Plague: Modern strategies for an ancient disease
Program leader: Virginia Miller
SE-RP-006: Yersinia autotransporters (Yaps): Structure, function and host response to Plague
Virginia Miller and Matt Redinbo
University of North Carolina
Autotransporter proteins (ATs) are exceptionally elegant yet complex proteins ideally positioned on the bacterial cell surface (or released) for interactions with the host. ATs consist of three basic domains: a N-terminal signal sequence, a “passenger domain” (PD) of variable size and finally a b-domain of 250-300 amino acids at the C-terminus that facilitates translocation of the PD across the outer membrane. Although more than 1000 have been identified by in silico analyses a relatively few have been studied in detail as to their molecular and biological function. The Yaps (predicted ATs) of Y. pestis represent an excellent opportunity to do this as (a) they do not appear to be closely related to the already well studied ATs and thus are likely to encode novel functions, and (b) Y. pestis is amenable to molecular, genetic and biological studies. We have preliminary data indicating that all ten of the yaps are expressed during infection. We also demonstrated that the Yaps are localized and exposed on the bacterial surface, while three Yaps appear to be released into the culture supernatant. In addition, we have constructed deletion mutations in all ten yaps in a fully virulent Y. pestis strain and have begun testing the effect of these mutations on virulence. While these tests are ongoing, four of these mutants clearly have phenotypes in a bubonic plague model of infection. These results are consistent with our hypothesis that the yaps play a role in pathogenesis. The studies proposed here to examine the host response to Y. pestis and the role of Yaps in that response (Aims 1 & 2) are based on the observation that many yap mutations appear to affect early events and/or dissemination of Y. pestis. These studies should inform us not only about key host responses during Y. pestis infection, but they should also give us important clues as to host targets of the Yaps. This will complement the structural work described in Aim 3 aimed at defining important functional domains of the Yaps and also at providing a foundation for future screening for broad spectrum small molecule inhibitors of ATs.
SE-RP-007: Controlling the progression of Pneumonic Plague
University of North Carolina
Our overall goal for this research plan is to use a mouse model system for pneumonic plague to discover and evaluate Y. pestis genes critical for the development and progression of disease. We will pinpoint these candidates using two methods: transcriptional profiling to reveal genes that are differentially regulated in the various stages of pneumonic plague; and forward genetics approaches to screen/select for Y. pestis genes that are indispensable for development of pulmonary disease.
Specific Aim 1. Comparative transcriptional responses by Y. pestis during the stages of pneumonic plague. We previously developed a whole genome microarray to characterize the bacterial transcriptome during pneumonic plague, but this analysis was technically limited to a late stage of infection. Therefore, we will use quantitative RT-PCR to examine a subset of Y. pestis genes throughout the entire time course of disease. This subset of 288 genes is based on genes that show evidence of differential expression during infection, as well as genes that were not sufficiently explored by microarray technology.
Specific Aim 2. Forward genetics to identify bacterial genes important in the development of pneumonic plague. Transposon site hybridization (TraSH) is a gene discovery strategy using negative selection to identify bacterial genes that are essential during infection. The microarrays we have constructed will allow us to take advantage of a TraSH-based approach using array hybridizations to identify Y. pestis genes implicated in various stages of the pulmonary infection. We will be comparing these results with experiments in which we use ultra-deep sequencing as an alternative method to pinpoint the relevant genes in our mutant pools.
Specific Aim 3. Analyzing the importance and role of candidate virulence-associated genes. The genes selected in the first two Aims will be targeted for further analysis by creating defined mutant strains of Y. pestis. Mutant and control strains will be tested for virulence in the murine model of pneumonic plague, monitoring bacterial proliferation in the lung, dissemination to the spleen, and histopathology to evaluate differences in the manifestation or kinetics of disease. The characterization of mutant strains will be extended to a microarray analysis of host transcriptional responses during infection, done in collaboration with Dr. Virginia Miller.
