Genetic Basis for Campylobacter Jejuni Induced Pathotypes in the Host

Non-Technical Summary:<br/>
Campylobacter jejuni is a common cause of bacterial foodborne diarrhea world-wide. Serious medical and economic losses are associated with this disease. Several autoimmune conditions have been definitively associated with recent C. jejuni infection including Guillain Barre syndrome (GBS) and Miller Fisher syndrome (MFS). GBS is the world's leading cause of acute neuromuscular paralysis. Five percent of people with GBS die and a significant proportion of the others with this disease have long term disabilities. It would be ideal to vaccinate to prevent disease due to C. jejuni. However, little is known about the virulence factors of this bacterium and how it causes autoimmune disease. Vaccines often target surface structures of bacterial cells, but vaccine development for C. jejuni has been difficult because surface structures of C. jejuni sometimes trigger paralysis and thus would pose a risk to vaccine recipients. The results from this project could lead to the development of vaccines that would not pose a risk of paralysis. Pathogens evolve and may become more virulent during infection of a single host. We will study the evolution of a gastrointestinal pathogen in mice to determine how host responses early in infection influence this evolutionary process. The knowledge gained could lead to development of new disease prevention and treatment strategies that do not promote evolution of increased virulence in pathogens or lead to autoimmunity.
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Approach:<br/>
Aim 1: Determine whether differences in innate immune responses produce different evolutionary outcomes in C. jejuni populations infecting C57BL/6 IL-10-/-, C57BL/6 NF-KB-/-, and C57BL/6 WT mice that exhibit different strengths of the inflammatory response (IL-10-/- > WT > NF-kappaB-/-). Hypothesis 1: During serial passage, virulence of four C. jejuni strains will increase most in mice experiencing the most inflammation; that is, virulence will increase most in IL-10-/- mice, to an intermediate level in WT mice, and least in NF-KB-/- mice.
<br/>Aim 2: Determine whether major differences in pathogen genotype/ phenotype produce similar or different evolutionary outcomes in populations of phylogenetically and phenotypically distinct C. jejuni strains infecting C57BL/6 IL-10-/-, NF-kappa-/-, and WT mice to test the generality of the results in specific aim 1. Hypothesis 2: In all C. jejuni strains, genes that show evidence of positive selection will include those encoding molecules known to interact with the innate immune system through Toll-like receptors and Nod-like receptors (e.g. lipopolysaccharide [LPS], lipooligosaccharide [LOS], lipoproteins, Type III secretion systems, and possibly others).
<br/>Aim 3: Identify contingency genes under selection during infection of IL-10-/- mice but not WT or NF-KB-/- mice by resequencing the evolved strains. Contingency genes contain homopolymeric nucleotide tracts subject to length variation by slip-strand mutagenesis, producing phase variation. Many C. jejuni genes involved in determination of surface structures important in interactions of pathogens with the innate immune system have such homopolymeric tracts. We expect to find changes in known and putative virulence factors, particularly in contingency genes. Hypothesis 3: In all C. jejuni strains, genes exhibiting change during evolutionary increase in virulence will include those encoding known virulence factors and secretion systems as well as those encoding surface molecules such as LOS, capsule, and flagella.
<br/>Aim 4: Identify host innate response genes mediating intrahost evolutionary increases in C. jejuni virulence using concurrently gathered immunological data. We expect innate immune responses to be progressively exacerbated as the number of passages increases, indicating the evolution of immune evasion in the pathogen mediated by an increased ability to activate host innate immune signaling pathways. Hypothesis 4: Innate immune inflammatory responses increase progressively during serial passage of C. jejuni in host animals. We further hypothesize that the resultant increase in tissue damage releases more resources for growth of the pathogen.
