Progress 10/01/16 to 09/30/19
Outputs
Target Audience:The target audience(s) of our efforts during this reporting period was undergraduate, graduate and professional students who would become scientists and health care providers who will play a critical role in providing the general public with new information and education on health and food safety associated with Salmonella infections. Of particular emphasis was on the alarming trend of the emergence and wide-spread of multidrug-resistant Salmonella strains in the US as well as in the globe. Examples of the efforts during this reporting period include formal classroom instruction, small group discussion, and panel discussion as part of undergraduate, graduate, and veterinary curriculum. It is important to note that, although the severity of typhoid fever, the disease caused by Salmonella Typhi, can vary among individuals, Salmonella Typhi infection occurs across gender, age, and ethnicity. Some minority groups, as well as people living in poorer conditions, are more at risk for severe symptoms of typhoid fever. The safer we can make the food, the safer these populations will be. Therefore, we foresee our investigations serving the interests of minority groups, families with children, people living in at risk conditions, and the elderly, who also tend to show more severe symptoms of the disease. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Sohyoung Lee, a postdoc associate in my laboratory, has been devoted 20% effort to this project. Dr. Lee whose doctoral training was focused on fungal pathogenesis who has published 22 original research and review articles in peer reviewed journals in the field of fungal pathogenesis. This Hatch fund, along with other funds available in my laboratory, offers Dr. Lee training and professional development opportunities in the field of bacterial pathogenesis. I meet with all trainees in my lab weekly at the focus, individual meetings. This enables me to manage their projects and gauge their progress. Should they require additional input or mentoring I set up additional individual meetings. All trainees present their research at more formal weekly lab meetings and at least once a year at the Department Work-in-Progress presentations. Moreover, all trainees are expected to present their research progress at one regional/national meeting per year. How have the results been disseminated to communities of interest?I have served as a panel for the panel discussion on Antimicrobial Resistance, which was held in November 2019 at the Cornell College of Veterinary Medicine to help raise awareness for antimicrobal resistance and proper usage of antimicrobials. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall goals of this research program are to better understand the host-Salmonella interactions, which may ultimately offer significant insights into the development of alternative intervention strategies applicable to antibiotic resistant Salmonella. Two objectives were proposed to define the mechanism underlying the massive replication and survival of Salmonella enterica serovar Typhi (S. Typhi) in plants. Objective 1: Investigate whether S. Typhi's known virulence genes essential during the human infectious cycle are also important during the plant infectious cycle. 1) Major activities completed/experiments conducted: For the three year funding period, we have completed the experiments proposed in Objective 1. In brief, we have successfully generated five S. Typhi mutants required for functional studies and completed experiments aimed to address a key scientific question whether these genes exerting an essential role in human infection of S. Typhi are also required for the bacterial replication in plants or whether the underlying mechanism is different between these two hosts. 2) Data collected: Five S. Typhi mutants were generated: (1) a phoP and phoQ gene deletion mutant (a two component transcriptional regulatory system for Salmonella virulence genes), (2) a tviB gene deletion mutant (Vi polysaccharide biosynthesis), (3) an invA gene deletion mutant (virulence genes clustered in the Salmonella pathogenicity island 1 required for invasion into human epithelial cells), (4) a ssaC gene deletion mutant (virulence genes clustered in the Salmonella pathogenicity island 2 (SPI-2) required for replication and survival in human cells), and (5) a typhoid toxin gene mutant (virulence genes playing an essential role in causing typhoid signs and symptoms and contributing to the establishment of persistent/chronic infection). We have conducted bacterial colony forming unit (c.f.u.) assays to compare infection and replication of wild-type and these five mutants in tomatoes and found that all S. Typhi strains replicated well in tomatoes. 3) Summary statistics and discussion of results: A select of S. Typhi virulence genes playing essential roles in human infection have been genetically engineered in wild-type S. Typhi, resulting in five S. Typhi mutants. These five S. Typhi mutant strains were validated via PCR-based application of the target locus on the S. Typhi genome, followed by Sanger sequencing. The c.f.u. experimental results indicate that these five genes do not contribute significantly to the massive replication of S. Typhi in tomatoes. 4) Key outcomes or other accomplishments realized: We found out that the molecular mechanism underlying massive replication in tomato, an environmental host for S. Typhi, is different from the mechanism used in humans. Objective 2: Identify and characterize S. Typhi's virulence genes contributing to the massive replication and survival of S. Typhi in plants. 1) Major activities completed/experiments conducted: For the three year funding period, we have completed the experiments proposed as part of Objective 2. In brief, to rapidly identify S. Typhi virulence genes required for its massive growth and survival in plant cells, we have successfully generated a genome-wide transposon disruption library in S. Typhi. This is a good resource for applications that would aim at identifying important bacterial determinants for Salmonella Typhi's pathogenesis and virulence in human and environmental hosts. We have optimized several experimental conditions for Tn-seq analysis. 