Progress 07/15/06 to 07/14/10
Outputs
OUTPUTS: We investigated the: 1) Avian immune response upon colonization with C. jejuni; 2) The role of the CadF protein in bacteria-host cell adhesion; 3) Effects of source (host species) on the pathogenic potential of C. jejuni strains; and 4) Bacterial proteins that promote C. jejuni colonization of chickens. We also performed experiments to assess the feasibility of using a C. crescentus recombinant strain as a potential intervention strategy to reduce the carriage of C. jejuni in poultry. We published five manuscripts. PARTICIPANTS: Dr. Konkel was the project director, and his lab has performed the majority of the experiments, as outlined in the proposal. Dr. John Smit, who is an expert in the area of Caulobacter research and has extensive experience in designing S-layer fusion proteins, generated the recombinant Caulobacter S-layer fusion strains. Dr. Call's expertise is in host immune responses and mechanisms of bacterial pathogenesis. Dr. Call assisted with studies to address the immune responses of chickens to C. jejuni. Dr. Singh was responsible for the C. jejuni inoculation and challenge studies with chickens. In addition to the personnel listed above, several other senior investigators (G. J. Haldorson, W.G. Miller, X. Tang), post-docs/research associates (S. Ahn, C. Davitt, D.H. Shah, K.D. Shoaf-Sweeney) and graduate students (C.L. Larson, R.C Flanagan, J.M. Neal-McKinney) assisted with this project. TARGET AUDIENCES: We published five articles in peer-reviewed journals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Studies are needed that explore C. jejuni-host cell interactions, and in particular, the interaction of C. jejuni within the natural host. We performed a study to identify the C. jejuni membrane-associated proteins recognized by the Campylobacter-specific maternal antibodies contained within the sera of the 2-day old chicks. Campylobacter-specific maternal antibodies, which are passed from hens to chicks, are partially protective against C. jejuni colonization of chicks. Immunoblot analysis was performed with the chick sera and the reactive C. jejuni proteins were identified by tandem mass spectrometry. In the published manuscript, we reported a list of C. jejuni proteins recognized by maternal antibodies. These data provide a foundation for a variety of future projects, including studies to determine the efficacy of various C. jejuni proteins as vaccine candidates. A manuscript describing the work was published in Applied and Environmental Microbiology. A second project was performed to address the characteristics that distinguish the commensal relationship of C. jejuni with chickens from its pathogenic relationship with humans. A hallmark of C. jejuni gastrointestinal infection in humans is an acute host inflammatory response. Central to this acute inflammatory response is the secretion of IL-8 from the cells lining the intestinal tract. In the study, we reported differences in the expression and secretion of IL-8, as well as differences in the functional activity of chemoattractants between chicken and humans cells inoculated with C. jejuni. These data revealed that chicken LMH cells respond differently to C. jejuni versus other bacteria that are pathogenic to chickens. A manuscript describing the work was published in Microbiology. We also undertook a study to examine the genetic relatedness of ninety-eight C. jejuni isolates recovered from human, poultry, bovine, swine, ovine, and canine sources by multilocus sequence typing and examined their profile of putative adhesin-encoding genes. This study led to the identification of a new bacterial adhesin, which we designated FlpA for Fibronectin-like protein A, required for C. jejuni to colonize chickens. A manuscript describing the work was published in Infection and Immunity. Finally, studies were performed to characterize the binding properties of the FlpA protein, and the cooperative activity of the FlpA and CadF adhesins. A manuscript describing the work was published in the Journal of Bacteriology.
Publications
- 4. Flanagan, R.C., Neal-McKinney, J.M., Dhillon, A.S., Miller, W.G., and M.E. Konkel. Examination of Campylobacter jejuni putative adhesins leads to the identification of a new protein, designated FlpA, required for chicken colonization. Infect. Immun. 77(6):2399-407, 2009.
- 1. Konkel, M.E., Christensen, J.E., Dhillon, A.S., Lane, A.B., Hare-Sanford, R., D. Schaberg, D., and C. Larson. 2007. Campylobacter jejuni strains compete for colonization of broiler chicks. Appl. Environ. Microbiol. 73:2297-2305.
- 2. Shoaf-Sweeney, K.D., Larson, C.L., Tang, X., and M.E. Konkel. Identification of Campylobacter jejuni proteins recognized by chicken maternal antibodies. Appl. Environ. Microbiol. 74:6867-6875, 2008.
- 3. Larson, C.L., Shah, D.H., A. Dhillon, A.S., Call, D.R., Ahn, S., Haldorson, G. J., Davitt, C., and M.E. Konkel. Campylobacter jejuni invade chicken LMH cells inefficiently and stimulate differential expression of the chicken CXCLi1 and CXCLi2 cytokines. Microbiology. 54:3835-3847, 2008.
