title: Concomitant endosome-phagosome fusion and lysis of endosomal membranes account for Pseudomonas aeruginosa survival in human endothelial cells.
abstract: P. aeruginosa is selectively internalized by human endothelial cells but is not efficiently killed in the intracellular (IC) compartment. To investigate whether IC survival is associated with failure in bacteria-containing endosome-lysosome (E-L) fusion, endothelial cells were exposed to albumin-colloidal gold complex and to bacterial suspension and submitted to transmission electron microscopy (TEM). Gold granules were detected in P. aeruginosa-containing vacuoles, indicating that E-L fusion had occurred. Bacteria were also seen apparently free in the cell cytoplasm, suggesting disruption of endosome membranes. To ascertain whether phospholipase C (PLC) could account for vacuolar lysis, PLC producing PAO1 and PAK strains were compared with a PLC deficient mutant (PLCN) in their IC survival. All three strains were equally uptaken by the endothelial cells, as determined by the gentamicin exclusion assay. After 3 h of infection, the IC concentration of PAK and PAO1 increased significantly while the concentration of the mutant decreased to 56.8 +/- 18.2% of the viable counts at 1 h of infection. After 5 h, the IC concentration of P. aeruginosa corresponded to 83.1 +/- 34.6%, 109 +/- 22.6% and 26.2 +/- 14.7% of the viable counts detected at 1 h, for PAK, PAO1 and the mutant, respectively. By TEM, while most PAO1-containing vacuoles presented partially lysed membranes, in cells infected with the PLCN mutant bacteria were most often observed in vacuoles with intact membranes. These observations suggest that the IC survival of P. aeruginosa results from a competition between the microbicidal activity of endothelial cells following E-L fusion and the capacity of bacteria to escape from endosomes.
authors.affiliation: Department of Microbiology and Immunology, State University of Rio de Janeiro, Brazil.
authors.forename: M C
authors.initials: MC
authors.lastname: Plotkowski
authors.forename: M N
authors.initials: MN
authors.lastname: Meirelles
mesh_terms: D002470:Cell Survival; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D003593:Cytoplasm / Q000382:microbiology / Q000648:ultrastructure; D011992:Endosomes / Q000502:physiology / Q000648:ultrastructure*; D004730:Endothelium, Vascular / Q000382:microbiology* / Q000502:physiology / Q000648:ultrastructure*; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology / Q000648:ultrastructure; D008561:Membrane Fusion; D008854:Microscopy, Electron; D010588:Phagosomes / Q000502:physiology / Q000648:ultrastructure*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D010738:Type C Phospholipases / Q000378:metabolism; D014617:Vacuoles / Q000382:microbiology / Q000502:physiology / Q000648:ultrastructure
chemical_list: D002738:Chloroquine; D010738:Type C Phospholipases