2) Data collected: We have created a genome-wide transposon disruption library in S. Typhi, by exploiting a sequencing adapted mariner transposon derivative tailored for minimizing hits associated with polar effects (that often result in false-positive hits of genes located in the same operon). This modified transposon derivative allows for implementing PCR-based molecular barcoding procedures, and for comparative analysis among S. Typhi transposon mutant populations harvested from plants. 3) Summary statistics and discussion of results: We have generated a comprehensive genome-wide transposon disruption library in S. Typhi. We have optimized several experimental conditions for Tn-seq analysis, which would be used for Tn-seq screen analysis in the future. 4) Key outcomes or other accomplishments realized: We have generated a genome-wide transposon disruption library in S. Typhi and optimized the experimental conditions required for successful Tn-seq screen analysis. Transposon sequencing (Tn-seq) will allow us to screen entire bacterial genomes to identify genes involved in its massive growth and survival in plants. Overall, we have successfully completed our research goal proposed in Objective 1 and part of Objective 2. With the grant support through the Hatch program, we have advanced the understanding of the similarities and differences of the pathogen's strategies that S. Typhi employs to survive and grow in human and environmental hosts. Furthermore, the genome-wide transposon disruption library in S. Typhi would serve as a useful resource for other studies aimed at identifying bacterial determinants responsible for certain phenotypes in human and environmental hosts.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience(s) of our efforts during this reporting period is the general public, who relies on scientists and agencies to provide new information and education on health and food safety. Toward this goal, we have published a review paper through an open-access, peer-reviewed journal, where we have discussed why eradicating Salmonella Typhi is so challenging and our insights into future vaccine and therapeutic development. It is important to note that, although the severity of typhoid fever, the disease caused by Salmonella Typhi, can vary among individuals, Salmonella Typhi infection occurs across gender, age, and ethnicity. Some minority groups, as well as people living in poorer conditions, are more at risk for severe symptoms of typhoid fever. The safer we can make the food, the safer these populations will be. Therefore, we foresee our investigations serving the interests of minority groups, families with children, people living in at risk conditions, and the elderly, who also tend to show more severe symptoms of the disease. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Sohyoung Lee, a postdoc associate in my laboratory, has been devoted 0.01% effort to this project. Dr. Lee whose doctoral training was focused on fungal pathogenesis who has published 19 original research and review articles in peer-reviewed journals in the field of fungal pathogenesis. This Hatch fund, along with other funds available in my laboratory, offers Dr. Lee training and professional development opportunities in the field of bacterial pathogenesis, where she has recently published 2 research and review articles. Dr. Yi-An (Amy) Yang, a postdoctoral associate, has also been devoted 0.01% effort to this project. Dr. Yang obtained a DVM degree from National Taiwan University with her focus on small animal practice and basic research in 2011, and MS from Michigan State University under the guidance of Dr. Shannon Manning in 2014. Since the opening of my laboratory in October 2014, Dr. Yang has been working with me on the interplay between Salmonella and hosts. As part of her training in my laboratory, Dr. Yang co-authors two Salmonella pathogenesis review articles and is the first author of our recent Salmonella toxin research paper. I meet with all postdocs and students in my lab weekly at the focus, individual meetings. This enables me to manage their projects and gauge their progress. Should they require additional input or mentoring I set up additional individual meetings as required. All trainees present their research at more formal weekly lab meetings and at least once a year at the Department Work-in-Progress presentations. Moreover, all trainees are expected to present their research progress at one regional/national meeting per year. How have the results been disseminated to communities of interest?The Hatch award (NYC-4333446), along with other funds available in the PI's laboratory, allowed us to make important progress in obtaining additional preliminary data that serves as a solid foundation of the ongoing research program focused on the typhoid fever-causing Salmonella. In addition to making research progress, to increase the general public and policy maker's awareness on the Salmonella-related health risk, during this funding period (Oct 1, 2017 - Sep 30, 2018), we have published a review paper through an open-access, peer-reviewed journal, where we have discussed why eradicating Salmonella Typhi is so challenging and our insights into future vaccine and therapeutic development. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 has been completed. In Objective 2, we will conduct Tn-seq to identify Salmonella Typhi genes required for massive growth and survival in tomato. Additionally, as the ultimate goals of this research program is to discoversignificant insights into the development of alternative intervention strategies applicable to antibiotic resistant Salmonella, we may tweak the direction of Objective 2 toward a more targetted approach to discover tomatogene/protein possessing strong bactericidal activities even against intracellular Salmonella and multi-drug resistant Salmonella. Current intervention strategies including antibiotics are effective against neitherintracellular bacteria nor multi-drug resistant bacteria. Therefore, this alternative approach may offer a solution to one of the major concerns with regard to food safety.
