Progress 09/01/02 to 08/31/07
Outputs
OUTPUTS: The long-range goal of this award was to understand how S. Typhimurium persists and causes disease in its bovine reservoir. The objectives of this award were to determine the mechanism by which the colonization factor ShdA mediates persistence of S. Typhimurium in the bovine intestine. We tested the central hypothesis that binding of extracellular matrix proteins by ShdA is a mechanism to colonize the bovine mucosa, thereby promoting persistent colonization. Our data indicate that the primary fibronectin-binding domain of ShdA is present in the repeat region of the passenger domain in the residues in a 97 residues region called repeat A3, which is associated with persistence of S. Typhimurium. These results were communicated at national meetings and scientific journals.
PARTICIPANTS: Professor A. J. Baumler, Medical Microbiology & Immunology, School of Medicine, University of CAlifornia, Davis, CA Dr. R. A. Kingsley, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom Dr. L. Berghman, Associate Professor, Poultry Science, Texas A&M University, College Station, TX Professor L. Garry Adams, Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, College Station, TX
Impacts
Knowledge of the fibronectin-binding domain of S. Typhimurium ShdA provides a basis to explore mechanisms for enhanced rational design of live vaccines and for identifying potential inhibitors to prevent S. Typhimurium colonization of the intestine of food-producing animals which serve as a reservoir for contamination of the human food and water supplies. These results are expected to have an impact on the overall strategy for improving the safety of food production systems.
Publications
- Kingsley, Robert A., Santos, Renato L, Keestra, A. Marijke, Adams, L. Garry and Baumler, Aandreas J. 2002. Salmonella enterica serovar Typhimurium ShaD is an outer membrane fibrinonectin-binding-protein that is expressed in the intestine. Molecular Microbiology 43:895-905.
- Rabsch, W., H.L. Andrews, R.A. Kingsley, R. Prager, H. Tschape, L.G. Adams, and A.J. Baumler. 2002. Salmonella enterica serotype Typhimurium and its host adapted variants. Infection & Immunity. 70:2249-2255.
- Humphries, A. D., Raffatellu, M, Winter, S., Weening, E. H., Kingsley, R. A., Droleskey, R., Zhang, S., Figueiredo, J., Khare, S., Nunes, J., Adams, L. G., Tsolis, R. M. and Baumler, A. J. 2003. The use of flow cytometry to detect in vitro expression of subunits encoded by 11 Salmonella enterica serotype Typhimurium fimbrial operons. Molecular Microbiology. 48:1357-1376.
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Progress 01/01/06 to 12/31/06
Outputs
The long-range goal of this award was to understand how S. Typhimurium persists and causes disease in its bovine reservoir. The objectives of this award were to determine the mechanism by which the colonization factor ShdA mediates persistence of S. Typhimurium in the bovine intestine. We tested the central hypothesis that binding of extracellular matrix proteins by ShdA is a mechanism to colonize the bovine mucosa, thereby promoting persistent colonization. The shdA gene of S. Typhimurium encodes an outer surface protein of the autotransporter-family that binds fibronectin through its passenger domain. To identify the residues in ShdA that are required for fibronectin binding we investigated the domain structure of the ShdA passenger domain. The ShdA passenger domain consists of approximately 1500 amino acid residues that can be divided into two regions based on features of the primary amino acid sequence; an N-terminal non-repeat region followed by a repeat region
composed of two types of imperfect direct amino acid repeats, denoted type A and type B. The repeat region bound bovine fibronectin with a similar estimated disassociation constant (Kd) as the complete ShdA passenger domain, while the non-repeat region exhibited comparatively low Fibronectin-binding activity. A number of fusion proteins containing truncated fragments of the repeat region did not bind bovine fibronectin. However, binding of the passenger to Fibronectin was inhibited in the presence of immune serum raised to one truncated fragment of the repeat region that contained repeats A2, B8, A3 and B9. Furthermore, a monoclonal antibody (mAb) that specifically recognized an epitope in a recombinant protein containing repeats the A3 repeat inhibited binding of ShdA to fibronectin. Together these data suggest that a the primary fibronectin-binding domain of ShdA is present in the repeat region of the passenger domain and may residues in a 97 residues region called repeat A3. To
determine the role of the fibronectin binding activity of ShdA in intestinal colonization of calves we investigated the role of shdA in colonization of the bovine intestine, and characterized the distribution of S. Typhimurium and fibronectin in the bovine cecal mucosa.
Impacts
The described work is innovative because it does for the first time explore molecular mechanisms of S. Typhimurium intestinal persistence. It is our expectation that our results will identify potential new targets for intervention strategies to reduce the prevalence of Salmonella serotypes in our food supply. This outcome will be significant since it will provide the basis for the development of knowledge-based approaches to improve public health.
