (white bars) and 4 hpi (black bars). Values are normalized to cytotoxicity caused by PA103 at each time point. Negative controls include uninfected HeLa cells and cells infected with the TTSS^– mutant, pscJ::Tn5. ΔrtsM attB::rtsM carries a copy of RtsM under control of its own promoter integrated into the chromosomal attB site, while ΔrtsM attB::Gm has a gentamicin resistance cassette inserted at the attB locus. Bars show the mean ± SD of an assay which is representative of three independent experiments.

Fig. 3

RtsM is required for virulence in a murine model of acute pneumonia. Eight to ten week old female C57Bl/6 mice were infected with 0.4–1.0 × 10⁶ cfu of PA103 (n = 8), ΔrtsM (n = 15) or ΔexsA (n = 10). Mice were euthanized at 18 hpi and the number of bacteria present in lungs, liver and spleen were determined as described in Experimental procedures. Results are expressed as the ratio of cfu recovered/g tissue (output) to cfu present in the inoculum (input); each animal is represented by a data point, while the bar shows the geometric mean for each group. The Mann–Whitney test was used to calculate P-values (two-tailed) for each pair-wise comparison indicated. No colonies of ΔrtsM or ΔexsA were recovered from spleen or liver in any of the animals; the values shown indicate the limit of detection of the assay.

Fig. 4

A. Western immunoblot analysis of whole-cell pellet lysates (P) and culture supernatants (S) of PA103 and mutant derivatives grown under TTSS-inducing conditions. Sample loading was normalized to total protein (15 μg lane⁻¹) or bacterial counts (5 × 10⁸ cfu lane⁻¹) for pellet and supernatant samples respectively. Polypeptides were separated by SDS-PAGE, transferred to PVDF and probed with polyclonal antiserum that recognizes TTSS effectors. The absence of ExoU or ExoT bands in supernants indicates a defect in secretion, while the absence of bands in the pellet indicates a defect in production. ΔrtsM pMMB (empty vector) and ΔrtsM attB::Gm serve as controls for the plasmid complemented strain ΔrtsM pMMB-RtsM and the strain carrying an ectopic copy of RtsM integrated at the attB site of ΔrtsM respectively. B. Northern blot analysis of RNA prepared from PA103, ΔrtsM or ΔexsA bacteria grown under TTSS-inducing conditions. Five microgram total RNA was separated by electrophoresis, blotted to nitrocellulose and probed with riboprobes specific for exoU or exoT mRNA. Equal loading was confirmed by methylene blue staining of rRNA (not shown).

Fig. 5

Transcriptional activation of exoT during exposure of bacteria to HeLa cells requires RtsM. Luminescence (RLU) produced by PA103 or ΔrtsM carrying a single copy of the exoT::lux transcriptional reporter was measured over 4 h following bacterial binding to HeLa cell monolayers or tissue culture plastic (media control). Results are reported as the fold increase in RLU relative to the reading obtained at t = 0 h. Bars represent the mean ± SD of triplicate samples, and are representative of two independent experiments.

Fig. 6

Western immunoblot analysis of whole-cell pellet lysates (P) and culture supernatants (S) of PA103 and mutant derivatives grown under TTSS-inducing conditions in the presence of carbenicillin (200 μg ml⁻¹) and 0.2% L-arabinose. Samples were normalized to total protein (15 μg lane⁻¹) or bacterial counts (5 × 10⁸ cfu lane⁻¹) for pellet and supernatant samples respectively. ExoU and ExoT were detected by Western blotting with polyclonal antiserum (as described for Fig. 2A). PA103 and ΔrtsM carry the empty vector pARA, allowing all strains to be grown under identical conditions.

Fig. 7

Detection of RtsM-BB2 in bacterial lysates grown under TTSS non-inducing and inducing conditions. ΔrtsM pRtsM-BB2 was grown in MinS media supplemented with 2.5 mM CaCl₂ (non-inducing, NI) or 10 mM NTA (inducing, I). Bacterial pellets were lysed, and 5 μg of total protein was separated by SDS-PAGE. Proteins were transferred to PVDF and detected by Western immunoblotting using an anti-BB2 antibody. The positions of the 81.2 and 113.9 kDa molecular weight markers are indicated.

Fig. 8

Predicted domain structure of RtsM. rtsM (PA4856) is predicted to encode a 942 aa protein, with a cleaved signal sequence, large periplasmic domain and seven transmembrane regions followed by a sensor kinase domain and two response regulator domains in tandem. Numbers in parentheses indicate the predicted boundaries of each domain (aa), while predicted sites of phosphorylation (H424, D713 and D858) are indicated for each domain.