Impacts
What was accomplished under these goals?
The overall goals of this research program are to better understand the host-Salmonella interactions, which may ultimately offer significant insights into the development of alternative intervention strategies applicable to antibiotic resistant Salmonella. Two objectives were proposed to define the mechanism underlying the massive replication and survival of Salmonella Typhi in plants. Objective 1: Investigate whether Salmonella Typhi's known virulence genes essential during the human infectious cycle are also important during the plant infectious cycle. 1) Major activities completed/experiments conducted: We have successfully generated five Salmonella Typhi mutants required for functional studies and completed experiments aimed to address a key scientific question whether these genes exerting an essential role in human infection of Salmonella Typhi are also required for the bacterial replication in plants or whether the underlying mechanism is different between these two hosts. 2) Data collected: Five S. Typhi mutants were generated: (1) a phoP and phoQ gene deletion mutant (a two component transcriptional regulatory system for Salmonella virulence genes), (2) a tviB gene deletion mutant (Vi polysaccharide biosynthesis), (3) an invA gene deletion mutant (virulence genes clustered in the Salmonella pathogenicity island 1 required for invasion into human epithelial cells), (4) a ssaC gene deletion mutant (virulence genes clustered in the Salmonella pathogenicity island 2 required for replication and survival in human cells), and (5) a typhoid toxin gene mutant (virulence genes playing an essential role in causing typhoid signs and symptoms and contributing to the establishment of persistent infection). We have conducted bacterial colony forming unit (c.f.u.) assays (at least three independent experiments with quadruplicate)to compare infection and replication of wild-type and these five mutants in tomatoes and found that all S. Typhi strains replicated well in tomatoes. 3) Summary statistics and discussion of results: A select of Salmonella Typhi virulence genes playing essential roles in human infection have been genetically engineered in wild-type Salmonella Typhi, resulting in five Salmonella Typhi mutants. These five S. Typhi mutant strains were validated via PCR-based application of the target locus on the S. Typhi genome, followed by Sanger sequencing. The c.f.u. experimental results indicate that these five genes do not contribute significantly to the massive replication of S. Typhi in tomatoes. Unpaired student t-test was used for statistic analyses. 4) Key outcomes or other accomplishments realized: Key outcomes from Objective 1 is obtaining evidence that the molecular mechanism underlying massive replication in tomato, an environmental host for S. Typhi, is different from the mechanism used in humans. Objective 2: Identify and characterize Salmonella Typhi's virulence genes contributing to the massive replication and survival of S. Typhi in plants. 1) Major activities completed/experiments conducted: To rapidly identify Salmonella Typhi virulence genes required for its massive growth and survival in plant cells, we have successfully generated a genome-wide transposon disruption library in S. Typhi. 2) Data collected: We have created a genome-wide transposon disruption library in S. Typhi, by exploiting a sequencing adapted mariner transposon derivative tailored for minimizing hits associated with polar effects (that often result in false positive hits of genes located in the same operon). This modified transposon derivative allows for implementing PCR-based molecular barcoding procedures, and for a comparative analysis among S. Typhi transposon mutant populations harvested from plants. 3) Summary statistics and discussion of results: Our 1st and 2nd year efforts toward the completion of Objective 2 have resulted in the establishment a comprehensive genome-wide transposon disruption library in S. Typhi. 4) Key outcomes or other accomplishments realized: We have generated a genome-wide transposon disruption library in S. Typhi.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Yang, Y-A, Chong, A, & Song, J. (2018). Why is eradicating typhoid fever so challenging: implications for vaccine and therapeutic design. Vaccines. 6(3): E45. PMC6160957. DOI: 10.3390/vaccines6030045.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The Hatch Award (2016-17-111), along with other funds available in the PI's laboratory, allowed us to make important discoveries about Salmonella Typhi, the typhoid fever-causing Salmonella. Our effortsduring this reporting period (October 2016 to September 2017) allowed us to providescience-based knowledge that typhoid toxin produced by Salmonella Typhi targets the sugar coatings of selected cells related to typhoid fever disease. The target audience(s) of our efforts during this reporting period is the general public, who relies on scientists and agencies to provide new information and education on health and food safety. Although the severity oftyphoid fever, the disease caused bySalmonella Typhi, can vary among individuals, Salmonella Typhi infection occurs across gender, age, and ethnicity. Some minority groups, as well as people living in poorer conditions, are more at risk for severe symptoms of typhoid fever. The safer we can make the food, the safer these populations will be. Thus, we foresee our investigations serving the interests of minority groups, families with children, people living in at-risk conditions, and the elderly, who also tend to show more severe symptoms of the disease. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Sohyoung Lee, a postdoc researcher in my laboratory, has been devoted 0.04% effort to this project. Dr. Lee whose doctoral training was focused on fungal pathogenesis who has published 19 original research and review articles in peer-reviewed journals in the field of fungal pathogenesis. This Hatch fund, along with other funds available in the PI's laboratory, offers Dr. Lee training and professional development opportunities in the field of bacterial pathogenesis (e.g., one-on-one meeting with a mentor in a weekly basis). How have the results been disseminated to communities of interest?The Hatch Award (2016-17-111) has allowed us to make important discoveries about Salmonella Typhi, with regard to the mechanism of how typhoid toxin causes typhoid symptoms. New information obtained from this workis important for performing the comparative analysis of a select of S. Typhi virulence genes, as proposed in Objective 1. To effectively disseminate this work to the general public, we have published this work on health and food safety issues in a peer-reviewed journal and presented at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?With the required research materials that we have generated during the first year of the research program (e.g., Salmonella Typhi mutants, a genome-wide transposon disruption library in Salmonella Typhi), we will move on to the next stage experiments. For objective 1, using the five Salmonella Typhi mutants generated for this objective, we will perform phenotype screens to address a fundamental scientific question ' Are Salmonella Typhi virulence genes essential in human infection also essential in plant infection?'. For objective 2, using the comprehensive Salmonella Typhi transposon library that we have generated for this objective,we will perform transposon sequencing (Tn-seq) to identify bacterial genes required for its massive growth and survival in plants.
