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<p>Campylobacter jejuni is the leading cause of food-borne illness in humans and sheep abortions in the U.S. In other bacterial pathogens, quorum sensing systems, such as the LuxS/autoinducer-2 (AI-2) system, are known to contribute to virulence. C. jejuni harbors a functional AI-2/LuxS system, where the LuxS enzyme recycles methionine in the activated methyl cycle (AMC), and also synthesizes the quorum signaling molecule AI-2. Previous publications have identified correlation of this enzyme with many important virulence factors, including colonization of the host, toxin production, and response to stress. Using a pregnant guinea pig model of abortion, we have demonstrated that the luxS mutation led to 0% abortion rate in pregnant guinea pigs, whereas wildtype C. jejuni caused an abortion rate of 50% in the guinea pigs. Chromosomal complementation of luxS gene in the luxS mutant restored its virulence. Additionally, our preliminary data shows that the luxS mutation is associated with efflux pump systems, which are one of the key factors responsible for rising antimicrobial resistance in Campylobacter. In addition, some Campylobacter strains that lack the luxS gene are unable to survive in conditions lacking methionine, which is an important amino acid for many cell biochemical functions. These results, and findings from previous publications, suggest luxS plays a central role in C. jejuni pathogenicity. Although the AI-2/LuxS system has been identified to be involved with many virulence factors, there is a fundamental gap in our understanding of the mechanisms responsible for its role in C. jejuni pathogenesis. Our primary goal, which fall in direct lines with two of the AFRI NIFA Program Area Priorities (animal health, food safety and human health), is to define the role of the LuxS/Autoinducer-2 (AI-2) quorum sensing system in Campylobacter jejuni pathogenesis. Our main objectives are to: </p>
<p>1) define the role of the LuxS/Autoinducer-2 (AI-2) quorum sensing system in Campylobacter jejuni phenotypes necessary for environmental adaptation, colonization, and invasion of the host intestinal environment, and </p>
<p>2) determine the role of AI-2/LuxS system in methionine synthesis and virulence of C. jejuni.</p>
<p>This project would set the foundation for developing novel therapeutic strategies against C. jejuni and also significantly expand the knowledge of AI-2/LuxS systems in general, both of which are major milestones of this project. As a fellow of the NIFA Pre-doc Fellowship, another major goal of this project is to train the project director (PD) to become a technically and functionally competent professional in the food and agricultural sciences. To do so, the PD will build experience in grantsmanship, research study design, and networks with other research scientists through university activities or national training conferences. In addition, the PD will gain exposure and skills to veterinary medicine through a variety of animal species used in disease models in the laboratory as well as observing the practice and academics of clinical medicine. These opportunities will collectively provide a strong foundation for the PD to develop into a translational research scientist and a leader addressing issues critical for food safety, animal health, and the future of agriculture in the U.S.</p>
<p>NON-TECHNICAL SUMMARY: <br/>Campylobacter jejuni is the leading cause of bacterial diarrhea in humans and sheep abortions in the U.S. Prevention of the diseases caused by Campylobacter are hampered by the lack of efficacious vaccines and lack of effective treatment protocols, particularly with the rising occurrence of antimicrobial resistance. To identify alternative therapeutic targets, our project focuses on identifying how bacterial communication systems (quorum sensing), more specifically the LuxS/autoinducer-2 (AI-2) system that C. jejuni possesses, contribute to bacterial virulence and disease development. Although the AI-2/LuxS system has been identified to be involved with many virulence factors, there is a fundamental gap in our understanding of the mechanisms responsible for its role in C. jejuni pathogenesis. The primary goal of this project is to define the
molecular mechanisms of C. jejuni pathogenesis and AI-2/LuxS system when the luxS gene is compromised. Methods we will use to achieve our objective is first to define the role of AI-2/LuxS system in C. jejuni traits necessary for environmental adaptation, colonization, and invasion of the host intestinal environment. To do so, we will conduct several in vitro and in vivo studies to define the role of luxS during the pathogenic stage of adherence and translocation through the intestinal epithelium. Secondly, we will determine the role of luxS in methionine synthesis and C. jejuni virulence. This will be done by testing the physiology and metabolism of C. jejuni strains with and without a functioning luxS gene in conditions rich in and restricting in methionine. We will also determine the role of functional methionine biosynthesis pathways in in vitro and in vivo virulence models.
