Related Experiment Videos
Type III secretion-mediated killing of endothelial cells by Pseudomonas aeruginosa
Alessandra Mattos Saliba1, Alain Filloux, Geneviève Ball
1Department of Microbiology and Immunology, State University of Rio de Janeiro, Brazil.
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 DiOC(6)(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.
Insights
Pseudomonas aeruginosa kills human endothelial cells via its type III secretion system (TTSS). This bacterial killing mechanism is distinct from apoptosis, despite initial signs of mitochondrial depolarization.
Area of Science:
- Microbiology
- Cell Biology
- Pathogenesis
Background:
- Pseudomonas aeruginosa is a significant cause of septicemia.
- Endothelial cell invasion by P. aeruginosa is a critical step in infection.
- The precise mechanisms of P. aeruginosa-induced endothelial cell death remain incompletely understood.
Purpose of the Study:
- To investigate the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa-mediated killing of human umbilical vein endothelial cells (HUVEC).
- To determine if P. aeruginosa-induced HUVEC death involves apoptotic pathways.
Main Methods:
- Infection of HUVEC with invasive (PAO1) and noninvasive (PA103) P. aeruginosa strains.
- Assessment of cell viability using the MTT assay.
- Construction and use of TTSS-defective mutants (exsA, pscC).
- Analysis of mitochondrial depolarization and apoptotic markers (ultrastructure, DNA fragmentation).
Main Results:
- Both P. aeruginosa strains exhibited cytotoxicity to HUVEC within 3 hours.
- TTSS-defective mutants were significantly less cytotoxic than parental strains.
- Inactivation of exsA restored HUVEC entry for PA103 but not PAO1 invasiveness.
- Cell killing correlated with mitochondrial depolarization but lacked other apoptotic features.
Conclusions:
- Type III secretion system (TTSS) effectors are key mediators of P. aeruginosa-induced HUVEC death.
- The mechanism of endothelial cell killing by P. aeruginosa is independent of classical apoptosis.
- This study elucidates a novel bacterial pathogenesis pathway involving TTSS effectors.