2012 Annual Report 1a.Objectives (from AD-416): Identify metabolic and other functional pathways, genes and gene families required for tick colonization. Specific aim 1: Using Dermacentor andersoni ticks for negative selection, screen pools of Francisella tularensis subsp. novicida mutants to identify those mutants that have reduced fitness in the midgut or salivary glands. Specific aim 2: Verify the phenotype of a subset of the F. novicida mutants with reduced fitness in the midgut or salivary glands using dual infection experiments. 1b.Approach (from AD-416): A variety of intracellular bacterial pathogens of both humans and animals are transmitted by ticks and include members of the genus Anaplasma, Ehrlichia, and Rickettsia. Not only are the molecular mechanisms by which these pathogens are able to colonize the tick largely unknown. But, identification of these mechanisms is difficult, if not impossible, as the techniques required for genetic manipulation of this group of pathogen are in the early stages of development. In contrast to this limitation for tick-borne bacterial pathogens, exploitation of genomic sequence data through mutant library screens has allowed for relatively rapid identification of genes required for specific functions in a broad array of bacterial pathogens. Similarly, a transposon mutant library of Francisella tularensis subsp. novicida has been developed and used to negatively select and identify genes required for pulmonary and systemic infection in mice. We have determined that F. novicida readily colonizes D. andersoni in a manner similar to other tick borne pathogens, including A. marginale. Through a negative selection screen and high throughput sequencing, we propose to use this mutant library to identify genes required for tick colonization. 3.Progress Report: This work relates to objective 1 of parent project by provision of pathogen genome data concerning genes required for efficient transmission. In collaboration with colleagues at Washington State University, ARS scientists in Pullman, Washington, have established that Francisella tularensis subsp. novicida colonize and are transmitted from the tick similarly to tick-borne bacterial pathogens. In order to identify genes required for tick colonization, we are screening a mutant library to identify those mutants which are unable to colonize the tick. Approximately 20% of the library has been tested in a primary screen. Approximately 60% of the mutants are covered from the midgut, which is the organ of interest. Thus, approximately 40% of the genes screened to date, 10% of which code for proteins, may be required for tick colonization. The negatively selected mutants include genes that encode hypothetical proteins as well as better characterized genes such as pdpD, frgA, pilD, and xerC, which are involved in a variety of cellular functions such as virulence, nutrient acquisition, motility, and DNA repair and recombination. A secondary screen to confirm these results is underway.