Source: UNIVERSITY OF MICHIGAN submitted to
IDENTIFICATION OF CAMPYLOBACTER JEJUNI FACTORS THAT INFLUENCE CHICKEN COMMENSALISM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1000302
Grant No.
2013-67012-21136
Project No.
MICW-2013-03955
Proposal No.
2013-03955
Multistate No.
(N/A)
Program Code
A7201
Project Start Date
Sep 1, 2013
Project End Date
Aug 31, 2015
Grant Year
2013
Project Director
Johnson, J. G.
Recipient Organization
UNIVERSITY OF MICHIGAN
(N/A)
ANN ARBOR,MI 48109
Performing Department
Microbiology & Immunology
Non Technical Summary
Campylobacter jejuni is one of the most common causes of bacterial-derived gastroenteritis in the United States, with a projected 2.4 million diagnosed and undiagnosed cases, annually. Infection often occurs through the ingestion of raw or undercooked chicken meat, which becomes contaminated with Campylobacter following dissemination from the chicken gastrointestinal tract. Due to this, much interest lies in understanding the factors of the bacterium that promote colonization of the chicken gut. Here we propose the use of a sensitive, novel screening technology to provide the most comprehensive accounting of C. jejunifactors involved in colonization of the chicken gastrointestinal tract. Also, we intend to screen for small molecule inhibitors of a previously identified colonization factor in an attempt to identify those that may be efficacious in reducing the numbers of C. jejuni in the chicken gastrointestinal tract. These aims will provide the basis for the establishment of a pipeline for the identification of non-antibiotic compounds that can reduce the numbers ofC. jejuniwithin chickens, increasing the safety of chicken-derived meat products for human consumption.
Animal Health Component
0%
Research Effort Categories
Basic
70%
Applied
10%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240101100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Specific aim 1: Identify mechanisms of C. jejuni that influence commensal colonization of the chicken gastrointestinal tract using a deep-sequencing-based mutagenesis approach. A method termed insertion sequencing (INSeq), was used to identify mutants of Bacteroides thetaiotaomicron that were affected in their ability to colonize the gastrointestinal tract of gnotobiotic mice. Applying this approach to C. jejuni will enable the most comprehensive and sensitive interrogation of the genome to date, to identify diverse C. jejuni traits that promote commensalism in the chicken gastrointestinal tract. Additionally, this approach allows for the construction of an ordered library of transposon mutants that will be an invaluable resource for the Campylobacter field. Specific aim 2: Identify small molecule inhibitors of a known C. jejuni colonization factor. Previous work supported by the USDA identified multiple flagellar mutants of C. jejuni that were unable to colonize the chicken gastrointestinal tract. Further work, also USDA-supported, elucidated the regulatory hierarchy of flagellar biogenesis, with the operon flgDE2 being expressed late in the biosynthetic pathway and essential for biogenesis. Using a flgDE2:cat reporter, we will screen approximately 174,000 diverse chemical compounds at the University of Michigan Center for Chemical Genomics for inhibitors that block flgDE2 expression. Those found to significantly reduce expression of this reporter, are likely to affect flagellar biosynthesis and will be studied further both to understand their mechanism of action and to determine whether they can alter the course of C. jejuni colonization in the chicken gastrointestinal tract. Similar drug discovery efforts can be applied to mechanisms identified in specific aim 1.
Project Methods
For specific aim 1, INSeq libraries will be generated from samples collected from both Mueller-Hinton agar grown cultures and bacteria enriched from chicken caeca. This protocol will be performed exactly as that done by Goodman et al (Nature Protocols, 2011) and sequence data will be analyzed by Dr. Jonathan Livny (Broad Institute-MIT and Harvard). Briefly, sequences will be enumerated and mapped to theC. jejuni genome, then normalized by the number of individual insertions mapped within each gene and expressed as the number of sequences per million reads. This data will be generated for both input and output samples followed by the calculation of fitness ratios (output:input) for each gene. These ratios will allow us to identify those mutants that are unable to colonize (ratios < 1), are unaffected during colonization (ratios = 1), or are enriched during colonization (ratios > 1). From this, we will take those loci that appear to be required for colonization and mutagenize them in order to confirm whether those genes are required for colonization. Afterwards, depending on the predicted functions of those genes identified, we can begin to characterize the mechanisms of those gene products and their roles in allowingC. jejunito establish itself within the chicken gastrointestinal tract. For specific aim 2, we will subject a previously contructedflgDE2:cat reporter strain to the entire 154,000 small molecule library housed at the Center for Chemical Genomics at the University of Michigan. Briefly, the reporter strain will be grown in the presence of each compound under standard growth conditions in chloramphenicol (CM)-containing media. Under these conditions, if a compound is able to inhibitflgDE2 expression, we would expect a decrease in the production of the Cat gene product and an increase in the sensitivity of the strain to CM. Compounds that are found to confer sensitivity to CM will be further assayed to eliminate indirect effects on CM sensitivity and to determine the minimum inhibitory concentration of the compound. Specific inhibition of flagellar biosynthesis by suspect compounds will be determined using compound-containing motility agar. Finally, following confirmation of a motility defect, remaining compounds will be used to inhibit colonization of the chicken gastrointestinal tract both pre- and post-inoculation of day-of-hatch chicks.

