Metabolic regulation of type III secretion gene expression in Pseudomonas aeruginosa

Abstract

Type III secretion-mediated cytotoxicity is one of the key virulence mechanisms of the opportunistic pathogen Pseudomonas aeruginosa. Prior data from several laboratories have established that metabolism is a key factor in the regulation of type III secretion gene expression in P. aeruginosa. Here we use a fluorescence-activated cell sorter (FACS)-based approach to investigate expression of type III secretion genes at a single-cell level. The data demonstrate that the metabolic state regulates the percentage of cells that are able to induce type III secretion gene expression under inducing conditions. We also present evidence that this regulation is the result of an effect of the growth conditions on the ability of P. aeruginosa to assemble a functional type III secretion apparatus. Preliminary data suggest that the metabolite that controls type III secretion gene expression is derived from acetyl-CoA and that this regulation may, in part, be mediated by changes in the intracellular concentration of cyclic-AMP.

Fig. 1

Induction of exoS expression monitored by FACS analysis using a GFP reporter. P. aeruginosa PAK (A, C and D) or PAK ΔpscC (B) harbouring the exoS∷GFP-lacZ reporter were grown in l-broth (A,B) or MOPS minimal media supplemented with 0.2% glucose (C) or 0.2% glucose and 5 mM glutamate (D). Relative fluorescence of individual bacteria is plotted on the x-axis. The y-axis denotes the number of bacteria of a given fluorescence intensity (10 000 bacteria were analysed). The grey overlay indicates the distribution for cultures in which calcium had been chelated by the addition of NTA to the media.

Fig. 2

Osmolarity controls the ability to induce exoS expression. Strain PAK harbouring the exoS∷GFP-lacZ reporter was grown in LB containing the indicated concentration of NaCl (A, B) or 5 mM NaCl and 271 mM sucrose (D). The grey overlay indicates the distribution for cultures in which calcium had been chelated by the addition of NTA to the media. Panel C, a graph plotting the percentage of cells that induce exoS expression after removal of calcium from the media (y-axis) when grown in LB with the indicated NaCl concentration (x-axis). Panel E depicts levels of exoS expression as measured using the lacZ reporter in LB with 5 mM or 200 mM NaCl in the absence or presence of chelator (NTA). The actual activities are noted above each column of the graph.

Fig. 3

Effect of regulatory mutations on induction in high- and low-osmolarity media. Indicated mutants of strain PAK were grown in LB with low (5 mM) or high (200 mM) salt and assayed, by FACS, for exoS expression in the presence (black) or absence (grey overlay) of calcium using a chromosomal exoS-GFP reporter.

Fig. 4

exoS expression in various regulatory mutations in the context of a defective type III secretion apparatus. Derivatives of strain PAK bearing the indicated deletion mutations were assayed, by FACS, for exoS expression in LB with 200 mM NaCl in the presence (black) or absence (grey overlay) of calcium.

Fig. 5

Assembly of the type III secretion machinery in low- and high-salt media. A. Bacteria grown in LB with 5 mM or 200 mM NaCl were stained with the indicated primary antibody (anti-PcrV or anti-OprH antiserum) and detected using a AlexaFluor 488 coupled secondary antibody. Phase contrast and fluorescence images of representative fields were overlayed. The pcrV and pscC mutants were only grown in LB with 200 mM NaCl. B. FACS analysis of antibody stained cells. All strains were stained with the PcrV and AlexaFluor-488 coupled secondary antibody as described above and analysed by FACS. The wild-type and exsE mutant strain were grown in LB with 5 mM NaCl (black) or 200 mM NaCl (grey). The pcrV and pscC mutant strains were only grown in LB with 200 mM NaCl.

Fig. 6

Metabolic mutations control exoS induction. Wild-type P. aeruginosa as well as the indicated mutants were grown in MOPS minimal media supplemented with 0.1% tryptone and 20 mM glutamate. exoS expression was induced by removal of calcium from the media and analysed by FACS in the presence (black) or absence (grey overlay) of calcium (aceE, pyruvate dehydrogenase subunit E1, gltA, citrate synthetase 1, prpC, citrate synthetase 2). The percentage of cells that induce exoS expression in the absence of calcium is indicated in the upper right-hand corner of each graph.