Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity - PubMed (original) (raw)

Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity

D J Evans et al. Infect Immun. 1998 Apr.

Abstract

Pseudomonas aeruginosa clinical isolates exhibit invasive or cytotoxic phenotypes. Cytotoxic strains acquire some of the characteristics of invasive strains when a regulatory gene, exsA, that controls the expression of several extracellular proteins, is inactivated. exsA mutants are not cytotoxic and can be detected within epithelial cells by gentamicin survival assays. The purpose of this study was to determine whether epithelial cell invasion precedes and/or is essential for cytotoxicity. This was tested by measuring invasion (gentamicin survival) and cytotoxicity (trypan blue staining) of PA103 mutants deficient in specific exsA-regulated proteins and by testing the effect of drugs that inhibit invasion for their effect on cytotoxicity. A transposon mutant in the exsA-regulated extracellular factor exoU was neither cytotoxic nor invasive. Furthermore, several of the drugs that inhibited invasion did not prevent cytotoxicity. These results show that invasion and cytotoxicity are mutually exclusive events, inversely regulated by an exsA-encoded invasion inhibitor(s). Both involve host cell protein tyrosine kinase (PTK) activity, but they differ in that invasion requires Src family tyrosine kinases and calcium-calmodulin activity. PTK inhibitor drugs such as genistein may have therapeutic potential through their ability to block both invasive and cytotoxicity pathways via an action on the host cell.

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Figures

FIG. 1

Cytotoxicity of various isogenic mutants of strain PA103 for corneal epithelial cells measured by trypan blue staining. (A) PA103 exoT::Tc (exoT); (B) PA103 exoU::Tn_5_Tc (exoU); (C) complementation of exoU by pUCP_exoU_ (PA103 exoU::Tn_5_Tc pUCP_exoU_); (D) vector control strain, PA103 exoU::Tn_5_Tc pUCP18.

FIG. 2

The effect of mutations in the ExsA-regulated cytotoxicity pathway on the invasion of strain PA103 into corneal epithelial cells. (A) Comparison between the parent strain PA103 and PA103 exsA::Ω (exsA), PA103 exoT::Tc (exoT), PA103 exoU::Tn_5_Tc (exoU), PA103 exoU::Tn_5_Tc pUCP18 (vector control), and PA103 exoU::Tn_5_Tc complemented with pUCP_exoU_ (exoU+). (B) Different experiment comparing PA103, PA103 exsA::Ω, and PA103 exoU::Tn_5_Tc with two mutants of PA103 exoU::Tn_5_Tc in which exsA was inactivated (exoU exsA double mutants).

FIG. 3

Effect of signal transduction inhibitors on invasion of P. aeruginosa 6294 into corneal epithelial cells. (A) Comparison of the effects of genistein, cytochalasin D, and herbimycin A. (B) Comparison of the effects of wortmannin, tyrphostin A47, and staurosporine with genistein.

FIG. 4

Effect of various concentrations of W-7 (calmodulin antagonist) and BAPTA-AM (intracellular calcium chelator) on invasion of P. aeruginosa 6294 into corneal epithelial cells.

FIG. 5

The effect of cytochalasin D on susceptibility of corneal epithelia to P. aeruginosa cytotoxicity. (A) Bacterium-induced cytotoxicity without drug treatment; (B) effect of cytochalasin D (10 μM) treatment of corneal cells before and during exposure to strain 6206 (cytotoxic).

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Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity - PubMed (original) (raw)