Cell death of human polymorphonuclear neutrophils induced by a Pseudomonas aeruginosa cystic fibrosis isolate requires a functional type III secretion system

Abstract

With a coincubation model incorporating Pseudomonas aeruginosa and human polymorphonuclear neutrophils (PMNs), a cystic fibrosis (CF) P. aeruginosa isolate has been shown to resist the bactericidal action of PMNs and to induce their cellular death. An isogenic mutant of this CF isolate in which the type III secretion system was rendered nonfunctional was unable to induce cellular death of PMNs.

FIG. 1

Kinetics of mortality of PMNs in coincubation experiments with P. aeruginosa strains. The percentages of cytotoxicity were calculated according to the release of LDH activity. Data are the means of results of at least three experiments. (A) Comparison between CHA (●) and PAO1 (⧫) and the effect of cytochalasin D on the cytotoxicity of CHA (■). ▴, PMNs only. (B) Cytotoxicities of CHA-D1 (○) and of the complemented strain CHA-D1(pDD2) (▾). Results obtained with CHA () are shown.

FIG. 2

Extracellular bacterial protein profiles. Silver nitrate-stained 0.1% sodium dodecyl sulfate–12% PAGE of supernatants of CHA (lanes 1 and 2), CHA-D1 (lanes 3 and 4), CHA-D1(pDD2) (lanes 5 and 6), and PAO1 (lanes 7 and 8) cultured with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) induction of the type III secretion system by calcium depletion. Molecular weight markers (MW) are shown, with weights being reported in thousands. The positions of ExoT, ExoS, PopB, and PopD are shown.