Orf1/SpcS chaperones ExoS for type three secretion by Pseudomonas aeruginosa.

International audienceOBJECTIVE: Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type III secretion system (TTSS) to inject effector proteins directly into the cytosol of target cells to subvert the host cell's functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P. aeruginosa, we analyzed the role of a postulated chaperone termed Orf1. METHODS: By allelic exchange, we constructed the mutant with the deletion of gene Orf1. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orf1, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF. The role of Orf1 in the expression of exoS was evaluated by gene reporter analysis. RESULTS: Pull-down assay showed that Orf1 binds to ExoS and ExoT. Secretion profile analysis showed that Orf1 was necessary for the optimal secretion of ExoS and ExoT. However, Orf1 had no effect on the expression of exoS. CONCLUSION: Orf1 is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orf1 as SpcS for "specific Pseudomonas chaperone for ExoS"

Yersinia pestis IS1541 Transposition Provides for Escape from Plague Immunity▿

Yersinia pestis is perhaps the most feared infectious agent due to its ability to cause epidemic outbreaks of plague disease in animals and humans with high mortality. Plague infections elicit strong humoral immune responses against the capsular antigen (fraction 1 [F1]) of Y. pestis, and F1-specific antibodies provide protective immunity. Here we asked whether Y. pestis generates mutations that enable bacterial escape from protective immunity and isolated a variant with an IS1541 insertion in caf1A encoding the F1 outer membrane usher. The caf1A::IS1541 insertion prevented assembly of F1 pili and provided escape from plague immunity via F1-specific antibodies without a reduction in virulence in mouse models of bubonic or pneumonic plague. F1-specific antibodies interfere with Y. pestis type III transport of effector proteins into host cells, an inhibitory effect that was overcome by the caf1A::IS1541 insertion. These findings suggest a model in which IS1541 insertion into caf1A provides for reversible changes in envelope structure, enabling Y. pestis to escape from adaptive immune responses and plague immunity

Immunization with Recombinant V10 Protects Cynomolgus Macaques from Lethal Pneumonic Plague▿

Vaccine and therapeutic strategies that prevent infections with Yersinia pestis have been sought for over a century. Immunization with live attenuated (nonpigmented) strains and immunization with subunit vaccines containing recombinant low-calcium-response V antigen (rLcrV) and recombinant F1 (rF1) antigens are considered effective in animal models. Current antiplague subunit vaccines in development for utilization in humans contain both antigens, either as equal concentrations of the two components (rF1 plus rLcrV) or as a fusion protein (rF1-rLcrV). Here, we show that immunization with either purified rLcrV (a protein at the tip of type III needles) or a variant of this protein, recombinant V10 (rV10) (lacking amino acid residues 271 to 300), alone or in combination with rF1, prevented pneumonic lesions and disease pathogenesis. In addition, passive immunization studies showed that specific antibodies of macaques immunized with rLcrV, rV10, or rF1, either alone or in combination, conferred protection against bubonic plague challenge in mice. Finally, we found that when we compared the reactivities of anti-rLcrV and anti-rV10 immune sera from cynomolgus macaques, BALB/c mice, and brown Norway rats with LcrV-derived peptides, rV10, but not rLcrV immune sera, lacked antibodies recognizing linear LcrV oligopeptides

Polymorphisms in the lcrV Gene of Yersinia enterocolitica and Their Effect on Plague Protective Immunity

Current efforts to develop plague vaccines focus on LcrV, a polypeptide that resides at the tip of type III secretion needles. LcrV-specific antibodies block Yersinia pestis type III injection of Yop effectors into host immune cells, thereby enabling phagocytes to kill the invading pathogen. Earlier work reported that antibodies against Y. pestis LcrV cannot block type III injection by Yersinia enterocolitica strains and suggested that lcrV polymorphisms may provide for escape from LcrV-mediated plague immunity. We show here that polyclonal or monoclonal antibodies raised against Y. pestis KIM D27 LcrV (LcrVD27) bind LcrV from Y. enterocolitica O:9 strain W22703 (LcrVW22703) or O:8 strain WA-314 (LcrVWA-314) but are otherwise unable to block type III injection by Y. enterocolitica strains. Replacing the lcrV gene on the pCD1 virulence plasmid of Y. pestis KIM D27 with either lcrVW22703 or lcrVWA-314 does not affect the ability of plague bacteria to secrete proteins via the type III pathway, to inject Yops into macrophages, or to cause lethal plague infections in mice. LcrVD27-specific antibodies blocked type III injection by Y. pestis expressing lcrVW22703 or lcrVWA-314 and protected mice against intravenous lethal plague challenge with these strains. Thus, although antibodies raised against LcrVD27 are unable to block the type III injection of Y. enterocolitica strains, expression of lcrVW22703 or lcrVWA-314 in Y. pestis did not allow these strains to escape LcrV-mediated plague protective immunity in the intravenous challenge model

Anti-tumor immunotherapy via antigen delivery from a live attenuated genetically engineered Pseudomonas aeruginosa type III secretion system-based vector.

Immunotherapy requiring an efficient T lymphocyte response is initiated by antigen delivery to antigen-presenting cells. Several studies have assessed the efficiency of various antigen loading procedures, including microbial vectors. Here a live strain of Pseudomonas aeruginosa was engineered to translocate a recombinant antigenic protein into mammalian cells via the type III secretion system, a bacterial device translocating effector proteins into host cells. Optimization of the vector included virulence attenuation and determination of the N-terminal sequence allowing translocation of fused antigens into cells. In vitro delivery of an ovalbumin fragment by the bacterial vector into dendritic cells induced the activation of ovalbumin-specific CD8(+) T lymphocytes. Mice injected with the ovalbumin-delivering vector developed ovalbumin-specific CD8(+) T lymphocytes and were resistant to a subsequent challenge with an ovalbumin-expressing melanoma. Moreover, in a curative assay, injection of the vaccine vector 5 and 12 days after tumor implantation led to a complete cure in five of six animals. These results highlight the utility of type III secretion system-based vectors for anti-tumor immunotherapy