Plague is a zoonosis that is present in wild rodent populations worldwide and is transmitted primarily by fleas. Yersinia pestis, the plague bacillus, is unique among the enteric group of gram-negative bacteria in having adopted an arthropod-borne route of transmission. Y. pestis has evolved in such a way as to be transmitted during the brief encounter between a feeding flea and a host. A transmissible infection primarily depends on the ability of Y. pestis to grow in the flea as a biofilm that is embedded in a complex extracellular matrix. Bacteria in the biofilm phenotype are deposited into the dermis together with flea saliva, elements which cannot be satisfactorily mimicked by needle-injection of Y. pestis from laboratory cultures. The objective of this project is to identify and determine the function of Y. pestis genes that mediate flea-borne transmission and the initial encounter with the host innate immune system at the infection site in the skin. We study the interaction of Y. pestis with its insect vector by using an artificial feeding apparatus to infect fleas with uniform doses of wild-type or specific Y. pestis mutants. We seek to identify Y. pestis genes that are required for the bacteria to infect the flea midgut and to produce a biofilm that blocks the flea foregut and that is required for efficient transmission. The strategy entails first identifying bacterial genes that are differentially expressed in the flea by gene expression analysis and other techniques. Specific mutations are then introduced into these genes, and the mutants tested for their ability to infect and block the flea vector. Identification of such transmission factors allows further studies into the molecular mechanisms of the bacterial infection of the flea vector. Detailed understanding of the interaction with the insect host may lead to novel strategies to interrupt the transmission cycle. During FY2014, we continued to investigate the regulation of Y. pestis genes required for the biofilm life stage in the flea vector. This year we reported that a major bacterial gene regulatory system (known as PhoP-PhoQ) is induced during infection of the flea vector and is required to produce a normal transmissible infection. Because PhoP-PhoQ upregulates several genes required for bacterial resistance to innate immunity, induction of the PhoP-PhoQ system in the arthropod vector prior to transmission may preadapt Y. pestis to resist the initial encounter with the mammalian innate immune response. We have completed microarray experiments to compare the in vitro and in vivo (i.e., in the flea) transcriptomes of wild-type and phoP-negative Y. pestis and are now analyzing the data. We anticipate that PhoP-regulated genes that are involved in development of the transmission stage in the flea will be revealed. We also continue to investigate the genetic changes that led to the evolutionarily recent transition of Y. pestis to an arthropod-borne transmission route. Many nonfunctional genes (pseudogenes) occur on the Y. pestis genome;some of them are implicated in regulating the biofilm phenotype that enhances transmission by fleas. In a 2014 publication we detailed the genetic mechanisms behind the evolution of the flea-borne transmission route of Y. pestis. Remarkably, only four minor genetic changes- one gene gain and three gene losses- were required. Restoration of three Y. pestis pseudogenes with the equivalent functional genes of Yersinia pseudotuberculosis, the recent ancestor of Y. pestis, eliminates transmissibility from the flea. Conversely, mutation of these genes in Y. pseudotuberculosis results in the gain of biofilm-forming ability in the flea and transmissibility. In addition, fleas experience acute toxicity and 30-40% mortality after they ingest Y. pseudotuberculosis in a blood meal. In contrast, Y. pestis is not orally toxic to fleas. In FY2014 we completed a project that identified the Yersinia urease enzyme as the toxic factor. All Y. pestis are urease-negative because ureD is a pseudogene;our work indicates that loss of urease activity was positively selected during the evolution of Y. pestis because it increased flea-borne transmission potential. Thus, gene loss appears to have played a significant role in Y. pestis evolution. In collaboration with the Genomics Unit of the RML Research Technologies Branch, we are examining and characterizing the transcriptomes of Y. pestis, Y. pseudotuberculosis, and our biofilm-producing Y. pseudotuberculosis mutant during growth in vitro and during infection of the flea, with the goal of identifying genes and gene regulatory pathways that are important for flea-borne transmission. The in vivo and in vitro transcriptomic comparisons between these three strains are designed to broadly identify candidate components of biofilm regulatory pathways and other genes important for the recent evolutionary adaptation to flea-borne transmission. Significant differences in the expression of orthologous genes in the flea might be indicative of evolutionary changes in gene regulatory pathways. We have also further developed models to examine host-parasite interactions in the dermis after transmission by flea bite, and completed a project on the host immune response to flea saliva and how this might influence transmission (in press). This year we have added a third flea species, the cat flea Ctenocephalides felis, to our studies. We have developed new experimental systems to quantitate the CFUs transmitted by cohorts of fleas as well as individual fleas over a four-week period following a standardized infectious blood meal. This will allow for quantitative comparison of two mechanisms by which fleas transmit Y. pestis: biofilm-independent early-phase transmission during the first few days after the infectious blood meal and proventricular biofilm-dependent transmission, which occurs from 1-4 weeks after the infectious blood meal. These data will improve mathematical models used to assess the relative importance of the two transmission mechanisms in plague ecology.
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