Publications
- Abd El Ghany, M., Jansen, A., Clare, S., Hall, L., Pickard, D., Kingsley, R. A. and Dougan, G.Candidate Live, Attenuated Salmonella enterica Serotype Typhimurium Vaccines with Reduced Fecal Shedding Are Immunogenic and Effective Oral Vaccines. Infection & Immunity 75:1835-42, 2007
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Progress 09/01/02 to 08/31/06
Outputs
The shdA gene of Salmonella enterica serotype Typhimurium (S. Typhimurium) encodes an outer surface protein of the autransporter-family that binds fibronectin. We investigated the role of shdA in colonization of the bovine and porcine intestine, and characterize the distribution of S. Typhimurium and fibronectin in the bovine cecal mucosa. A S. Typhimurium strain containing a shdA deletion was recovered from the feces in significantly lower numbers on several occasions in the first eight days after oral inoculation. On day eight post inoculation, following sacrifice, significantly lower numbers of the shdA deletion strain compared with the wild type were found in organ homogenates of the ileum, Peyers patch and cecum. No significance difference was observed in colon, mesenteric lymph nodes and liver homogenates. The distribution of S. Typhimurium and fibronectin was investigated using immunofluorescence microscopy of thin sections of bovine cecum from a calf 3 days
after oral inoculation with S. Typhimurium RAK55 (wild type). Following inoculation with 1.5x109 cfu of S. Typhimurium RAK55 the calf developed profuse watery-diarrhoea and became moribund on day 3 post inoculation. H&E stained intestinal sections revealed histopathology typical of Salmonella colitis including neutrophil influx and epithelial erosion. Immunofluorescence microscopy revealed that Salmonella were not detected uniformly within the lamina propria but rather in discrete regions and that these regions of salmonella colonization coincided with areas of increased signal from anti-fibronectin antibody. We investigated the domain structure of the ShdA passenger domain to identify the primary fibronectin-binding domain. The ShdA passenger domain consists of approximately 2000 amino acid residues that can be divided into two regions based on features of the primary amino acid sequence; an N-terminal non-repeat region followed by a repeat region composed of two types of imperfect
direct amino acid repeats, denoted type A and type B. The repeat region bound bovine fibronectin with a similar estimated disassociation constant (Kd) as the complete ShdA passenger domain, while the non-repeat region exhibited comparatively low Fibronectin-binding activity. A number of fusion proteins containing truncated fragments of the repeat region did not bind bovine fibronectin. However, binding of the passenger to Fibronectin was inhibited in the presence of immune serum raised to one truncated fragment of the repeat region that contained repeats A2, B8, A3 and B9. Furthermore, a monoclonal antibody (mAb) that specifically recognized an epitope in a recombinant protein containing repeats the A3 repeat inhibited binding of ShdA to fibronectin. Together these data suggest that the primary fibronectin-binding domain of ShdA is present in the repeat region of the passenger domain and may reside in residues in a 97 residues region called repeat A3 or a region in close proximity in
the mature folded protein. To delineate the fibronectin binding site with higher resolution, four monoclonal antibodies able to entirely block the interaction between ShdA and fibronectin were produced.
Impacts
The described work is innovative because it does for the first time explore molecular mechanisms of S. Typhimurium intestinal persistence. It is our expectation that our results will identify potential new targets for intervention strategies to reduce the prevalence of Salmonella serotypes in our food supply. This outcome will be significant since it will provide the basis for the development of knowledge-based approaches to improve public health.
Publications
- Kingsley, R.A., A. M. Keestra, M. R. de Zoete and A. J. Baumler. 2004. The ShdA adhesin binds to the cationic cradle of the fibronectin 13FnIII repeat module: evidence for molecular mimicry of heparin binding. Mol. Microbiol. 52:345-355.
- Kingsley, R.A., D.A. Ghanem, N. Puebla-Osorio, A. M. Keestra, L. Berghman and A. J. Baumler. 2004. Fibronectin binding of the Salmonella enterica serotype Typhimurium ShdA aurotransporter protein is inhibited by a monoclonal antibody recognizing the A3 repeat. J. Bacteriol. 186:4931-4939.
- Baumler, A.J. Food-borne Salmonella Infections. 2004. In. Beier, R.C., S. D. Pillai, T. D. Phillips and R. L. Ziprin (eds.). Pre-harvest and post-harvest food safety: contemporary issues and future directions. Blackwell Publishing, Ames, IA. p. 3-12.
