The V antigen of Pseudomonas aeruginosa is required for assembly of the functional PopB/PopD translocation pore in host cell membranes.
Pseudomonas aeruginosa efficiently intoxicates eukaryotic cells through the activity of the type III secretion-translocation system (TTSS). Gene deletions within the translocation operon pcrGVH-popBD abolish pore-forming activity of P. aeruginosa strains with macrophages and TTSS-dependent hemolysis. Here we investigated the requirements for PcrV, PopB, and PopD in pore formation by analyzing specific mutants using red blood cells (RBCs) and fibroblasts expressing green fluorescent protein fused to actin. Simultaneous secretion of three proteins, PopB, PopD, and PcrV, was required to achieve wild-type hemolysis and effector translocation. Deletion of pcrV in a cytotoxic strain did not affect secretion of PopB and PopD but abolished hemolytic activity and translocation of effectors into fibroblasts. Notably, the PcrV-deficient mutant was not capable of inserting PopD into host cell membranes, whereas PopB and PopD, but not PcrV, were readily found within membranes of wild-type-infected RBCs. Immunoprecipitation experiments performed by using a liposome model of pore assembly revealed a direct interaction between PopD and PopB but not between PopD and PcrV. Consequently, PcrV is necessary for the functional assembly of the PopB/D translocon complex but does not interact directly with pore-forming Pop proteins.
Goure
Julien
J
Biochimie et Biophysique des Systèmes Intégrés, UMR 5092 CNRS/CEA/UJF, DRDC, CEA, Grenoble, France.
Pastor
Alexandrine
A
Faudry
Eric
E
Chabert
Jacqueline
J
Dessen
Andréa
A
Attree
Ina
I
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000382:microbiology; D004912:Erythrocytes / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial; D006461:Hemolysis; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D052899:Pore Forming Cytotoxic Proteins; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C412007:PopB protein, Ralstonia solanacearum; C412006:PopC protein, Ralstonia solanacearum; D052899:Pore Forming Cytotoxic Proteins; C118546:antigen V, Pseudomonas