The V Antigen ofPseudomonas aeruginosaIs Required for Assembly of the Functional PopB/PopD Translocation Pore in Host Cell Membranes

Abstract

Pseudomonas aeruginosa efficiently intoxicates eukaryotic cells through the activity of the type III secretiontranslocation system (TTSS). Gene deletions within the translocation operon pcrGVH-popBD abolish poreforming 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.

Yersinia pestis YopD 150–287 fragment is partially unfolded in the native state

“…The criteria by which Y. pestis selects the YopB and YopD translocators to be delivered first are not known. The LcrV protein is localized on the tip of needle [5] and most likely is not in the pore, similar to the data for Pseudomonas aeruginosa ortholog PcrV [6], as it lacks a defined transmembrane domain. It is not excluded, however, that it may be associated with a pore structure.…”

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

“…Here, we employed two strains that insert PopB into the membrane: P. aeruginosa ⌬BD complemented with both PopB-␤-Lac and PopD (⌬BD/B-␤-Lac/D) and ⌬PcrV complemented with the same constructs (⌬V/B-␤-Lac/D). This latter strain, despite not being able to express PcrV, is able to form the translocon, although it is non-infectious (49). ␤-Lactamase cleavage of nitrocefin could be measured for both transfected constructs.…”

Gamma-aminobutyric acid acts as a specific virulence regulator in Pseudomonas aeruginosa

“…MALDI-TOF/TOF identification of the proteins in Fig. 5 whose expression was modified by GABA in P. aeruginosa induced by a type III secretion system (Goure et al, 2004) or phospholipase C (Ostroff et al, 1989). The tests done on blood-supplemented agar plates showed no variation in bacterial haemolytic activity.…”