- Andrews, H.L. and A.J. Baumler. Salmonella spp. 2005. In. Fratamico, P.M and A.K. Bhunia (ed.). Food-borne Pathogens: Microbiology and Molecular Biology. Horizon Scientific Press, Ltd., Norfolk, UK, p327-339.
- Figueiredo, J. F, Barhoumi, R., Raffatellu, Lawhon, S. D., Robert C. Burghardt, R. C., Tsolis, R. M., Baumler, A. J. and Adams, L. G. 2006. Salmonella typhimurium induced internalization and IL-8 gene expression do not correlate with intracellular Ca2+ levels. In Press.
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Progress 10/01/03 to 09/30/04
Outputs
The shdA gene of S. Typhimurium encodes an outer surface protein of the autransporter-family that binds fibronectin. We investigated the role of shdA in colonization of the bovine and porcine intestine, and characterize the distribution of S. Typhimurium and fibronectin in the bovine cecal mucosa. A S. Typhimurium strain containing a shdA deletion was recovered from the feces in significantly lower numbers on eight days after oral inoculation. On day eight post inoculation, following sacrifice, significantly lower numbers of the shdA deletion strain compared with the wild type were found in organ homogenates of the ileum, Peyer s patch and cecum. No significance difference was observed in colon, mesenteric lymph nodes and liver homogenates. The distribution of S. Typhimurium and fibronectin was investigated using immunofluorescence microscopy of thin sections of bovine cecum from a calf 3 days after oral inoculation with S. Typhimurium RAK55 (wild type). Following
inoculation with 1.5x109 cfu of S. Typhimurium RAK55 the calf developed profuse watery-diarrhoea and became moribund on day 3 post inoculation at which stage it was sacrificed and cecal tissue fixed in cold ethanol overnight. Paraffin embedded thin sections (5mm) were stained Haemotoxylin and Eosin (H&E) or with DAPI, anti O4 (somatic antigen) antibody (alexafluor-488 conjugated secondary) and anti fibronectin antibody (alexafluor 594-conjugated secondary). H&E stained sections revealed histopathology typical of Salmonella colitis including neutrophil influx and epithelial erosion. Immunofluorescence microscopy revealed that Salmonella were not detected uniformly within the lamina propria but rather in discrete regions and that these regions of salmonella colonization coincided with areas of increased signal from anti-fibronectin antibody. We further investigated the domain structure of the ShdA passenger domain to identify the primary fibronectin-binding domain. The ShdA passenger
domain consists of approximately 1500 amino acid residues that can be divided into two regions based on features of the primary amino acid sequence; an N-terminal non-repeat region followed by a repeat region composed of two types of imperfect direct amino acid repeats, denoted type A and type B. The repeat region bound bovine fibronectin with a similar estimated disassociation constant (Kd) as the complete ShdA passenger domain, while the non-repeat region exhibited comparatively low Fibronectin-binding activity. A number of fusion proteins containing truncated fragments of the repeat region did not bind bovine fibronectin. However, binding of the passenger to Fibronectin was inhibited in the presence of immune serum raised to one truncated fragment of the repeat region that contained repeats A2, B8, A3 and B9. Furthermore, a monoclonal antibody (mAb) that specifically recognized an epitope in a recombinant protein containing repeats the A3 repeat inhibited binding of ShdA to
fibronectin. Together these data suggest that a the primary fibronectin-binding domain of ShdA is present in the repeat region of the passenger domain and may residues in a 97 residues region called repeat A3.
Impacts
The described work is innovative because it does for the first time explore molecular mechanisms of S. Typhimurium intestinal persistence. It is our expectation that our results will identify potential new targets for intervention strategies to reduce the prevalence of Salmonella serotypes in our food supply. This outcome will be significant since it will provide the basis for the development of knowledge-based approaches to improve public health.
Publications
- Kingsley, R.A., A. M. Keestra, M. R. de Zoete and A. J. Baumler. 2004. The ShdA adhesin binds to the cationic cradle of the fibronectin 13FnIII repeat module: evidence for molecular mimicry of heparin binding. Mol. Microbiol. 52:345-355.
- Kingsley, R.A., D.A. Ghanem, N. Puebla-Osorio, A. M. Keestra, L. Berghman and A. J. Baumler. 2004. Fibronectin binding of the Salmonella enterica serotype Typhimurium ShdA aurotransporter protein is inhibited by a monoclonal antibody recognizing the A3 repeat. J. Bacteriol. 186:4931-4939.
- Baumler, A.J. Food-borne Salmonella Infections. 2004. In. Beier, R.C., S. D. Pillai, T. D. Phillips and R. L. Ziprin (eds.). Pre-harvest and post-harvest food safety: contemporary issues and future directions. Blackwell Publishing, Ames, IA. p. 3-12.