- 5. Konkel, M.E., Larson, C.L., and R.C. Flanagan. The food-borne pathogen Campylobacter jejuni synthesizes a novel fibronectin-binding protein termed FlpA that facilitates adherence to human epithelial cells. J. Bacteriol. 192:68-76, 2010.
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Progress 07/15/08 to 07/14/09
Outputs
OUTPUTS: This past year we investigated the: 1) Avian immune response upon colonization with C. jejuni; 2) The role of the CadF protein in bacteria-host cell adhesion; and 3) Effects of source (host species) on the pathogenic potential of C. jejuni strains. Work continues to further characterize a bacterial protein that promotes C. jejuni binding to chicken cells and to assess the feasibility of using a C. crescentus recombinant strain as a potential intervention strategy to reduce the carriage of C. jejuni in poultry. We published three papers last year. PARTICIPANTS: Dr. Konkel is the project director, and his lab has performed the majority of the experiments, as outlined in the proposal. Dr. John Smit, who is an expert in the area of Caulobacter research and has extensive experience in designing S-layer fusion proteins, has generated recombinant Caulobacter S-layer fusion strains. Dr. Call has expertise in host immune responses and mechanisms of bacterial pathogenesis. Dr. Call has assisted with studies to address the immune responses of chickens and humans to C. jejuni. Dr. Singh has been responsible for the C. jejuni inoculation and challenge studies with chickens. In addition to the personnel listed above, several other senior investigators (G. J. Haldorson, W.G. Miller, X. Tang), post-docs/research associates (S. Ahn, C. Davitt, D.H. Shah, K.D. Shoaf-Sweeney) and graduate students (C.L. Larson, R.C Flanagan, J.M. Neal-McKinney) have assisted with this project. TARGET AUDIENCES: We have published three articles in peer-reviewed journals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Studies are needed that explore C. jejuni-host cell interactions, and in particular, the interaction of C. jejuni in its natural hosts. We performed a study to identify the C. jejuni membrane-associated proteins recognized by the Campylobacter-specific maternal antibodies contained within the sera of the 2-day old chicks. Campylobacter-specific maternal antibodies, which are passed from hens to chicks, are partially protective against C. jejuni colonization of chicks. Immunoblot analysis was performed with the chick sera and the reactive C. jejuni proteins were identified by tandem mass spectrometry. In the published manuscript, we reported a list of C. jejuni proteins recognized by maternal antibodies. These data provide a foundation for a variety of future projects, including studies to determine the efficacy of various C. jejuni proteins as vaccine candidates. A manuscript describing the work was published in Applied and Environmental Microbiology. A second project was performed to address the characteristics that distinguish the commensal relationship of C. jejuni with chickens from its pathogenic relationship with humans are unclear. A hallmark of C. jejuni gastrointestinal infection in humans is an acute host inflammatory response. Central to this acute inflammatory response is the secretion of IL-8 from the cells lining the intestinal tract. In the study, we reported differences in the expression and secretion of IL-8, as well as functional activity of chemoattractants between chicken and humans cells inoculated with C. jejuni. These data provided insight regarding the differences that distinguish the interactions of C. jejuni with chicken and human cells, and show that chicken LMH cells respond differently to C. jejuni versus other bacteria that are pathogenic to chickens. A manuscript describing the work was published in Microbiology. This past year, we also undertook a study to examine the genetic relatedness of ninety-eight C. jejuni isolates recovered from human, poultry, bovine, swine, ovine, and canine sources by multilocus sequence typing and examined their profile of putative adhesin-encoding genes. This study led to the identification of a new bacterial adhesin, which we designated FlpA for Fibronectin-like protein A, required for C. jejuni to colonize chickens. A manuscript describing the work was published in Infection and Immunity.
Publications
- 1. Shoaf-Sweeney, K.D., Larson, C.L., Tang, X., and M.E. Konkel. Identification of Campylobacter jejuni proteins recognized by chicken maternal antibodies. Appl. Environ. Microbiol. 74:6867-6875, 2008.
- 2. Larson, C.L., Shah, D.H., A. Dhillon, A.S., Call, D.R., Ahn, S., Haldorson, G. J., Davitt, C., and M.E. Konkel. Campylobacter jejuni invade chicken LMH cells inefficiently and stimulate differential expression of the chicken CXCLi1 and CXCLi2 cytokines. Microbiology. 54:3835-3847, 2008.
- 3. Flanagan, R.C., Neal-McKinney, J.M., Dhillon, A.S., Miller, W.G., and M.E. Konkel. Examination of Campylobacter jejuni putative adhesins leads to the identification of a new protein, designated FlpA, required for chicken colonization. Infection and Immunity. 77(6):2399-407, 2009.
