Transcriptional induction of the Pseudomonas aeruginosa type III secretion system by low Ca2+ and host cell contact proceeds through two distinct signaling pathways - PubMed (original) (raw)

Transcriptional induction of the Pseudomonas aeruginosa type III secretion system by low Ca2+ and host cell contact proceeds through two distinct signaling pathways

Nandini Dasgupta et al. Infect Immun. 2006 Jun.

Abstract

The opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to intoxicate eukaryotic host cells. Transcription of the T3SS is induced under calcium-limited growth conditions or following intimate contact of P. aeruginosa with host cells. In the present study, we demonstrate that expression of the T3SS is controlled by two distinct regulatory mechanisms and that these mechanisms are differentially activated in a host cell-dependent manner. The first mechanism is dependent upon ExsC, a regulatory protein that couples transcription of the T3SS to the activity of the type III secretion machinery. ExsC is essential for induction of the T3SS under low-calcium-growth conditions and for T3SS-dependent cytotoxicity towards social amoebae, insect cells, and erythrocytes. The second regulatory mechanism functions independently of ExsC and is sufficient to elicit T3SS-dependent cytotoxicity towards certain types of mammalian cells. Although this second pathway (ExsC independent) is sufficient, an exsC mutant demonstrates a lag in the induction of cytotoxicity towards Chinese hamster ovary cells and is attenuated for virulence in a mouse pneumonia model. We propose that the ExsC-dependent pathway is required for full cytotoxicity towards all host cell types tested whereas the ExsC-independent pathway may represent an adaptation that allows P. aeruginosa to increase expression of the T3SS in response to specific types of mammalian cells.

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Figures

FIG. 1.

Genes required for T3SS expression in response to low-Ca2+ and CHO cell signals. (A) The indicated strains carrying the P_exsD_-lacZ reporter were grown under noninducing (lacking EGTA; white bars) or inducing (with EGTA; hatched bars) conditions for the expression of the T3SS and assayed for β-galactosidase (β-gal) activity (reported in Miller units). (B) P. aeruginosa strains were cultured in Ham's F12 medium alone (white bars) or in the presence (hatched bars) of CHO cells (10:1 MOI) for 4 h and assayed for the expression of the P_exsD_-lacZ reporter and for T3SS-dependent cytotoxicity, respectively. Percent cytotoxicity (based on the release of LDH) was calculated relative to an uninfected control (0% cytotoxicity) and the amount of LDH released by coculture with the wild-type (wt) strain (100% cytotoxicity). Under these conditions, the wild-type strain released 72% of the LDH compared to cells completely lysed with Triton X-100. (C) Time course analysis of T3SS-dependent cytotoxicity towards CHO cells. P. aeruginosa strains were cocultured with CHO cells (10:1 MOI) for the indicated times and then assayed for LDH release. The reported values represent averages from at least three independent experiments, and error bars indicate the standard errors of the means.

FIG. 2.

Cytotoxicity of the exsC mutant towards mammalian and nonmammalian cells. (A-B) P. aeruginosa strains were cocultured (10:1 MOI) with the indicated eukaryotic cells for 4 h and assayed for LDH release (A549, HeLa, RAW, and Sf9 cells) or hemolysis (erythrocytes). Percent cytotoxicity was calculated relative to an uninfected control (0% cytotoxicity) and to the amount of LDH or hemoglobin released following coculture with wild-type (wt) P. aeruginosa (100% cytotoxicity). The reported values represent averages from at least three independent experiments, and error bars indicate the standard errors of the means. The statistical significance (one-way ANOVA test, 95% confidence interval) between the cytotoxicities elicited by the exsA and exsC mutants for each cell type is indicated (*, P < 0.01; **, P < 0.001). (C) Dictyostelium discoideum plaquing assay. The indicated P. aeruginosa strains were mixed with D. discoideum and plated on nutrient agar. Whereas D. discoideum does not form plaques on a wild-type lawn of P. aeruginosa, plaques (indicated by arrowheads) are readily formed on the lawn formed by strains lacking the expression of the T3SS.

FIG. 3.

Complementation analysis of regulatory mutants. P. aeruginosa strains were transformed with pUCP18 (vector control), an ExsC expression plasmid (p_exsC_), or a Vfr expression plasmid (p_vfr_) and assayed for the expression of the P_exsD_-lacZ reporter (A) or T3SS-dependent cytotoxicity towards Sf9 (B) and CHO (C) cells. The reported values represent averages from at least three independent experiments, and error bars indicate the standard errors of the means. wt, wild type; β-gal, β-galactosidase.

FIG. 4.

ExsC is required for full virulence in a murine pneumonia model. Groups (n = 4) of C57B/6 mice were infected with the indicated P. aeruginosa strains (5 × 105 CFU). At 6 or 16 h postinfection, the animals were euthanized and the bacterial loads in the lung (white bars), blood (hatched bars), and liver (black bars) samples were determined by plate counting. The reported values are numbers of CFU per ml of blood or per 1 g of lung or liver tissue. The statistical significance (one-way ANOVA test) between the bacterial loads in mice infected with the wild-type (wt) strain and the exsC mutant are indicated.

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Transcriptional induction of the Pseudomonas aeruginosa type III secretion system by low Ca2+ and host cell contact proceeds through two distinct signaling pathways - PubMed (original) (raw)