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The broad objectives are to understand the mechanisms by which the human pathogen Campylobacter jejuni colonizes chickens. The original proposal had three aims: i) to study the role of of a newly identified glycoprotein, Cj1496c, and other glycoproteins on colonization in the chick model, ii) to determine whether a system of DNA uptake, called Cts, is required for DNA exchange between strains during chick infection and iii) to re-visit signature tagged mutagenesis screening for mutants with a focus on sites other than the ceca. Based on the comments of the review panel and a recommendation for funding less than the original request, we will focus largely on aims ii and iii. The role of protein glycosylation remains very important to us and we will continue analyzing Cj1496c, a glycoprotein required for wild type levels of chick colonization, but a comprehensive analysis of the N-linked protein glycome will be a lower priority in favor of focusing on aims ii and iii. Genetic exchange mechanisms that lead to acquisition of new colonization and virulence traits or antibiotic resistance are important targets for investigation. My lab discovered the Cts competence system in C. jejuni and our goal is to determine whether this system operates for acquisition of antibiotic resistance during chick infection. Work in the third aim will enable us to learn whether specific sets of genes are required by C. jejuni to colonize different parts of the gastrointestinal tract. In work to date we have focused primarily on genes required for cecal colonization. As we ultimately wish to use our knowledge from this work to control chick infection in C. jejuni, a broader understanding of factors used by the microbe to colonize sites in the gastrointestinal tract other than the ceca is essential.
NON-TECHNICAL SUMMARY: The human bacterial pathogen Campylobacter jejuni lives in the gastrointestinal tract of chickens as a harmless, commensal. Because of this, humans are at risk for illness caused by Campylobacter infection from eating improperly prepared chicken or from contamination of other foods during the preparation of meals that include chicken. The purpose of this project is to identify potential targets for vaccine or drug development that will enable us to eliminate the threat of Campylobacter infection in chickens that are coming to market.
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APPROACH: We will use molecular and genetic approaches to study Campylobacter jejuni infection in newly-hatched chickens. We developed a number of tools to discover and analyze genes for chicken colonization and other aspects of C. jejuni biology, and this set of tools is the basis of many of our approaches. For aim i I proposed making various alleles of the the Cj1496c gene to determine what role, if any, glycosylation plays in the activity of this protein. In the interim between writing the proposal and now, we submitted a manuscript showing that Cj1496c is required for wild type chick colonization levels, but its glycosylation is not. Pursuit of further knowledge on glycosylation will be a lower priority in the period of this funding. To study the role of Cts in genetic exchange in vivo for aim ii, will involve chicken co-infection with two genetically marked strains, followed by intestinal dissection and scoring for newly arising strains with the resistance alleles of each of the co-infecting strains. For aim iii we will use our tagged transposon library to identify mutants that are unable to colonize in the gastrointestinal tract, screening in sites other than the cecum. Newly hatched chicks will be gavaged with mixtures from the mutant library and after three or seven days, bacteria will be recovered from sites along the gastrointestinal tract. Tags from these collected, pooled bacteria will be amplified by PCR and used to probe a master filter arrayed with individually tagged strains used to make up the infection pools. In this way we were successful isolating cecal-colonization mutants.
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PROGRESS: 2006/09 TO 2007/08 <BR>
OUTPUTS: The Output activities include the following: 1. Experiments: A prominent outcome from this year of funding is our careful analysis of experimental Campylobacter jejuni colonization in the chicken gastrointestinal tract for the first 14 days post-hatch. We are assessing bacterial and host factors that contribute to long term colonization. These experiments include colony counts, microscopy and qRT-PCR analysis of inflammatory responses by the host. We are using wild type C. jejuni as well as selected mutant strains isolated as part of this project. We have collected a great deal of data regarding the dynamics of wild type Campylobacter infection after oral gavage in day-of-hatch chicks, as well those of a mutant strain lacking the pglB gene, responsible for N-linked protein glycosylation. Another mutant, lacking the cj0143c gene, which encodes a component of a putative ion transport system, has also been characterized in some detail. Cj0143c is a glycoprotein and analysis of the role in chicken colonization of glycosylation on this protein has been carried out. Also extensive in vitro work to study the physiology of a cj0143c mutant relative to wild type has also been undertaken. <BR> 2. Teaching/mentoring: A graduate student, Lindsay Davis, is carrying out many of these studies. This has included training in electron microscopy, pathology, microbial physiology and genetics, and molecular biology. <BR> Dissemination of the outcomes has been carried out by seminars at three significant meetings: 1. the Campylobacter/Helicobacter Research meeting in Amsterdam, 2. the FASEB Host-Pathogen Conference in Snowmass, Colorado and 3. the Cold Spring Harbor Pathogenesis meeting in Cold Spring Harbor, NY. <BR> PARTICIPANTS: Lindsay Davis - carried out experiments related to Cj0143c and the chick colonization dynamics. She is a graduate student partially funded by a Genetics Training Grant to the University of Michigan. <BR> TARGET AUDIENCES: The general bacterial pathogenesis research community is the target audience of our work thus far. The efforts to reach these individuals include presentation of the work at international meetings such as FASEB and CHRO. <BR> PROJECT MODIFICATIONS: No major changes are anticipated. As yet we have not carried out experiments to analyze the role of natural transformation in acquisition of new traits during chick colonization. A post-doctoral fellow who will carry out that work has been identified and will join the group once his Ph.D. studies are completed.
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IMPACT: 2006/09 TO 2007/08<BR>
This work has advanced our knowledge of Campylobacter jejuni colonization in two ways. First, we have learned a great deal about the earliest events in colonization from the perspective of the host. As a frequent and benign colonizer of chickens, Campylobacter was hypothesized to colonize in the absence of significant immune stimulation of the animals. Contrary to this hypothesis, our results indicate that immediately after infection there is a transient inflammatory response associated with elevated cytokine production and influx of heterophils to the cecum. This response is evidently unable to clear the infection, as cecal colony counts of Campylobacter continue to increase during this period, leading to very high loads of bacteria in the cecum at 14 days post-gavage. Unlike wild type, pglB mutant C. jejuni are poor colonizers and induce a very weak heterophil response. We intend to carry out further analyses using older animals in which the innate immune response is more fully developed. These experiments are ongoing and are being developed more fully into another proposal for USDA. We also carried out a good deal of investigation of cj0143c, which we showed is essential for chick colonization. By RT-PCR we showed that this is part of a three-gene operon. Its similarity to periplasmic components of zinc transport systems from other bacteria led us to investigate whether it plays a role in zinc-dependent growth. This work showed that a mutant lacking cj0143c is unable to grow in media with limiting zinc concentrations, whereas wild type cells grow well. Preliminary experiments using extracts enriched for the Cj0143c protein demonstrate that the protein binds zinc. The protein is glycosylated at a single residue (Asparagine-28) but glycosylation is not required for its role in zinc-limiting growth or chick colonization. The very poor chick colonization levels exhibited by the cj0143c mutant strain compared to wild type suggest that this system is a good target for developing anti-Campylobacter therapeutics aimed at reducing the load of C. jejuni in chicken colonization.