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LcrV delivered via type III secretion system of live attenuated Yersinia pseudotuberculosis enhances immunogenicity against pneumonic plague.

ABSTRACT: Here, we constructed a Yersinia pseudotuberculosis mutant strain with arabinose-dependent regulated and delayed shutoff of crp expression (araC P(BAD) crp) and replacement of the msbB gene with the Escherichia coli msbB gene to attenuate it. Then, we inserted the asd mutation into this construction to form ?10057 [?asd-206 ?msbB868::P(msbB) msbB(EC) ?P(crp21)::TT araC P(BAD) crp] for use with a balanced-lethal Asd-positive (Asd(+)) plasmid to facilitate antigen synthesis. A hybrid protein composed of YopE (amino acids [aa]1 to 138) fused with full-length LcrV (YopE(Nt138)-LcrV) was synthesized in ?10057 harboring an Asd(+) plasmid (pYA5199, yopE(Nt138)-lcrV) and could be secreted through a type III secretion system (T3SS) in vitro and in vivo. Animal studies indicated that mice orally immunized with ?10057(pYA5199) developed titers of IgG response to whole-cell lysates of Y. pestis (YpL) and subunit LcrV similar to those seen with ?10057(pYA3332) (?10057 plus an empty plasmid). However, only immunization of mice with ?10057(pYA5199) resulted in a significant secretory IgA response to LcrV. ?10057(pYA5199) induced a higher level of protection (80% survival) against intranasal (i.n.) challenge with ~240 median lethal doses (LD50) (2.4 × 10(4) CFU) of Y. pestis KIM6+(pCD1Ap) than ?10057(pYA3332) (40% survival). Splenocytes from mice vaccinated with ?10057(pYA5199) produced significant levels of gamma interferon (IFN-?), tumor necrosis factor alpha (TNF-?), and interleukin-17 (IL-17) after restimulation with LcrV and YpL antigens. Our results suggest that it is possible to use an attenuated Y. pseudotuberculosis strain delivering the LcrV antigen via the T3SS as a potential vaccine candidate against pneumonic plague.

SUBMITTER: Sun W

PROVIDER: S-EPMC4187879 | biostudies-other | 2014 Oct

REPOSITORIES: biostudies-other

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LcrV delivered via type III secretion system of live attenuated Yersinia pseudotuberculosis enhances immunogenicity against pneumonic plague.

Sun Wei W Sanapala Shilpa S Henderson Jeremy C JC Sam Shandiin S Olinzock Joseph J Trent M Stephen MS Curtiss Roy R

Infection and immunity 20140811 10

Here, we constructed a Yersinia pseudotuberculosis mutant strain with arabinose-dependent regulated and delayed shutoff of crp expression (araC P(BAD) crp) and replacement of the msbB gene with the Escherichia coli msbB gene to attenuate it. Then, we inserted the asd mutation into this construction to form χ10057 [Δasd-206 ΔmsbB868::P(msbB) msbB(EC) ΔP(crp21)::TT araC P(BAD) crp] for use with a balanced-lethal Asd-positive (Asd(+)) plasmid to facilitate antigen synthesis. A hybrid protein compos ...[more]

PMID: 25114109

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Project description:Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and ?lpp ?msbB double mutants. While the ?ail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the ?lpp ?ail double mutant and the ?lpp ?msbB ?ail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the ?lpp ?msbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection.

Dataset Information

LcrV delivered via type III secretion system of live attenuated Yersinia pseudotuberculosis enhances immunogenicity against pneumonic plague.

Publications

LcrV delivered via type III secretion system of live attenuated Yersinia pseudotuberculosis enhances immunogenicity against pneumonic plague.

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