Progress 09/01/13 to 08/31/15

Outputs
Target Audience:Through the publications and presentations I gave during this period, I reached out to scientists with broad interests. I gave talks to general biology departments at state universities as well as food science departments at elite institutions. In many of these situations, I was able to reach out to everyone from undergraduates to senior faculty, describing how my research aims to increase the safety of food. I also was able to connect with individuals in animal health pharmaceuticals and discuss myvisions for my research program. Changes/Problems:The original goal of the small molecule inhibitor screen was to identify flagellar inhibitors inC. jejunias compounds that may prevent colonization of the chicken. None of the inhibitors that were identified robustly inhibited motility and were unlikely to affect colonization. As such, we changed the focus of the research to develop genus-specific inhibitors of growth and chicken colonization. What opportunities for training and professional development has the project provided?I was able to train undergraduate students during the course of the project, as well as teach a course relavent to my field. I also was able to make valuable connections in the field of food science and have begun to attract interest as a faculty member. How have the results been disseminated to communities of interest?Yes. The results from this work have resulted in multiple publications and several presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The original proposal outlined two specific aims: 1. To construct and screen an INSeq-compatible transposon mutant library of Campylobacter jejuniin chickens, in order to identify genetic determinants that are required for full colonization of the chicken. 2. To screen a small molecule library against a constructed C. jejuni reporter strain in order to identify molecules that inhibit flagellar biosynthesis - a known colonization factor. 1. I was able to construct the INSeq-compatible mutant library, which consists of approximately 8,500 C. jejuni mutants. Illumina sequencing of this library showed that approximately 60% of the genome was successfully mutated with the remaining 40% consisting, primarily, of genes that are predicted to be essential. This library was passed through day-of-hatch chickens and, following sequencing of output pools, we identified approx. 140 genetic determinants that are required for full colonization of the chicken gastrointestinal tract. This led to a publication in the Journal of Bacteriology and is leading to further study on mechanisms that govern C. jejuni capsule production and lipoprotein sorting. There are also other determinants that we will need to follow up on, as they may also be important for chicken colonization. 2. I performed the small molecule inhibitor screen using a C. jejuni flgDE2::cat-kan reporter strain and identified several molecules that were able to modestly inhibit motility in C. jejuni. Since a greater effect on motility is required to affect chicken colonization, I turned my attention toward small molecules that appeared to specifically inhibit growth of C. jejuni. I verified that these compounds inhibited growth of C. jejuni, but not other Gram-negative bacteria, including the closely related organism, Helicobacter pylori. These results were recently published in Antimicrobial Agents and Chemotherapy. It has become our hypothesis that these compounds may be efficacious at reducing C. jejuni carriage in chickens without using antibiotics that are used in human medicine. We are currently writing a proposal based on identifying the targets of these compounds and optimizing the molecules for use as agricultural feed additives.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Cold Spring Harbor Laboratory Microbial Pathogenesis and Host Response September 2013 Johnson, J., Livny, J., and V. DiRita. Poster: High-throughput approaches to identifying and targeting Campylobacter jejuni factors that influence chicken commensalism.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Research Seminar: Indiana State University  Department of Biology April 2014 Johnson, J. Talk: Using omics approaches to identify factors of Campylobacter jejuni that influence chicken commensalism
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 21st Annual Midwest Microbial Pathogenesis Conference, Chicago, IL September 2014 Johnson, J. and V. DiRita. Poster: Tryptophan confers hyperosmotic stress resistance to Campylobacter jejuni during colonization of the chicken gastrointestinal tract
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Research Seminar: University of Delaware  Dept. of Animal and Food Sciences December 2014 Johnson, J. Talk: Using omics approaches to identify factors of Campylobacter jejuni that influence chicken commensalism
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Research Seminar: Iowa State University  Dept. of Food Science and Human Nutrition April 2015 Johnson, J. Talk: Using omics approaches to identify factors of Campylobacter jejuni that influence chicken commensalism
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Frirdich, E., Vermeulen, J., Biboy, J., Soares, F., Taveirne, M.E., Johnson, J.G., DiRita, V.J., Girardin, S.E., Vollmer, W., and Erin C. Gaynor. 2014. Peptidoglycan LD-Carboxypeptidase Pgp2 Influences Campylobacter jejuni Helical Cell Shape and Pathogenic Properties, and Provides the Substrate for the DL-Carboxypeptidase Pgp1. J. Biol. Chem. 289(12):8007-18.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Johnson, J.G., Carpentier, S., Spurbeck, R., Sandhu, S., and Victor J. DiRita. 2014. Genome sequences of Campylobacter jejuni 81-176 variants with enhanced fitness relative to the parental strain in the chicken gastrointestinal tract. Genome Announc. 2(1):e00006-14.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Gordon Research Conference: Microbial Toxins and Pathogenicity July 2014 Johnson, J. and V. DiRita. Poster: Tryptophan accumulation confers hyperosmotic stress resistance to Campylobacter jejuni during colonization of the chicken gastrointestinal tract
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Johnson, J.G., Livny, J., and Victor J. DiRita. 2014. High-throughput sequencing of Campylobacter jejuni transposon mutant libraries reveals mapA as a fitness factor for chicken colonization. J. Bacteriol. 196(11):1958-67.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Johnson, J.G., Yuhas, C., McQuade, T., Larsen, M. and Victor J. DiRita. 2015. Narrow-spectrum inhibitors of Campylobacter jejuni flagellar expression and growth. Antimicrob. Agents Chemother. 59(7):3880-6.
  • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Johnson, J.G. and V.J. DiRita. 2015. Construction and analysis of Campylobacter jejuni TnSeq mutant libraries. Method Mol. Biol. [Submitted]