Progress 01/01/12 to 12/31/16
Outputs
Target Audience:The target audiences include scientists (both in academia and industry), and graduate students in the filed of Salmonella functional genomics and animal vaccine development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided various opportunities to train a technician and graduate students in both knowledge and experimental skills in the areas of Salmonella pathogenesis, genetics, genomics, next generation sequencing technology, and bioinformatics. How have the results been disseminated to communities of interest?Publications. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We developed a powerful functional genomics method called, Tn-seq (transposon-sequencing), for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This method was applied to a genome-saturating Salmonella Typhimurium mutant library recovered from selection under various in vitro growth conditions (low-nutrients, bile salts, chicken carcass, refrigeration, and freezing) mimicking various aspects ofenvironmental stressors found in poultry production and processing. Numerous genes have been identified, revealing previous unknown mechanisms Salmonella uses to survive against the stressors in poultry production and processing. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes in Salmonella and the other important foodborne bacterial pathogens. These experimental approaches represent powerful means to study Salmonella gene functions, which is readily applicable to explore complex genetic mechanisms of this important foodborne pathogen to survive and persist in poultry production as well as human food chains in general. We also used Tn-seq method and bioinformatics analysis to identify the genes in Salmonella Typhimurium that are required for growth or survival under various in vitro conditions mimicking certain aspects of environmental stress and host infection, including starvation, desiccation, mildly acidic pH, propionic acid, bile acid or cold temperature. The mutants recovered after each selection were used to create Tn-seq profiles and the bioinformatics analysis identified the genes that are significantly underrepresented in each of the selectiveconditions. The information was used in conjunction with the previously identified gene required for host infection to construct gene-condition interaction maps, which provides valuable insights into the mechanisms used by this pathogen to cope with the stressors during host infection. In addition, we performed similar studies using Campylobacter jejuni to identify the genes conditionally requried for growth or survival under diverse growth or selection conditions. Salmonella spp., one of the most common foodborne bacterial pathogens, has the ability to survive under desiccation condition in foods and food processing facilities for years and is the most notable and frequent cause of contamination in low water activity foods. The response of Salmonella to desiccation stress is complex involving immediate physiological actions as well as coordinated genetic responses. However, the exact mechanisms of Salmonella to resist desiccation stress remain to be fully elucidated. For the first time to our knowledge, we screened a genome-saturating transposon (Tn5) library of Salmonella Typhimurium (S. Typhimurium) 14028S under the in vitro desiccation stress using transposon sequencing (Tn-seq). We identified 61 genes and 6 intergenic regions required to overcome desiccation stress. Salmonella desiccation resistance genes were mostly related to energy production and conversion; cell wall/membrane/envelope biogenesis, inorganic ion transport and metabolism; regulation of biological process; DNA metabolic process; ABC transporters; and two component system.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Khatiwara, A., T. Jiang, S. S. Sung, T. Dawoud, J. N. Kim, D. Bhattacharya, H. B. Kim, S. C. Ricke, and Y. M. Kwon. 2012.
Genome scanning for conditionally essential genes in Salmonella. Appl. Environ. Microbiol. 78, 3098-3107.
Dawoud, T., T. Jiang, R. K. Mandal, S. C. Ricke, and Y. M. Kwon. 2014. Improving the efficiency of transposon mutagenesis in Salmonella enteritidis by overcoming host-restriction barriers. Mol. Biotechnol. 56, 1004-1010
Wolfenden. A. D., Y. Yang, O.B. Faulkner, R. K. Mandal, B. M. Hargis, L. R. Berghman, Y. M. Kwon, and L. R. Bielke. 2016. Evaluation of recombinant Salmonella Typhimurium and Enteritidis vaccines to protect against Salmonella Heidelberg infection. Poultry Science Association, New Orleans, LA.
