Source: MICHIGAN STATE UNIV submitted to
IDENTIFICATION OF CAMPYLOBACTER JEJUNI FACTORS THAT INFLUENCE CHICKEN COMMENSALISM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1009602
Grant No.
2013-67012-25269
Cumulative Award Amt.
$21,096.50
Proposal No.
2016-05008
Multistate No.
(N/A)
Project Start Date
Jun 1, 2016
Project End Date
May 31, 2017
Grant Year
2016
Program Code
[A7201]- AFRI Post Doctoral Fellowships
Project Director
Johnson, J. G.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Microbiology & Molec. Genetics
Non Technical Summary
We will be using cutting-edge approaches to identify genes that allow the bacteriumCampylobacter jejuni, an importantfoodborne pathogen, to colonize its natural host, the chicken. We will be leveraging this information to create protocols or develop compounds that can reduce the carriageof thebacterium in chickens, without using conventional antibiotics, making food safer as a result.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71232601100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3260 - Poultry meat;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
During the project period I will be continuing two projects that arose from the original NIFA funding:1. From the TnSeq screen that was conducted in chickens, I identified a regulator that was significantly decreased for colonization. I have identified, by RNAseq analysis, that this regulator is involved in controlling expression of the heme uptake genes in Ccampylobacter jejuni. I will be working to further charactrize this regulator by looking at direct interactions of the protein with the heme uptake gene promoter and am collaborating with groups at Vanderbilt and the University of Chicago to look at effects on heme uptake and the structure of the regulator, respectively.2. From the small molecule inhibitor screen, I identified several compounds that specifically inhibited growth of C. jejuni in vitro. Recently, I generated mutants to these compounds and have sequenced the genomes to identify single nucleotide polymorphisms (SNPs) that may be responsible for resistance. I will be working to determine whether these mutated genes are in fact the targets of thesmall molecule inhibitors.
Project Methods
1. Binding of the heme uptake regulator to the promoter of the gene cluster will be evaluated by electrophoretic mobility shift assay. Purified regulator will be added to reactions containing radiolabeled promoter fragments and subsequently separated on tris-glycine gels. Phosphoimaging will be used to visualize whether promoter fragments are retarded in response to the addition of regulator.2. Heme utilizationassays will be performed in iron depleted media with HPLC purified heme or hemoglobin. If the regulator truly increases expression of the heme utilization gene cluster, we would expect mutants of this gene cannot use the heme in iron depleted media to restore growth.3. Small molecule resistant strains will be sequenced using an Illlumina MiSeq and genome assembly will be performed using Geneious. Assembled genomes will then be compared to strains that are sensitive to the compounds in order to identify SNP candidates. These genes will then be compared to Campylobacter coli and Helicobacter pylori homologs since C. coli is similarly affected by these compounds and H. pylori is not.

Progress 06/01/16 to 05/31/17

Outputs
Target Audience:During this reporting period, I presented my work in research seminars and poster sessions. This was done at individual universities, one research group at the USDA, and international scientific conferences. The audiences at the venues were generally academic reseachers interested in basic science related to bacterial infection. The USDA presentation was to academic researchers as well as government researchers within the poultry production and product safety group. I also published two open access publications during this reporting period, meaning that some of this research is available to the international community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I have been able to work with protein crystallographers at the University of Chicago and Vanderbilt University to solve the structure of our regulator. This has provided be with experience in the tools they use to characterize proteins at the molecular level. How have the results been disseminated to communities of interest?We have published research articles and have presented to various groups, the results of this work. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. We have been continuously working on characterizing the regulator described in this aim. The investigator we initially worked with at the University of Chicago left academia and we located another collaborator at Vanderbilt University whois currently purifiying various truncations of the regulator to determine whether they will form crystals to solve the structures - we tried to crystallize the various domains of the protein, but none of those worked as the protein isremarkablysoluble. Theyperformed proteolytic analysis to identify the regions of potential truncation and are convince their current work will result in a structure of the ligand binding domain. Additionally, we have identified tandem repeats within the promoter region of the heme utilization genes that may be used for regulator binding. We are currently creating promoter truncations to look at the ability of the regulator to specifically bind these regions. Since these repeats overlap with the ferric uptake regulator (Fur) binding sites, we are also looking at interations between Fur and our regulator (HeuR). 2. We have shown that genotypically pure mutants of the gene Cjj0812 are resistant to the small molecular inhibitors we identified in our earlier work. Additionally, we also found mutants that were resistant to these compounds that were not represented by any particular genotype. To understand the mechanism of resistance in these strains, we recently performed transcriptomics on cultures and have identified approximately three dozen genes that are differentially expressed in these mutants. We are currently following up on these various pathways, e.g. Cjj0812 is involved in processing cysteine into sulfur and alanine, to see how they impact Campylobacter resistance to our growth inhibitors.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni. Ha R, Frirdich E, Sychantha D, Biboy J, Taveirne ME, Johnson JG, DiRita VJ, Vollmer W, Clarke AJ, Gaynor EC. J Biol Chem. 2016 Oct 21;291(43):22686-22702. Epub 2016 Jul 29. PMID: 27474744
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Generation and Screening of an Insertion Sequencing-Compatible Mutant Library of Campylobacter jejuni. Johnson JG, DiRita VJ. Methods Mol Biol. 2017;1512:257-272. PMID: 27885613
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: The PAS Domain-Containing Protein HeuR Regulates Heme Uptake in Campylobacter jejuni. Johnson JG, Gaddy JA, DiRita VJ. MBio. 2016 Nov 15;7(6). pii: e01691-16. doi: 10.1128/mBio.01691-16. PMID: 27935836