Background: The availability of hundreds of bacterial genomes allowed a comparative genomic study of the Type VI Secretion System (T6SS), recently discovered as being involved in pathogenesis. By combining comparative and phylogenetic approaches using more than 500 prokaryotic genomes, we characterized the global T6SS genetic structure in terms of conservation, evolution and genomic organization. Results: This genome wide analysis allowed the identification of a set of 13 proteins constituting the T6SS protein core and a set of conserved accessory proteins. 176 T6SS loci (encompassing 92 different bacteria) were identified and their comparison revealed that T6SS-encoded genes have a specific conserved genetic organization. Phylogenetic reconstruction based on the core genes showed that lateral transfer of the T6SS is probably its major way of dissemination among pathogenic and non-pathogenic bacteria. Furthermore, the sequence analysis of the VgrG proteins, proposed to be exported in a T6SS-dependent way, confirmed that some C-terminal regions possess domains showing similarities with adhesins or proteins with enzymatic functions. Conclusion: The core of T6SS is composed of 13 proteins, conserved in both pathogenic and non-pathogenic bacteria. Subclasses of T6SS differ in regulatory and accessory protein content suggesting that T6SS has evolved to adapt to various microenvironments and specialized functions. Based on these results, new functional hypotheses concerning the assembly and function of T6SS proteins are proposed.

The Pseudomonas aeruginosa cystic fibrosis isolate CHA induces type III secretion system‐dependent but ExoU‐independent oncosis of neutrophils and macrophages. Time‐lapse microscopy of the infection process revealed the rapid accumulation of motile bacteria around infected cells undergoing the process of oncosis, a phenomenon we termed pack swarming. Characterization of the non‐chemotactic CHAcheZ mutant showed that pack swarming is a bacterial chemotactic response to infected macrophages. A non‐cytotoxic mutant, lacking the type III‐secreted proteins PcrV, PopB and PopD, was able to pack swarm only in the presence of the parental strain CHA or when macrophages were pretreated with the pore‐forming toxin streptolysin O. Interaction of P. aeruginosa with red blood cells (RBCs) showed that the contact‐dependent haemolysis provoked by CHA requires secretion via the type III system and the PcrV, PopB/PopD proteins. The pore inserted into RBC membrane was estimated from osmoprotection experiments to be between 2.8 and 3.5 nm. CHA‐infected macrophages could be protected from cell lysis with PEG3350, indicating that the pore introduced into RBC and macrophage membranes is of similar size. The time course uptake of the vital fluorescent dye, Yo‐Pro‐1, into infected macrophages confirmed that the formation of transmembrane pores by CHA precedes cellular oncosis. Therefore, CHA‐induced macrophage death results from a pore‐forming activity that is dependent on the intact pcrGVHpopBD operon.

Pseudomonas aeruginosaefficiently intoxicates eukaryotic cells through the activity of the type III secretion-translocation system (TTSS). Gene deletions within the translocation operonpcrGVH-popBDabolish pore-forming activity ofP. aeruginosastrains 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 ofpcrVin 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.

Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the ‘translocon’). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol‐rich membranes, which occurs within a pH range of 5–7 in the case of PopB/PcrH, and only at acidic pH for PcrH‐free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 Å wide rings which encircle 40 Å wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.