Activation of the Pseudomonas aeruginosa type III secretion system requires an intact pyruvate dehydrogenase aceAB operon

Abstract

Pseudomonas aeruginosa clinical cystic fibrosis isolate CHA was mutagenized with Tn5Tc to identify new genes involved in type III secretion system (TTSS)-dependent cytotoxicity toward human polymorphonuclear neutrophils. Among 25 mutants affected in TTSS function, 14 contained the insertion at different positions in the aceAB operon encoding the PDH-E1 and -E2 subunits of pyruvate dehydrogenase. In PDH mutants, no transcriptional activation of TTSS genes in response to calcium depletion occurred. Expression in trans of ExsA restored TTSS function and cytotoxicity.

FIG. 1.

Analysis of TTSS function by electrophoretic analysis of P. aeruginosa proteins secreted into extracellular media under TTSS-inducing calcium depletion conditions (5 mM EGTA, 20 mM MgCl₂). Secreted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. In each lane, the same amount of supernatants collected from culture grown to the same optical density at 600 nm was used. (A) Profile obtained with CHA, CHA-D1, and noncytotoxic mutants cultured in inducing conditions. (B) Profile obtained either in noninducing (−) or inducing (+) conditions with mutant 44 (aceA) and mutant 9 (aceB). (ExsA), the strain contained the pDD2 plasmid expressing ExsA. The CHA wild type was used as a control for both panels.

FIG. 2.

GFP fluorescence in CHA and PDH mutants transformed with plasmid pIApC grown in LB medium (white bars) or in LB medium supplemented with 5 mM EGTA and 20 mM MgCl₂ (inducing conditions) (hatched bars). The final concentration of sodium acetate in the culture medium is indicated. Error bars represent the standard deviations. Data are from at least three independent experiments.