Ultimately, the acquisition of such knowledge is critical for developing novel therapeutic strategies against diseases associated with C. jejuni, thus reaching our long term research goals of protecting human and animal health, and improving food safety. This work will improve our basic understanding of how luxS attributes to the success of C. jejuni virulence and pathogenesis. We also expect results from this research to greatly expand the knowledge base for various other bacterial pathogens possessing the AI-2/LuxS system, due to the conserved nature of luxS.
<p>APPROACH: <br/>The goals of this project will be accomplished through two objectives. In the first objective, we will define the role of AI-2/LuxS system in C. jejuni phenotypes necessary for environmental adaptation, colonization, and invasion of the host intestinal environment. The first set of experiments will test the role of luxS in this pathogenic stage using polarized intestinal cells in vitro to analyze cellular adhesion and invasion of several C. jejuni strains. Next, in vivo adherence and translocation of the same strains using intestinal tract explants from chickens and guinea pigs will be evaluated. The use of both animal model systems will allow the evaluation of C. jejuni in different host species whereby C. jejuni is either pathogenic (guinea pigs) or nonpathogenic (chickens). Tissue explants are used because they possess much of the original tissue
properties (e.g. mucin and host-specific receptors on intestinal epithelia) which are very difficult to mimic through conventional in vitro tissue culture methods. The second objective will determine the role of luxS in methionine synthesis and C. jejuni virulence. We will first use unique methods to assess the physiology and metabolism of C. jejuni strains with or without a functioning luxS gene under methionine-restricted and methionine-rich conditions. In addition to comparing the strains with their wildtype counterparts in their ability to grow in the varying concentrations of methionine medias, we will also profile the metabolites from the AI-2/LuxS system of these strains using liquid chromatography-tandem mass spectrometry. Secondly, we will conduct in vitro and in vivo studies to determine the role of a functional methionine biosynthesis pathway. This will be done using in vitro
cellular invasion assays like the first aim and in an in vivo guinea pig abortion virulence model. Research progress will be verified through several milestones at the institutional, national and international level. As a fellow of the NIFA Pre-doc Fellowship, the PD will give presentations at the national meetings annually, andan international meeting in 2015. The PD will continually update her LinkedIn profile documenting career progress and achievements. Within the university, the PD is required to present on a quarterly-annual basis her current research progress at departmental and ASM Student Chapter Work-In-Progress meetings. The PD is also required to submit annual reports to both the Interdepartmental Microbiology program and the Veterinary Microbiology and Preventive Medicine department, which are assessed by third party members of the program. The first paper will be submitted
by the end of year 1 which will include the first experiment from the first objective. The second and third papers, which will discuss results from the first and second objectives, respectively, will be finished by the end of the second year, along with the PD's dissertation. As stated before, this project will significantly increase our knowledge base of the role that AI-2/LuxS systems play in C. jejuni disease, as well as contribute to general knowledge of other bacterial AI-2/LuxS systems. In addition, understanding these mechanisms will allow for exploitation of new therapeutic targets. The outcomes of this project will greatly assist in the development of novel therapeutic strategies against C. jejuni in domestic animals and humans. The highly interactive mentoring and training activities from this fellowship will significantly increase the technical and functional competence of
the PD to better prepare her for a professional research career in the agricultural sciences field. This research will impact numerous individuals in the biomedical science community, especially those studying food-borne pathogen biology and Campylobacter; vaccine producers, food animal producers, and the general public. We hope to develop collaborative relationships with such investigators wishing to address similar issues based on scientific and societal importance. Strains will also be available to interested parties.