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Regulation of Capsular Polysaccharide Production in Camplyobacter Jejuni and Its Role in Host Interactions

Institutions
University of Texas Southwestern Medical Center
Start date
2009
End date
2012
Objective
Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans in the United States. In contrast, C. jejuni promotes a natural, harmless colonization of the gastrointestinal tracts of poultry and other animals. Association of C. jejuni with animals results in contamination of the human food supply and exposure to humans. Identifying and understanding factors of C. jejuni involved in colonization of poultry are required for development of new interventions to eradicate C. jejuni from poultry to make safer meat products for human consumption. C. jejuni serogroups each produce a structurally different capsular polysaccharide (CPS) necessary for colonization of the avian gastrointestinal tract, interaction with human intestinal epithelial cells, and resistance to human serum. We have identified BocR as a transcriptional regulator required for expression of multiple genes of the CPS biosynthesis locus. Mutation of BocR or two genes within the CPS biosynthesis locus results in loss of CPS production and over 1000-fold reduction in colonization of chicks. The objectives of this proposal are to examine BocR-dependent expression of CPS biosynthesis genes in different C. jejuni serogroups and understand the specific requirements of each gene in the BocR regulon for CPS production, colonization of chicks, and virulence properties. Completion of research goals will impact our understanding of how CPS production, a proven colonization and virulence factor, is regulated in a broad range of C. jejuni serogroups. Furthermore, we will understand how C. jejuni serogroups have evolved diverse CPS structures while potentially maintaining BocR-dependence for expression of these genes.
More information
NON-TECHNICAL SUMMARY: Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans in the United States. In contrast, C. jejuni promotes a natural, harmless colonization of the gastrointestinal tracts of poultry and other animals. Association of C. jejuni with animals results in contamination of the human food supply and exposure to humans. Identifying and understanding factors of C. jejuni that are involved in colonization of poultry and causing disease in humans would contribute to potential antimicrobial strategies to inhibit the bacterium upon infection of animals or humans. C. jejuni produces a capsular polysaccharide that is required for wild-type levels of colonization of chickens and virulence properties such as invasion of epithelial cells and serum resistance. We have found that the BocR protein of C. jejuni is a transcriptional regulator of a section of the capsule gene locus. The proposed work will examine the BocR-dependent transcription of the capsule locus and determine which genes of the capsule locus are required for capsule production, colonization, and virulence properties. We will then determine if BocR is required for expression of the capsule locus and production of the capsular polysaccharide in strains of C. jejuni from other serogroups. This work will allow us to better understand how C. jejuni controls production of an important colonization and virulence factor required for interactions with both humans and animals. Future broad-range goals from this work may allow for the development of an antimicrobial to inhibit the production of capsule, and thus, the growth of C. jejuni in chickens or humans.

APPROACH: To define the transcriptional organization of the BocR-dependent regulon of the capsule locus, we will perform Northern blot analysis and reverse transcriptase-PCR to determine how many BocR-dependent mRNAs are within the operon. Then, we will perform primer extension analysis to determine the start sites of transcription. DNA containing putative promoter elements will be examined by deletion analysis and gel-shift assays to determine the identify of the BocR-dependent promoter. BocR-dependent expression of the capsule locus will be evaluated in other C. jejuni serogroups by making a mutation in bocR and then examining expression and production of capsule by real-time RT-PCR analysis and immunoblotting analysis, respectively. Each gene of the BocR-dependent capsule locus will be deleted by site-specific mutagenesis to determine ones required for production of capsular polysaccharide, colonization of chicks, interactions with human intestinal epithelial cells, and serum-resistance assays. Capsule production will be monitored by immunoblotting analysis and high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Colonization assays will be performed with 1-day old chicks and the bacterial loads of the wild-type and mutant strains will be determined seven days post-infection. Adherence and invasion assays will be performed with the INT407 intestinal epithelial cells. Serum resistance assasy will use pooled normal human serum. All assays described above will be performed with a positive control (wild-type strain) so that each mutant can be evaluated conclusively for any defects.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
TEXR-2008-01453
Accession number
215324
Categories
Food Defense and Integrity
Bacterial Pathogens
Campylobacter
Viruses and Prions
Commodities
Meat, Poultry, Game