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Continuing Activities as Follows: Additional experiments completed to prepare for an NCSU publication for this project. We now have two strains with the gene mutation that we prepared based on our C. jejuni microarray studies. Characterization of the second mutant strain as well as confirmation of the results from the first mutant strain has been the primary focus. E. coli strains that have a matching gene mutation are being used to underscore the importance of the results in C. jejuni and to help in the determination of mechanism underlying the phenotypes. <P>The research has been presented at both state and international meetings. The manuscript is nearing completion and should be submitted before the fiscal year end. Through our collaboration with researchers at UNC we have modeled C. jejuni disease in a small animal (mouse). This collaboration has resulted in a recent publication.
Non-Technical Summary:<br/>
Campylobacter jejuni is the leading cause of severe bacterial gastroenteritis in the U.S., and has been classified as a Category B Bioterrorism agent by the NIH. In addition to the tremendous burden of disease due to severe gastroenteritis, C. jejuni infection is highly associated with the development of Guillain-Barre syndrome (GBS), an acute motor paralysis that has been linked to the production of autoimmune antibodies directed against C. jejuni glycolipid antigens. In particular, glycosylated structures such as glycoproteins, lipooligosaccharide and capsule are suggested to be the major antigens important for GBS induction following C. jejuni infection. Understanding the regulation of the production of these molecules and their effects on the host response will provide new strategies for the control and treatment of C. jejuni infection. The combined use of mutagenesis of specific bacterial genes and the characterization of those mutant strains in vitro (cell culture models) and in vivo (animal models) will provide insight into the mechanisms leading to C. jejuni induced gastroenteritis and subsequent GBS induction.
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Approach:<br/>
Using molecular biological techniques we are generating mutations in bacterial genes that we identified as being differentially regulated at two growth temperatures, 37 C (human) and 42 C (chicken). Our preliminary studies suggested that specific glycosylation was reduced at 42 C and we reasoned this might impact the host response and be one of the reasons that chickens are not sickened by C. jejuni whereas humans are. Using the techniques of allelic replacement, we have generated two mutant strains lacking a single gene function. We have characterized the strains through the use of invasion assays, electron microscopy, biofilm growth, and motility assays. We have cloned the putative enzyme in E. coli and prepared antibodies to both the wild-type enzyme as well as the upstream protein to verify that both proteins are produced in the wild-type strain, but that the mutant strain lacks the protein we knocked out and still produces the upstream protein. We are also using E. coli mutant strains to compare phenotypes for the gene knock-out in the E. coli genetic background to those seen in C. jejuni to aid in suggesting mechanisms/pathways that lead to the phenotypes.
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Progress:<br/>
2011/10 TO 2012/09<br/>
OUTPUTS: Activities were as follows: Additional experiments were completed to finalize a publication for this project. We have obtained a second C. jejuni strain mutated for the Cj 0372 gene. We have obtained E. coli mutants for a similarly annotated gene and expressed both Cj 0372 and Cj 0371. Antibodies to both of these proteins have been generated. We have verified that Cj0371 is produced at normal levels in the Cj 0372 mutant and thus demonstrated that the loss of Cj 0372 is responsible for the phenotypes we have measured. Posters on the research were presented at the NC ASM meeting Oct. 2011 by both a postdoc and undergraduate student. The postdoc also presented a poster on this work at the 16th Intl. Campylobacter, Helicobacter and Related Organisms Mtg, 2011. Our own efforts have led to a soon to be completed independent publication. Through our collaborators at UNC a related publication was successfully completed.
<br/>PARTICIPANTS: Participants: PI: Dr. Deborah Threadgill, Post-doctoral fellow: Dr. Kristen Delaney, Research Associate: Ms. Erin Harrell (MS) and NCSU undergraduate student Ms. Bridget Conley. Collaborators: Dr. Christian Jobin, and Dr. Xiaolun Sun, UNC Chapel Hill. Training opportunities for Dr. Kristen Delaney as well as Ms. Bridget Conley.
<br/>TARGET AUDIENCES: Target Audiences: The NC ASM and CHRO are public meetings where any interested party may register to attend. The knowledge gained is therefore available to the public prior to publication of completed research.
<br/>PROJECT MODIFICATIONS: We are now able to work with two mutant C. jejuni strains that we created, and also to work with E. coli mutants to determine the precise function of the annotated genes. We have established new connections with labs that work on the E. coli enzyme to obtain reagents and protocols so that we can complete our C. jejuni studies.
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IMPACT: Change in knowledge: We have come to a clearer understanding about a previously unknown gene in C. jejuni that we saw was elevated in expression at the chicken body temperature. This gene appears to be a conserved gene related to coping with oxidative stress that is also present in E. coli and we are using E. coli mutants as well as gene expression clones from E. coli to verify the C. jejuni studies. Change in action: Recent publications from groups working with E. coli have indicated a clear path forward for our research, and we are confident our revised and expanded publication will now be accepted at a ranked journal. We can now assay the function of the C. jejuni gene using clear protocols for E. coli, and can also show that the gene has similar functions in C. jejuni to what it has in E. coli.