Source: AGRICULTURAL RESEARCH SERVICE submitted to
AN IN VIVO NEGATIVE SELECTION SCREEN TO IDENTIFY GENES AND GENE FAMILIES REQUIRED FOR TICK COLONIZATION
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
NEW
Funding Source
USDA INHOUSE
Reporting Frequency
Annual
Accession No.
0421445
Grant No.
(N/A)
Project No.
5348-32000-033-02R
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Mar 15, 2011
Project End Date
Feb 28, 2014
Grant Year
(N/A)
Project Director
NOH S M
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
PULLMAN,WA 99164
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31133101040100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
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.
Project Methods
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.

Progress 10/01/12 to 09/30/13

Outputs
Progress Report 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. 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. 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.

Impacts
(N/A)

Publications


    Progress 10/01/11 to 09/30/12

    Outputs
    Progress Report 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. 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. 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.

    Impacts
    (N/A)

    Publications


      Progress 10/01/10 to 09/30/11

      Outputs
      Progress Report 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. 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. In collaboration with colleagues at Washington State University, ARS scientists in Pullman, WA have established that Francisella tularensis subsp. novicida initially infects the midgut of 100% of acquisition fed Dermacentor andersoni nymphs with stable colonization and replication during a subsequent molt. Increased dissemination to and florid replication within the salivary gland was closely linked to a second (transmission) feed and culminated in bacterial secretion into the saliva and successful transmission. Testing multiple mutants resulted in total bacterial levels similar to that observed for single mutants. However, there was evidence of a bottleneck during colonization resulting in a founder effect in which the most successful mutant varied when comparing individual ticks. Thus, it is essential to assess mutant success at the level of the tick population rather than in individual ticks. The ability of F. novicida to recapitulate the key physiological events by which bacteria colonize and are transmitted by ixodid ticks provides a new genome-wide approach to identify required pathogen molecules and pathways. A manuscript describing these experiments and results is in review.

      Impacts
      (N/A)

      Publications