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Progress 07/15/07 to 07/14/08
Outputs
OUTPUTS: This past year we investigated the: 1) Avian immune response upon colonization with C. jejuni; 2) The role of the CadF protein in bacteria-host cell adhesion; 3) Effects of source (host species) on the pathogenic potential of C. jejuni strains; and 4) Use of a C. crescentus recombinant strain as a potential intervention strategy to reduce the carriage of C. jejuni in poultry. We published two papers, and plan to submit another paper for publication in October 2008. PARTICIPANTS: Graduate students and postdocs participated in these projects. TARGET AUDIENCES: We published two papers, and a third paper has been submitted for publication. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Studies are needed that explore C. jejuni-host cell interactions, and in particular, the interaction of C. jejuni in its natural hosts. We performed a study to identify the C. jejuni membrane-associated proteins recognized by the Campylobacter-specific maternal antibodies contained within the sera of the 2-day old chicks. Campylobacter-specific maternal antibodies, which are passed from hens to chicks are partially protective against C. jejuni colonization of chicks. Immunoblot analysis was performed with the chick sera and the reactive C. jejuni proteins were identified by tandem mass spectrometry. In the published manuscript, we reported a list of C. jejuni proteins recognized by maternal antibodies. These data provide a foundation for a variety of future projects, including studies to determine the efficacy of various C. jejuni proteins as vaccine candidates. A hallmark of C. jejuni gastrointestinal infection in humans is an acute host inflammatory response. Central to this acute inflammatory response is the secretion of IL-8 from the cells lining the intestinal tract. In this study, we reported differences in the expression and secretion of IL-8, as well as functional activity of chemoattractants between chicken and humans cells inoculated with C. jejuni. These data provide insight regarding the differences that distinguish the interactions of C. jejuni with chicken and human cells, and show that chicken LMH cells respond differently to C. jejuni versus other bacteria that are pathogenic to chickens. This past year, we also undertook a study to examine the genetic relatedness of ninety-eight C. jejuni isolates recovered from human, poultry, bovine, swine, ovine, and canine sources by multilocus sequence typing and examined their profile of putative adhesin-encoding genes. This study led to the identification of an adhesin required for C. jejuni to colonize chickens. This work will be submitted for publication in fall 2008.
Publications
- 1. Shoaf-Sweeney, K.D., Larson, C., Tang, X., and M.E. Konkel. Identification of Campylobacter jejuni proteins recognized by chicken maternal antibodies. Appl. Environ. Microbiol. 2008. In press.
- 2. Larson, C.L., Shah, D.H., A. Dhillon, A.S., Call, D.R., Ahn, S., Haldorson, G. J., Davitt, C., and M.E. Konkel. Campylobacter jejuni invade chicken LMH cells inefficiently and stimulate differential expression of the chicken CXCLi1 and CXCLi2 cytokines. Microbiology. 2008. In press.
- 3. Flanagan, R.C., Neal-McKinney, J.M., Dhillon, A.S., Miller, W.G., and M.E. Konkel. Examination of Campylobacter jejuni putative adhesins leads to the identification of a new protein, designated FlpA, required for chicken colonization. 2008. Manuscript submitted.
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Progress 07/15/06 to 07/14/07
Outputs
Campylobacter jejuni is one of the leading causes of bacterial food-borne disease in the United States. CadF is an outer membrane fibronectin (Fn)-binding protein that mediates adherence of C. jejuni to cells lining the intestinal tract of humans and chickens. Caulobacter crescentus is an innocuous bacterium that secretes a surface (S)-layer composed of a protein monomer - RsaA. We hypothesize that C. crescentus displaying the CadF Fn-binding domain in the S-layer will possess Fn-binding activity and competitively inhibit the binding of C. jejuni to Fn. To test this hypothesis, the nucleotide sequence encoding the CadF Fn-binding domain was inserted into rsaA. Immunofluorescence microscopy demonstrated the CadF Fn-binding domain of C. crescentus RsaA-CadF strains is surface exposed. Moreover, purified RsaA-CadF demonstrated Fn-binding activity and competitively inhibited binding to Fn by purified CadF as judged by ELISAs. Together, these data suggest the RsaA-CadF
fusion protein is surface exposed and capable of binding Fn.
Impacts
Campylobacter jejuni is one of the leading causes of bacterial food-borne disease in the United States. Most cases of campylobacteriosis result from consumption of foods contaminated with undercooked chicken products. The goal of this proposal is to develop a new intervention strategy to reduce the carriage of Campylobacter jejuni in poultry. More specifically, we are performing experiments to generate a C. jejuni-blocking agent that will be used in broiler chicks.
Publications
- Submitted for publication: Larson, C.L., Ahn, S., Shah, D., Christensen, J.E., Dhillon, A.S., Haldorson, G.J., Call, D.R., and M.E. Konkel. 2007. Campylobacter jejuni invasion and induction of cytokine release is host origin specific. Submitted to Infect. Immun.
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