Acquisition of omptin reveals cryptic virulence function of autotransporter YapE in Yersinia pestis.

Autotransporters, the largest family of secreted proteins in Gram-negative bacteria, perform a variety of functions, including adherence, cytotoxicity and immune evasion. In Yersinia pestis the autotransporter YapE has adhesive properties and contributes to disease in the mouse model of bubonic plague. Here, we demonstrate that omptin cleavage of Y. pestis YapE is required to mediate bacterial aggregation and adherence to eukaryotic cells. We demonstrate that omptin cleavage is specific for the Y. pestis and Y. pseudotuberculosis YapE orthologues but is not conserved in the Yersinia enterocolitica protein. We also show that cleavage of YapE occurs in Y. pestis but not in the enteric Yersinia species, and requires the omptin Pla (plasminogen activator protease), which is encoded on the Y. pestis-specific plasmid pPCP1. Together, these data show that post-translation modification of YapE appears to be specific to Y. pestis, was acquired along with the acquisition of pPCP1 during the divergence of Y. pestis from Y. pseudotuberculosis, and are the first evidence of a novel mechanism to regulate bacterial adherence.

Improving the Th1 cellular efficacy of the lead Yersinia pestis rF1-V subunit vaccine using SA-4-1BBL as a novel adjuvant.

The lead candidate plague subunit vaccine is the recombinant fusion protein rF1-V adjuvanted with alum. While alum generates Th2 regulated robust humoral responses, immune protection against Yersinia pestis has been shown to also involve Th1 driven cellular responses. Therefore, the rF1-V-based subunit vaccine may benefit from an adjuvant system that generates a mixed Th1 and humoral immune response. We herein assessed the efficacy of a novel SA-4-1BBL costimulatory molecule as a Th1 adjuvant to improve cellular responses generated by the rF1-V vaccine. SA-4-1BBL as a single adjuvant had better efficacy than alum in generating CD4(+) and CD8(+) T cells producing TNFα and IFNγ, signature cytokines for Th1 responses. The combination of SA-4-1BBL with alum further increased this Th1 response as compared with the individual adjuvants. Analysis of the humoral response revealed that SA-4-1BBL as a single adjuvant did not generate a significant Ab response against rF1-V, and SA-4-1BBL in combination with alum did not improve Ab titers. However, the combined adjuvants significantly increased the ratio of Th1 regulated IgG2c in C57BL/6 mice to the Th2 regulated IgG1. Finally, a single vaccination with rF1-V adjuvanted with SA-4-1BBL+alum had better protective efficacy than vaccines containing individual adjuvants. Taken together, these results demonstrate that SA-4-1BBL improves the protective efficacy of the alum adjuvanted lead rF1-V subunit vaccine by generating a more balanced Th1 cellular and humoral immune response. As such, this adjuvant platform may prove efficacious not only for the rF1-V vaccine but also against other infections that require both cellular and humoral immune responses for protection.

Development of bioluminescent bioreporters for in vitro and in vivo tracking of Yersinia pestis.

Yersinia pestis causes an acute infection known as the plague. Conventional techniques to enumerate Y. pestis can be labor intensive and do not lend themselves to high throughput assays. In contrast, bioluminescent bioreporters produce light that can be detected using plate readers or optical imaging platforms to monitor bacterial populations as a function of luminescence. Here, we describe the development of two Y. pestis chromosomal-based luxCDABE bioreporters, Lux(PtolC) and Lux(PcysZK). These bioreporters use constitutive promoters to drive expression of luxCDABE that allow for sensitive detection of bacteria via bioluminescence in vitro. Importantly, both bioreporters demonstrate a direct correlation between bacterial numbers and bioluminescence, which allows for bioluminescence to be used to compare bacterial numbers. We demonstrate the use of these bioreporters to test antimicrobial inhibitors (Lux(PtolC)) and monitor intracellular survival (Lux(PtolC) and Lux(PcysZK)) in vitro. Furthermore, we show that Y. pestis infection of the mouse model can be monitored using whole animal optical imaging in real time. Using optical imaging, we observed Y. pestis dissemination and differentiated between virulence phenotypes in live animals via bioluminescence. Finally, we demonstrate that whole animal optical imaging can identify unexpected colonization patterns in mutant-infected animals.