Kwon, Y.M., S.C. Ricke, and R.K. Mandal. 2016. Transposon sequencing: methods and expanding applications. Applied Microbiology and Biotechnology. 100, 31-43 (invited review)
Mandal, R.K., and Y.M. Kwon. 2016. Global screening of Salmonella genes for desiccation survival. Applied and Environmental Microbiol. (submitted)
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audiences include the technician, graduate students in my research program, who were directly involved in the project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided various opportunities to train a technician and graduate students in both knowledge and experimental skills in the areas of Salmonella pathogenesis, genetics, genomics, next generation sequencing technology, bioinformatics, and vaccinology How have the results been disseminated to communities of interest?The major methods to disseminate the research results were to present the research at professional conferences and to publish in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?We will use the functional genomics data obtained from Tn-seq experiments to design and construct attenuate Salmonella vaccine candidates.
Impacts
What was accomplished under these goals?
We used Tn-seq method and bioinformatics analysis to identify the genes in Salmonella Typhimurium that are required for growth or survival under various in vitro conditions mimicking certain aspects of environmental stress and host infection.These conditions include starvation, desiccation, mildly acidic pH, propionic acid, bile acid or cold temperature. The mutants recovered after each selection were used to create Tn-seq profiles and the bioinformatics analysis identified the genes that are significantly underrepresented in each of the selectiveconditions. The information was used in conjunction with the previously identified gene required for host infection to construct gene-condition interaction maps, which provides valuable insights into the mechanisms used by this pathogen to cope with the stressors during host infection. In addition, we performed similar studies using Campylobacter jejuni to identify the genes conditionally requried for growth or survival under diverse growth or selection conditions.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Kwon, Y. M., S. C. Ricke, and R. K. Mandal. 2015. Transposon sequencing: methods and expanding applications. Applied Microbiology and Biotechnology [Epub ahead of print]
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided various opportunities to train a technician and graduate students in both knowledge and experimental skills in the areas of Salmonella genetics, bioinformatics, and vaccinology. How have the results been disseminated to communities of interest? The major methods to disseminate the results was to present the research at professional conferences and to publish in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? We will continue to characterize the genome of Salmonella Typhimurium for the genes important for colonization and virulence during chicken infection.
Impacts
What was accomplished under these goals?
We used Tn-seq method and bioinformatics analysis to identify the genes in Salmonella Typhimurium that are required for growth or survival under various in vitro conditions mimicking certain aspects of environmental stress and host infection. Briefly, a complex library of Tn5 insertions were created and subjected to selection under starvation, desiccation, mildly acidic pH, propionic acid, bile acid or cold temperature. The mutants recovered after each selection were used to create Tn-seq profiles and the bioinformatics analysis identified the genes that are significantly underrepresented in each of the selective conditions. The genes of significance will be selected and further characterized to validate the functions of the identified genes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Kim, J.N., and Y. M. Kwon. 2014. Phenotypic characterization of Salmonella RyhB-1 mutations that modulate target regulation. Current Microbiol. 69, 212-217.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Dawoud, T., T. Jiang, R. K. Mandal, S. C. Ricke, and Y. M. Kwon. 2014. Improving the efficiency of transposon mutagenesis in Salmonella enteritidis by overcoming host-restriction barriers. Mol. Biotechnol. 56, 1004-1010.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audiences include the technician, graduate students, and undergraduate students in my research program, who were directly involved in the project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided various opportunites to train a technician, graduate students, and an undergraduate student in both knowledge and experimental skills in the area of Salmonella genetics, bioinformatics, and vaccinology. How have the results been disseminated to communities of interest? The main routes to disseminate the results obtained from this project is presentation at conferences and to publish the results in peer-reviewed journals. The results were presented at 2 different national conferences in 2013 and manuscripts are currently in preparation for publication in near future. What do you plan to do during the next reporting period to accomplish the goals? We will focus on genome-wide screening of Tn5 mutants of Salmonella Typhimurium using the new Tn-seq method developed in our lab to identify genetic determinants important for fitness in vitro and in vivo. At the same time, the Tn-seq method will be combined with modified transposon for comprehensive identification of promising carrier proteins in Salmonella for development of more effective Salmonella-based vaccines.
