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ARS Home » Pacific West Area » Pullman, Washington » Animal Disease Research » Research » Research Project #421445

Research Project: An in Vivo Negative Selection Screen to Identify Genes and Gene Families Required for Tick Colonization

Location: Animal Disease Research

2013 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 the identification of genes required for transmission of tick borne pathogens. 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, F. novicida mutants are being used in a negative selection screen for their ability to colonize the tick. Approximately 200 F. novicida mutants have been tested, and four genes have been identified that are involved in tick colonization. One of these genes encodes for an outer membrane protein of unknown function. The other three genes are involved in a variety of cellular functions including iron acquisition, regulation of gene expression and cell wall biosynthesis. The iron acquisition pathway is of particular interest because iron metabolism is also likely important in tick colonization of A. marginale.

4. Accomplishments