To identify Yersinia pestis and Francisella tularensis surface proteins required for invasion and survival in human cells. The modification of surface proteins of Y. enterocolitica and Y. pseudotuberculosis by temperature and limiting calcium ion has led to the discovery of key facets involved in microbial pathogenesis. The outermembrane changes regulated by temperature and calcium ion led to the discovery of type III systems. Analysis of the InvA surface protein, elucidated the first mechanism of bacterial cell invasion. Young et al. 1999) were also able to show phospholipase A secretion is via the flagellum. This led to the identification of the first virulence protein using the flagellum as an export apparatus. Therefore, it is clear that the study of surface changes in yersiniae have provided essential clues and insights into pathogenicity. Simple one dimensional protein gels of membrane proteins isolated from Y. enterocolitica cells cultured at low and high temperature suggest a greater change than what has been reported. Y. pestis cycles between two very different hosts, the flea vector and its mammalian reservoir. Temperature is a key environmental cue used by Y. pestis to modulate gene expression. Phenotypically this is manifested in a dramatic phasing of surface proteins, relatively few of which have been characterized. Our goal is to compile a complete catalog of surface proteins using phoA fusions. This data will provide significant insight into targets for vaccine development.
NON-TECHNICAL SUMMARY: Yersinia pestis, the bacterium that causes bubonic and pneumonic plague, and Francisella tularensis responsible for tularemia are potential WMD agents that can be used against man and animals. At present there are no vaccines for either agent and we know little about their mechanisms by which they cause disease The overall purpose of this proposal is to make a complete panel of Y. pestis and F. tularensis surface proteins genetically fused to the enzymatic reporter molecule alkaline phosphotase gene(phoA). Such fusions will permit gene regulatory studies and determine individual gene contributions to invasion and growth within white blood cells. These data are critical in dissecting the disease process of each organism, and the information gained from these investigations will lead to new strategies for vaccine development and therapeutic interdiction.
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APPROACH: Experimental Design and Preliminary work: We have past success with this approach in Y. enterocolitica. Using phoA and lacZ fusions we assembled a precise picture of flagellar type III gene expression under various environmental conditions (Kapatral and Minnich, Kapatral et al. , Young et al. and our unpublished results). We are in the process of isolating mini-Tn5phoA fusions in Y. pestis in sufficient numbers to statistically cover all exported proteins and surface proteins. These fusions are generated using a pir-dependent suicide plasmid bearing mini-TnphoA. Independent pools of ex-conjugants have been isolated under different temperatures (25oC and 37oC) and high salt conditions, environmental conditions known to affect surface protein profiles. We will class these fusions as either constitutive or under environmental induction. Those fusions expressed at 37oC will be subjected to identification by DNA sequencing to give an exact profile of surface proteins under host conditions. This will be accomplished by cloning the insertions and sequencing across the transposon junctions followed by data base analysis of the generated sequences. Using PCR, we have adapted a rapid DNA sequencing method to identify the insertion junctions. Basically, the known transposon sequence end is amplified with degenerate primers to the unknown chromosomal sequence. The degenerate primers have an inside constant sequence which is then amplified in a second round of PCR reactions. This DNA is then subjected to PCR DNA sequencing. Using this method we have identified to the nucleotide, phoA insertions for 67 surface proteins. Within this panel, we have identified several fusions to caf1, pla protease, invasin, murine toxin, several ABC transporters, homoserine lactone transporter, several peroxidase genes, ompC, a serine protease, pesticin immunity protein, lipase, just to name a few. Having worked out the conditions for mutant screening, DNA isolation, insertion mapping and sequencing, we are now identifying about 50 insertions per week. Assuming 10% of proteins are surface associated, we need approximately 1,000 mutants to cover 3X saturation. The same approach will be used for Francisella tularensis. Expected results: These fusions will also provide a means to examine global regulation and potential roles of surface components during infection. Mutations that subsequently modify phoA expression will lead us to master control circuits. Hence, we can take individual phoA fusions and look for spontaneous suppressors for expression at normally nonpermissive conditions as one example. Additionally, identification of these proteins and their regulation may provide new insights for potential vaccine targets. Analysis of surface proteins contributing to intracellular invasion and survival will be identified. We have initiated extensive studies on the uptake and survival of Y. pestis in epithelial cells. Pools of phoA fusions will be formed to identify sets of clones that have lost the ability to invade host cells or show decreased survival within host cells. We have initiated these experiments with a subset of our identified fusions.