Impacts
What was accomplished under these goals?
1. The central tool to accomplish Aim1 is transposon-sequencing (Tn-seq) method. Since our previous version Tn-seq method had a major limitation, we have modified and evaluated the new Tn-seq method. The new Tn-seq method based on the use of terminal transferase to attach C-tails to transposon juction sequences allowed more comprehensive and quantitatively accurate determination of all transposon insertions in a transposon insertion library. 2. We significantly improved the efficiency of Tn5 mutagenesis in Salmonella enterica serovar Enteritidis by using host-adapted Tn5 fragment. Then, the new Tn-seq method was used to determine all Tn5 insertion sites in the constructed library. 3. We identified the genes in Salmonella enterica serovar Typhimurium genome that are required for growth or survival during cold storage using Tn5 mutagenesis and Tn-seq method. 4. We have constructed and evaluated serveral candidate vaccines based on Salmonella Enteritidis strain to control Salmonella and Campylobacter species in poultry.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Dawoud, T., T. Jiang, S. C. Ricke, and Y. M. Kwon. 2013. Identification of Salmonella Typhimurium genes essential for cold temperature survival on a chicken carcass using a Tn-seq method. 113th American Society for Microbiology General meeting, Denver, CO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Faulkner, O. B., Y. Yang, V.A. Kuttappan, L.R. Bielke, A.D. Wolfenden, G. Tellez, L.R. Berghman, Y.M. Kwon, and B.M. Hargis. 2013. Recognition of recombinant bacterial surface-expression of CD154 or HMGB1 by a macrophage cell line. Southern Poultry Science Society (SPSS), Athens, GA.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The use of virulence-attenuated Salmonella as a vaccine vector has been established as a promising vaccination strategy. We have developed a novel vaccination strategy using a LamB protein for efficient surface-display of foreign epitopes in Salmonella. We will maximize the efficacy of this vaccination strategy by identifying and employing the most suitable carrier proteins, and epitopes. The candidate vaccine vectors will be tested and the most promising Salmonella vector system will be used for construction and evaluation of Salmonella-based vaccines for various infectious agents of importance in poultry as well as in human. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We developed a powerful functional genomics method called, Tn-seq (transposon-sequencing), for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This method was applied to a genome-saturating Salmonella Typhimurium mutant library recovered from selection under various in vitro growth conditions (low-nutrients, bile salts, chicken carcass, refrigeration, and freezing) mimicking various aspects of environmental stressors found in poultry production and processing. Numerous genes have been identified, revealing previous unknown mechanisms Salmonella uses to survive against the stressors in poultry production and processing. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes in Salmonella and the other important foodborne bacterial pathogens. These experimental approaches represent powerful means to study Salmonella gene functions, which is readily applicable to explore complex genetic mechanisms of this important foodborne pathogen to survive and persist in poultry production as well as human food chains in general. Understanding the genetic factors and survival mechanisms will provide valuable and practical insights for development of effective strategies to reduce Salmonella in poultry and poultry products. We also continued development of Salmonella-based vaccines targeting CfrA and CfrB protein epitopes to control Campylobacter species. Three candidate vaccines have been constructed - Live CfrA/CD154(H), Live CfrA/CD154(C), and Live CfrB. These vaccine candidates will be evaluated for protection against Campylobacter challenge using chicken model of cecal colonization.
Publications
- Khatiwara, A., T. Jiang, S. S. Sung, T. Dawoud, J. N. Kim, D. Bhattacharya, H. B. Kim, S. C. Ricke, and Y. M. Kwon. 2012. Genome scanning for conditionally essential genes in Salmonella. Appl. Environ. Microbiol. 78, 3098-3107.
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