- Andrews, H.L. and A.J. Baumler. Salmonella spp. 2005. In. Fratamico, P.M and A.K. Bhunia (ed.). Food-borne Pathogens: Microbiology and Molecular Biology. Horizon Scientific Press, Ltd., Norfolk, UK, In press.
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Progress 10/01/02 to 09/30/03
Outputs
To characterize the molecular mechanism of ShdA-mediated adherence we determined its binding site in fibronectin. Analysis of ShdA binding to fibronectin proteolytic fragments and to recombinant fibronectin fusion proteins identified the 13FnIII repeat module of the Hep-2 domain as the primary binding site for this adhesin. The 13FnIII repeat module of fibronectin contains the cationic cradle, a high affinity heparin-binding site formed by six basic residues (R6, R7, R9, R23, K25 and R54) that are conserved among fibronectin sequences from frogs to man. Binding of ShdA to the 13FnIII repeat module of fibronectin could be inhibited by heparin. We therefore determined whether amino acid substitutions of basic residues in the cationic cradle of the Hep-2 domain that inhibit heparin binding would also abrogated binding of ShdA. Substitutions of R6S, R7S, R9S and R23S strongly inhibited ShdA binding to 13FnIII, while substitutions K25S and R54S caused a small but
significant reduction in binding. These data suggest that ShdA binds the Hep-2 domain of fibronectin by a mechanism that mimics binding of the host polysaccharide heparin. We next investigated the domain structure of the ShdA passenger domain. The ShdA repeat region bound bovine Fn with a similar disassociation constant (Kd) to that of the complete ShdA passenger domain. Specificity of these interactions was demonstrated by inhibition studies using biotin labeled and unlabelled ligand. Recombinant proteins truncates, together containing all of the repeats within the repeat region, did not bind the bovine Fn and did not exhibit biological activity in inhibition assays alone or in combination. However, immune serum raised to one of these proteins, containing repeat A2, B8, A3 and B9, reduced binding of the passenger domain to 40% of that in the presence of naive serum. Together these data suggest that the primary ligand binding domain is present in the repeat region of the passenger
domain of ShdA. Further, while one or more epitopes that contribute to binding are present in repeats A2, B8, A3 and B9 it is likely that additional sites elsewhere in the repeat region contribute to full binding activity. To establish that shdA is also required for intestinal persistence and efficient fecal shedding in a relevant host species, a group a 4 calves was infected orally with a sublethal dose (5x10e6 colony forming units /animal) containing equal numbers of the S. Typhimurium wild type and an isogenic shdA deletion mutant. Animals developed diarrhea within 48 hours after inoculation that lasted for 3 to 5 days. The S. Typhimurium wild type was recovered in significantly higher numbers than the shdA deletion mutant from the feces at day 5 and subsequent days. Animals were euthanized at day 8 after inoculation and bacteria recovered from intestinal tissues. The S. Typhimurium wild type was recovered in significantly higher numbers than the shdA deletion mutant from the cecal
wall, the mesenteric lymph node, the ileum and the ileal Peyer's patches.
Impacts
The described work is innovative because it does for the first time explore molecular mechanisms of S. Typhimurium intestinal persistence. It is our expectation that our results will identify potential new targets for intervention strategies to reduce the prevalence of Salmonella serotypes in our food supply. This outcome will be significant since it will provide the basis for the development of knowledge-based approaches to improve public health.
Publications
- Rabsch, W., C. Altier, H. Tschape, and A.J. Baumler. Food-borne Salmonella Infections. In. Torrence, M. and R. Isaacson (ed.). 2003. Microbial Food Safety in Animal Agriculture. Iowa State Press. 97-107.
- Baumler, A.J. 2004. Food-borne Salmonella Infections. In. Beier, R.C. (ed.). Pre-harvest and post-harvest food safety: contemporary issues and future directions. Iowa State Press. Ames, Iowa. In press.
- Kingsley, R.A., A. M. Keestra, M. R. de Zoete and A. J. Baumler. 2004. The ShdA adhesin binds to the cationic cradle of the fibronectin 13FnIII repeat module: evidence for molecular mimicry of heparin binding. Mol. Microbial. In press.
- Kingsley, R.A., A. M. Keestra, and A. J. Baumler. 2004. Characterization of the primary fibronectin-binding region of the Salmonella enterica serotype Typhimurium ShdA autotransporter-family adhesin. Submitted for publication.
