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Molecular Mechanisms of Pathogenesis and Antibiotic Resistance in Camplyobacter Jejuni

Lin, Jun
University of Tennessee
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C. jejun is the leading foodbome human pathogen in the United States and many other industrialized countries. This pathogenic organism not only causes watery diarrhea and inflammatory colitis, but also is associated with Guillain-Barre syndrome, an acute disease of peripheral nerves that is characterized by ascending paralysis, which may lead to respiratory muscle compromise and death. The estimated cases of campylobacteriosis in the United States are more than 2 million per year. The medical and productivity costs resulting from C. jejuni infection are estimated at 1.5 to 8.0 billion dollars each year in the United States. Epidemiologic studies have revealed that poultry are the major reservoir of C. jejuni, and consumption of undercooked chickens or food contaminated by poultry products are responsible for the majority of human Campylobacter enteritis. Despite the significance of C. jejuni in animal health, food safety and public health, no effective means are available to control Campylobacter to date because of lack of information on the pathogenesis and antibiotic resistance mechanisms in Campylobacter.

Overall objective of this project is to study molecular mechanisms of pathogenesis and antibiotic resistance in Campylobacter, consequently leading to the development of innovative intervention strategies to control Campylobacter infections in animal reservoirs and in humans and improving the safety of our food suppliers and reduce foodborne illness in the U.S.

We plan to achieve the goal of this application by pursuing the following specific objectives:

  1. To systematically screen genetic loci required for antimicrobial peptide (AMP) resistance in C. jejuni using random transposon mutagenesis and whole genome microarray.
  2. To identify and characterize genes contributing to AMP resistance in C. jejuni.
  3. To examine sequence polymorphism, antigenic homology, and in vitro immune protection of CmeC in primary Campylobacter isolates.
  4. To evaluate the immunogenicity and protective efficacy of a novel subunit CmeC vaccine in a chicken model of C. jejuni infection.
  5. To determine the role of CmeC vaccine in potentiating the activity of clinical antibiotics.
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NON-TECHNICAL SUMMARY: C. jejun is the leading foodbome human pathogen in the United States. Poultry is a major reservoir for Campylobacter infections in humans. Thus, on-farm control of C. jejuni is urgently needed to reduce foodborne illness in the U.S. To achieve this goal, searching antimicrobial alternatives and identifying novel virulence determinants have been two major efforts for developing effective intervention strategies to control C. jejuni on poultry farm. Antimicrobial peptides (AMPs) are a key component of host innate immunity to limit bacterial colonization. The AMPs are also recognized as a novel class of antibiotics to control pathogens including C. jejuni. As an important strategy to evade killing by future peptide antibiotics used in poultry and by avian innate immunity, the mechanisms of AMP resistance are still unknown in C. jejuni. Lack of this information and the appreciated magnitude of the role of AMP in antibiotic development and in innate immune defense have provided a strong rationale for us to conduct the proposed studies. In addition, our proposed studies will also elucidate critical information on the development of a novel subunit vaccine against C. jejuni infections in humans and animals. Once we have accomplished this project, we may develop a novel, bifunctional vaccine that not only directly reduce C. jejuni colonization but also combat antibiotic resistance in C. jejuni. In summary, the proposed studies have significant impact on animal health, food safety, public health and biodefense.

APPROACH: This hatch project uses fundamental and contemporary approaches to study Campylobacter jejuni, the leading foodbome human pathogen in the United States. To study the mechanisms of AMP resistance in Campylobacter, we will build a larger mutant library using random transposon mutagenesis and screen for C. jejuni mutants with increased sensitivity to AMPs. We will also use DNA microarray to profile gene expression in wild-type AMP-susceptible C. jejuni and three different AMP-resistant derivatives (mutants with resistance to PM, bacteriocin, or Fowlicidin-1, a host defense AMP from chicken) and screen promising gene targets based on array data analysis. The proposed microarray work will complement and strengthen the results from random mutagenesis study. After identifying candidate genes using above genomics approaches, we will conduct further functional studies to confirm phenotypes of the mutants. First, the mutants from random mutagenesis study will be complemented to confirm if the interrupted genes are indeed responsible for PM-resistance. Second, the selected mutants from random transposon mutagenesis and microarray analysis (generated by site-directed mutagenesis) together with parent strain and complemented constructs will be subjected to AMP killing assay using different AMPs including bacteriocins and chicken host defense peptides. Finally, we will investigate if some identified genes are required for C. jejuni colonization in chickens. To develop CmeC-based subunit vaccine against Campylobacter, we will sequence and align cmeC gene from a number of primary isolates of C. jejuni. Full-length recombinant CmeC will be produced and purified for generation of polyclonal anti-CmeC serum. Antigenic homology will be evaluated by immnoblotting using anti-CmeC antibodies. Effect of affinity-purified anti-CmeC IgG on the function of CmeABC pump will be evaluated. To evaluate protective efficacy of CmeC, we will orally immunize chickens with different doses of purified rCmeC with an effective mucosal adjuvant. Systemic and mucosal immune responses (serum IgA and IgG and intestinal secretory IgA) elicited by the vaccination will be determined by ELISA. Vaccinated chickens will be orally challenged with C. jejuni and protective efficacy will be assessed by measuring intestinal colonization of C. jejuni. Furthermore, to determine if CmeC also functions as a novel vaccine against antibiotic resistance, we will immunize chickens with CmeC vaccine, followed by challenging chickens with C. jejuni and evaluating Campylobacter colonization level in response to medicated feed containing macrolide, major drug of choice for treating human Campylobacteriosis.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Chemical Contaminants
Bacterial Pathogens
Viruses and Prions
Meat, Poultry, Game