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: 1) Draft Genome Sequences of Two Campylobacter jejuni Clinical Isolates, NW and 2600. Whole-genome sequences of C. jejuni isolates NW and D2600 were determined by Illumina sequencing at the Genomics Technology Support Facility at Michigan State Univ. NW was isolated at Sparrow Hospital in Lansing, MI, from a patient returning from Kenya with acute gastroenteritis. Strain D2600 was also isolated from a patient with gastroenteritis provided to us by the Centers for Disease Control and Prevention. These isolates colonized the C57BL/6 interleukin 10 knockout mouse model stably for 30 days but did not produce enteritis (1). Three other C. jejuni strains, including NCTC11168, that were tested in parallel both produced enteritis during initial infection and increased in virulence after a single passage in mice, while strains NW and D2600 did not (1). Virulent strains (such as NCTC11168) induce an inflammatory response that results in diarrhea, enlarged ileocecocolic lymph node, thickened colon and cecum wall, and occasional hemorrhagic enteritis (1, 7). The increased virulence of NCTC11168 during serial passage in mice was associated with mutations in genes containing hypermutable homopolymeric tracts, or contingency loci (6). Paired-end, 75-cycle reads were assembled using Velvet v1.1.04 (12) to generate 1,622,415 bases in 56 contigs for D2600 and 1,652,701 bases in 60 contigs for NW. Paired-end, 75-cycle reads were assembled using Velvet v1.1.04 (12) to generate 1,622,415 bases in 56 contigs for D2600 and 1,652,701 bases in 60 contigs for NW. The average G C contents were 30.31% and 30.46% for NW and D2600, respectively. Annotation of the contigs was done using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline and yielded 1,619 and 1,690 potential protein-coding regions in the D2600 and NW genomes, respectively. The NW genome is similar in gene content and order to those of the sequenced strains RM1221 and S3, which were both isolated from chickens in the US. RM1221 contains four C. jejuni insertion elements (CJIE1 through CJIE4) (3, 9), of which S3 contains parts of CJIE1 and CJIE4 (2). The NW genome contains a sequence from only one insertion element, CJIE3. Contingency loci are defined by homopolymeric guanine tracts greater than seven bases in length in the C. jejuni genome (6,10), and an initial search for these tracts showed that the genomes of NW and D2600 contain 21 and 26 contingency loci, respectively. Notably, strain D2600 contains the genes encoding a class B lipooligosaccharide (LOS) structure (4, 8), and a homopolymeric guanine tract is present within the 2,3-sialyltransferase gene cstII. This tract leads to the variable expression of sialylated LOS (5) and is intriguing considering the association of LOS sialylation and the development of post infection autoimmune neuropathy (5, 11). An analysis of changes to the NW and D2600 genomes resulting from mouse passage is ongoing. This whole-genome shotgun project was deposited at DDBJ/EMBL/GenBank. Illumina reads were deposited at the NCBI Sequence Read Archive.
<br/>PARTICIPANTS: Several students were mentored for study of enteric diseases of food animals. John Paul Jerome (PhD candidate) successfully defended his PhD thesis and has gone on to a job as a postdoc with Professor Sheng Yang He who has an endowed chair to study food borne pathogens in plants. Four other students are pursuing PhD degrees in food safety related to these enteric pathogens including Jessica St. Charles, Ankit Malik, Barbie Gadsden, DVM, and Phillip Brooks. Two of these students have passed their preliminary exams. Dr. Gadsden is preparing for this exam. Two of the graduate students are underrepresented minorities (African American). Four undergraduate students are working in the lab on these projects including Hahyung Kim, Elizabeth Gensterbloom, Julian Yu, and Anthony Brooks. Anthony was accepted to medical school and will start in 2013. Dr. Mansfield organized a seminar in Food and Waterborne Diseases for the faculty and students of Michigan State University on October 19, 2012. People attending came from the Agricultural, Veterinary Medicine, Human Medicine, Microbiology and Food Science, and Human Nutrition departments. The speakers included Shannon D. Manning, PhD, MPH Michigan State University (Evolution and Pathogenesis of E. coli O104:H4 infections) and Kathryn Eaton DVM, PhD, University of Michigan (Host, pathogen, and microbiota contribute to disease severity in EHEC-infected mice). Dr. Mansfield helped to organize and attended the USDA Enteric Diseases Meeting NC1041 in Chicago, Illinois on December 1st and 2nd as part of the Conference of Research Workers on Animal Diseases. Her graduate student Jessica St. Charles gave a talk at CRWAD about her work entitled "Campylobacter jejuni isolates from calves have A, B and C lipooligosaccharide (LOS) biosynthetic locus classes similar to human Guillain Barre syndrome associated strains". For the same CRWAD meeting, Dr. Mansfield gave a talk at CRWAD entitled "Comparison of induced small animal models for Guillain Barre syndrome (GBS) as post infectious sequelae to Campylobacter jejuni infection". Dr. Mansfield gave an invited talk entitled "Murine models of the autoimmune neuropathy Guillain Barre Syndrome" for the Small Animal Models of Enteric Diseases, NIAID, National Institutes of Health, September 13-14, 2012. She also gave an invited talk entitled "Campylobacter jejuni induces mixed Type 1 and 17 responses in acute and chronic disease" for the Enterics Research Investigational Network, Cooperative Research Centers Annual Meeting, National Institutes of Health, Seattle, Washington, May 22-23, 2012.
<br/>TARGET AUDIENCES: The main target audience is the scientific community studying food safety and medical interventions for food borne pathogens. We reach this audience with high quality research publications in scientific journals as well as talks to scientific meetings and producer groups for food animals. We also have a long term effect by teaching undergraduate, graduate students and postdoctoral candidates these advances and strategies for studying food borne pathogens. The ultimate goals of this work are to translate this information into prevnetatives and treatments for food borne diseases.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.
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IMPACT: Study Impact: The C. jejuni human clinical isolates NW and D2600 colonized C57BL/6 interleukin 10 knockout mice without inducing a robust inflammatory response (J. A. Bell et al., BMC Microbiol. 9:57, 2009). We announce draft genome sequences of NW and D2600 to facilitate comparisons with strains that induce gastrointestinal inflammation in this mouse model. Whole genome sequences for two human clinical isolates of C. jejuni are provided as a resource to the scientific community. Important differences in the genomes illustrate the diversity of these strains.