- Kingsley, R.A., A.D. Humphries, E.H. Weening, M.R. de Zoete, S. Winter, A. Papaconstantinopoulou, G. Dougan and A.J. Baumler. 2003. Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype Typhimurium: identification of intestinal colonization and persistence determinants. Infect. Immun. 71:629-640.
- Zhang, S., R.A. Kingsley, R.L. Santos, H. Andrews-Polymenis, M. Raffatellu, J. Figueiredo, J. Nunes, R.M. Tsolis, L.G. Adams and A.J. Baumler. 2003. Molecular Pathogenesis of Salmonella enterica serotype Typhimurium-induced diarrhea. Infect. Immun. 71:1-12.
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Progress 10/01/01 to 09/30/02
Outputs
To characterize the molecular mechanism of ShdA-mediated adherence we determined its binding site in fibronectin (Fn). Analysis of ShdA binding to Fn proteolytic fragments and to recombinant Fn fusion proteins identified the 13FnIII repeat module of the Hep-2 domain as the primary binding site for this adhesin. The 13FnIII repeat module of Fn contains the cationic cradle, a high affinity heparin-binding site formed by six basic residues (R6, R7, R9, R23, K25 and R54) that are conserved among Fn sequences from frogs to man. Binding of ShdA to the 13FnIII repeat module of Fn and to a second extracellular matrix protein, Collagen I, was found to be heparin-sensitive. Furthermore, binding of ShdA to the Hep-2 domain was sensitive to the ionic buffer strength, suggesting that binding involved ionic interactions. We thus determined whether amino acid substitutions of basic residues in the cationic cradle of the Hep-2 domain that inhibit heparin binding would also abrogated
binding of ShdA. Substitutions of R6S, R7S, R9S and R23S strongly inhibited ShdA binding to 13FnIII, while substitutions K25S and R54S caused a small but significant reduction in binding. These data demonstrate that ShdA binds the Hep-2 domain of fibronectin by a mechanism that mimics binding of the host polysaccharide heparin. We next investigated the domain structure of the ShdA passenger domain. The ShdA passenger domain consists of two regions, an N-terminal non-repeat region, followed by a repeat region that is composed of two types of imperfect direct amino acid repeats, type A and type B. While the non-repeat region retained low Fn-binding activity, the repeat region bound bovine Fn with a similar disassociation constant (Kd) to that of the complete ShdA passenger domain. Specificity of these interactions was demonstrated by inhibition studies using biotin labeled and unlabelled ligand. Immune serum raised against repeat A2, B8, A3 and B9 reduced binding of the passenger domain
to 40 percent of that in the presence of naive serum. Together these data suggest that one or more epitopes that contribute to binding are present in repeats A2, B8, A3 and B9. To establish that shdA is also required for intestinal persistence and efficient fecal shedding in a relevant host species, a group a 4 calves was infected orally with a sublethal dose (5x10e6 colony forming units /animal) containing equal numbers of the S. Typhimurium wild type and an isogenic shdA deletion mutant. Animals developed diarrhea within 48 hours after inoculation that lasted for 3 to 5 days. The S. Typhimurium wild type was recovered in significantly higher numbers than the shdA deletion mutant from the feces at day 5 and subsequent days. Animals were euthanized at day 8 after inoculation and bacteria recovered from intestinal tissues. The S. Typhimurium wild type was recovered in significantly higher numbers than the shdA deletion mutant from the cecal wall, the mesenteric lymph node, the ileum
and the ileal Peyer's patches.
Impacts
The described work is innovative because it does for the first time explore molecular mechanisms of S. Typhimurium intestinal persistence. It is our expectation that our results will identify potential new targets for intervention strategies to reduce the prevalence of Salmonella serotypes in our food supply. This outcome will be significant since it will provide the basis for the development of knowledge-based approaches to improve public health.
Publications
- Kingsley, R.A., A.D. Humphries, E.H. Weening, M.R. de Zoete, S. Winter, A. Papaconstantinopoulou, G. Dougan and A.J. Baumler. 2003. Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype Typhimurium: identification of intestinal colonization and persistence determinants. Infect. Immun. 71:629-640.
- Zhang, S., R.A. Kingsley, R.L. Santos, H. Andrews-Polymenis, M. Raffatellu, J. Figueiredo, J. Nunes, R.M. Tsolis, L.G. Adams and A.J. Baumler. 2003. Molecular Pathogenesis of Salmonella enterica serotype Typhimurium-induced diarrhea. Infect. Immun. 71:1-12.
- Rabsch, W., C. Altier, H. Tschape, and A.J. Baumler. Food-borne Salmonella Infections. In. Torrence, M. and R. Isaacson (ed.). 2003. Microbial Food Safety in Animal Agriculture. Iowa State Press. 97-107.
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