Type III secretion-mediated killing of endothelial cells by Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa, a common agent of septicemia, enters into human endothelial cellsin vitro but the effects of bacterial infection have not been addressed properly. In this study, human umbilical vein endothelial cells (HUVEC) were infected by the noninvasive PA103 and the invasive PAO1 P. aeruginosa strains and the viability of infected cells was assessed by the methyltiazole tetrazolium (MTT) assay. Both strains were cytotoxic within 3h of infection. To ascertain the role of proteins secreted by the type III secretion system (TTSS) in HUVEC killing, defective mutants of PAO1 and PA103 were constructed by plasmid insertion in exsA or pscC genes. ExsA is a transcriptional regulator that controls the expression of most TTSS related genes whereas pscC encodes a protein from the secretion machinery. Parental bacteria were significantly more cytotoxic to HUVEC than the mutants. Inactivation ofexsA reverted the inability of PA103 to enter into HUVEC but did not modify the invasiveness of PAO1. Cytofluorometric analysis of infected HUVEC labeled by DiOC6(3) showed that cell killing was associated with mitochondrial depolarization, an early event reported in apoptosis. However, infected cells did not show ultrastructural or DNA fragmentation features of apoptosis. Our results suggest that TTSS effectors mediate P. aeruginosa killing of HUVEC by a mechanism distinct from apoptosis.