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Characterization of Genes Involved in teh Production of Pili by Campylobacter Jejuni

Investigators
Cooper, Margarethe
Institutions
University of Arizona
Start date
2010
End date
2012
Objective
Previous work in our laboratory on biofilms has demonstrated the presence of fibers (pili) disseminating from the cell wall of C. jejuni isolates not expressing flagella (flaAB mutants). To further investigate this finding, annotation analysis was initiated of a recently sequenced C. jejuni strain M129 and a gene with nucleotide sequence homology to the pilT gene present in several bacterial genomes was identified. Reverse BLAST searches and further examination against the sequence of the type strain C. jejuni NCTC 11168 identified the formation and function of a type IV pilus. We hypothesize that C. jejuni produces a type IV pilus, and that Cj1343c is the major pilin subunit.

The following specific aims are proposed to test the above hypothesis:

  1. Determine the role of putative pilA (Cj1343c) gene in expression of pilus fibers. An isogenic mutant of the putative pilA (Cj1343c) gene in NCTC 11168 flaAB mutant (NCTC 11168 flaAB:pilA double mutant) will be made and examined by scanning electron microscopy (SEM) to confirm the loss of pili. Additionally, an isogenic mutant of putative pilA (Cj1343c) gene in the wild-type NCTC 11168 (NCTC 11168 putative pilA (Cj1343c) mutant) will be generated to investigate the gene's role in various virulence assays.
  2. Demonstrate surface localization of putative PilA (Cj1343c) protein. Antibody will be produced against the recombinant protein and used to attach gold beads to the expressed pilus from the NCTC 11168 flaAB mutant. This will confirm that the putative pilA (Cj1343c) gene is the major pilin subunit.
  3. Characterize the role of the putative pilA (Cj1343c) gene in virulence assays. The NCTC 11168 putative pilA (Cj1343c) mutant and NCTC 11168 (wild-type strain) will be used to characterize the role of the putative pilA (Cj1343c) gene in biofilm production and attachment/invasion assays. In vivo studies will be performed to assess the role of the putative pilA (Cj1343c) gene in the intestinal colonization of poultry.
More information
NON-TECHNICAL SUMMARY: Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the U.S., with an estimated cost of treatment and loss of productivity exceeding $1 billion annually. Epidemiologic case studies have linked campylobacteriosis to the handling and consumption of contaminated poultry. Despite many advances in the understanding of C. jejuni virulence, there is still much that is not understood about the mechanisms of pathogenesis. Because of this, there is still no effective vaccine in poultry against C. jejuni. Interventions at the level of the farm, poultry processing plant, and consumer have failed to eliminate the risk of C. jejuni infection from raw chicken. Thus the development of a vaccine is critical to improve food safety. Pili play an important role in the attachment of the bacterium to the host cell and are therefore considered important vaccine targets. Previous work in the laboratory indicates the presence of fibers disseminating from a strain of C. jejuni incapable of producing flagella. Further investigation and annotation analysis of sequences identified the formation and function of a type IV pilus, leading to the hypothesis that Cj1343c gene is the major pilin subunit. The purpose of this project is to test the hypothesis by 1) generating an isogenic mutant of the putative pilA (Cj1343c) gene in NCTC 11168 flaAB mutant (NCTC 11168 flaAB:pilA double mutant) to confirm the loss of pili by scanning electron microscopy, 2) by generating an isogenic mutant of putative pilA (Cj1343c) gene in the wild-type NCTC 11168 (NCTC 11168 putative pilA (Cj1343c) mutant) to investigate the gene's role in various virulence assays, 3) to attach gold beads to the expressed pilus from the NCTC 11168 flaAB mutant to confirm that the putative pilA (Cj1343c) gene is the major pilin subunit and 4) to characterize the role of the putative pilA (Cj1343c) gene in various virulence assays. Assessing the role of the putative pili in the colonization of poultry will contribute significant knowledge regarding virulence in C. jejuni, and determine whether Cj1343c would be a viable vaccine target. If so, pili vaccines have proved successful for other pathogens and an effective C. jejuni vaccine for poultry would be significant in preventing campylobacteriosis.

APPROACH:
Specific Aim 1. Determine role of putative pilA (Cj1343c) gene in expression of pilus fibers. An isogenic mutant of the putative major pilin subunit gene, pilA (Cj1343c), will be created in an existing NCTC 11168 flaAB mutant strain made in the Joens laboratory and in the wild-type NCTC 11168. C. jejuni isolates (NCTC 11168 flaAB mutant and NCTC 11168 flaAB:pilA double mutant) will be grown and prepared for SEM. The samples will be observed and digital images recorded using an Ultra-High Resolution Field Emission-SEM S-4800 (University Spectroscopy and Imaging Facilities, University of Arizona).
Specific Aim 2. Demonstrate surface localization of putative PilA (Cj1343c) protein. A hexahistidine tagged (HIS-tagged) recombinant protein will be made of the putative pilA (Cj1343c) gene. Eluted protein will be shipped to a commercial company for production of a rabbit anti-pilin PilA (Cj1343c)-specific antibody. This antibody will be used for ImmunoGold labeling. The C. jejuni flagella and putative pilA (Cj1343c) double mutant (NCTC 11168 flaAB:pilA), the parent strain (NCTC 11168 flaAB), and the NCTC 11168 (wild-type strain) will be grown in a biofilm preparation and prepared for transmission electron microscopy (TEM). Immunostaining will be performed, and the samples will then be viewed and digital images recorded using a Hitachi H-8100 TEM.
Specific Aim 3. Characterize the role of the putative pilA (Cj1343c) gene in virulence assays. Both in vitro and in vivo experiments will be conducted. The role of C. jejuni pili will be assessed using two in vitro assays. In the biofilm assay, the ability of the putative pilA (CJ1343c) mutant to form a biofilm will be compared to wild-type NCTC 11168. The putative pilA (Cj1343c) mutant will also be assessed for its ability to attach to and invade host cells in vitro using INT 407 cells. Additionally, the role of the C. jejuni NCTC 11168 putative pilA (Cj1343c) mutant will be compared to the wild-type NCTC 11168 to assess the role of putative pilus in vivo in colonization of poultry. All of the virulence assays will be done in triplicate to allow for standard deviation and statistical analysis of the data. Statistical significance will be determined for individual samples by using an unpaired Student t test, and assays with bacterial counts will be calculated following log10 transformation of the data. P values of <0.05 will be considered significant.
This is a post-doc grant and the efforts put forth into these experiments and in coordinating these experiments along with the collaboration of a mentor and other individuals at expert core facilities such as the University Spectroscopy and Imaging Facilities and Animal Facilities will allow the post-doc to develop the knowledge and skills in advanced molecular techniques, immunology, and research with animals to be a competent scientist and to be competitive. Evaluation and success will be measured by publications in scientific journals and presentations at scientific meetings.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
ARZT-321810G-02-533
Accession number
220698
Categories
Viruses and Prions
Bacterial Pathogens
Campylobacter
Commodities
Meat, Poultry, Game