Identification of New Virulence Factors and Vaccine Candidates for .

Earlier, we reported the identification of new virulence factors/mechanisms of using an signature-tagged mutagenesis (STM) screening approach. From this screen, the role of , which encodes an ATP-binding protein of ribose transport system, and , an essential component of the type VI secretion system (T6SS), were evaluated in mouse models of plague and confirmed to be important during infection. However, many of the identified genes from the screen remained uncharacterized. In this study, in-frame deletion mutants of , and , identified from the STM screen, were generated. While codes for a general secretion pathway protein E (GspE) of the T2SS, the -encoded protein has homology to the βγ crystallin superfamily, codes for the cytochrome o oxidase operon, and the genes are within the Tol-Pal system which has multiple functions. Additionally, as our STM screen identified three T6SS-associated genes, and, based on analysis, six T6SS clusters and multiple homologs of the T6SS effector hemolysin-coregulated protein (Hcp) exist in CO92, we also targeted these T6SS clusters and effectors for generating deletion mutants. These deletion mutant strains exhibited varying levels of attenuation (up to 100%), in bubonic or pneumonic murine infection models. The attenuation could be further augmented by generation of combinatorial deletion mutants, namely ΔΔ, ΔΔ, ΔΔ, ΔΔ, and ΔΔ. We earlier showed that deletion of the gene, which encodes Braun lipoprotein (Lpp) and activates Toll-like receptor-2, reduced virulence of CO92 in murine models of bubonic and pneumonic plague. The surviving mice infected with ΔΔ, ΔΔ, and ΔΔ mutant strains were 55-100% protected upon subsequent re-challenge with wild-type CO92 in a pneumonic model. Further, evaluation of the attenuated T6SS mutant strains revealed significant alterations in phagocytosis, intracellular survival in murine macrophages, and their ability to induce cytotoxic effects on macrophages. The results reported here provide further evidence of the utility of the STM screening approach for the identification of novel virulence factors and to possibly target such genes for the development of novel live-attenuated vaccine candidates for plague.