Earlier, we reported the identification of new
virulence factors/mechanisms of Yersinia pestis using an in vivo signature-tagged mutagenesis (STM) screening approach. From this screen, the role of rbsA, which encodes an
ATP-
binding protein of
ribose transport system, and vasK, an essential component of the
type VI secretion system (T6SS), were evaluated in mouse models of
plague and confirmed to be important during Y. pestis
infection. However, many of the identified genes from the screen remained uncharacterized. In this study, in-frame deletion mutants of ypo0815, ypo2884, ypo3614-3168 (cyoABCDE), and ypo1119-1120, identified from the STM screen, were generated. While ypo0815 codes for a general secretion pathway
protein E (GspE) of the T2SS, the ypo2884-encoded
protein has homology to the βγ
crystallin superfamily, cyoABCDE codes for the
cytochrome o oxidase operon, and the ypo1119-1120 genes are within the Tol-Pal system which has multiple functions. Additionally, as our STM screen identified three T6SS-associated genes, and, based on in silico analysis, six T6SS clusters and multiple homologs of the T6SS effector
hemolysin-coregulated
protein (Hcp) exist in Y. pestis 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 ΔlppΔypo0815, ΔlppΔypo2884, ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3. We earlier showed that deletion of the lpp gene, which encodes Braun
lipoprotein (Lpp) and activates Toll-like receptor-2, reduced virulence of Y. pestis CO92 in murine models of bubonic and
pneumonic plague. The surviving mice infected with ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3 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 in vitro 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.