Impacts
What was accomplished under these goals?
The overall impacts and key outcomes of the proposed research include (1) gaining a good understanding of the molecular and cellular mechanism underlying the interaction between the plant host and Salmonella Typhi, and (2) disseminating new knowledge obtained from the proposed study to the general public. To accomplish the goal, we have proposed addressing two fundamental scientific questions (two objectives) that will allow forbetter understanding of the mechanism underlying the massive replication and survival of Salmonella Typhi in plants. First, are S. Typhi's known virulence genes essential for human infection are also important for plant infection (Objective 1)? Second, what virulence genes of Salmonella Typhi contribute to its massive replication and survival in plants, a hallmark of S. Typhi during plant infection (Objective 2). Objective 1: Investigate whether Salmonella Typhi's known virulence genes essential during the human infectious cycle are also important during the plant infectious cycle. 1) Major activities completed/experiments conducted: We have successfully generated five Salmonella Typhi mutants required for functional studies that will be performed in the second year of this Hatch program. These genes play essential roles in human infection of Salmonella Typhi. 2) Data collected: In brief, five S. Typhi mutants were generated: (1) a phoP and phoQ gene deletion mutant (a two component transcriptional regulatory system for Salmonella virulence genes), (2) a tviB gene deletion mutant (Vi polysaccharide biosynthesis), (3) an invA gene deletion mutant (virulence genes clustered in Salmonella pathogenicity island 1 required for invasion into human epithelial cells), (4) a ssaC gene deletion mutant (virulence genes clustered in Salmonella pathogenicity island 2 required for replication and survival in human cells), and (5) a typhoid toxin gene mutant (virulence genes playing essential roles in causing typhoid symptoms and persistent infection). 3) Summary statistics and discussion of results: A select of Salmonella Typhi virulence genes playing essential roles in human infection have been genetically engineered in wild-type Salmonella Typhi, resulting in five Salmonella Typhi mutants. These five S. Typhi mutant strains were validated via PCR-basedapplicationof the target locus on the S. Typhi genome, followed by Sanger sequencing. 4) Key outcomes or other accomplishments realized: The function of typhoid toxin and how it works are less known among the five genes whose contributions to human and plant infection will be investigated in the second and third years of this Hatch program. As part of ourfirst yearefforts, we have also elucidated how typhoid toxin works in an animal model mimicking human infection, which has beendisseminatedto the general public, by publishing it in a peer-reviewed journal and presenting it at conferences. Overall, this new information and others, along with the five S. Typhi mutants, will facilitate the progress of research performed in the next funding years. Objective 2: Identify and characterizeSalmonella Typhi's virulence genes contributing to the massive replication and survival of S. Typhi in plants. 1) Major activities completed/experiments conducted: To rapidly identify Salmonella Typhi virulence genes required for its massive growth and survival in plant cells, we have successfully generated a genome-wide transposon disruption library in S. Typhi. 2) Data collected:We have created a genome-wide transposon disruption library in S. Typhi, by exploiting a sequencing-adapted mariner transposon derivative tailored for minimizing hits associated with polar effects (thatoftenresult in false-positive hits of genes located in the same operon). This modified transposon derivative allows for implementing PCR-based molecular barcoding procedures, and for a comparative analysis among S. Typhi transposon mutant populations harvested from plants. 3) Summary statistics and discussion of results: Ourfirst yearefforts toward the completion of Objective 2 have resulted in the establishment a comprehensive genome-wide transposon disruption library in S. Typhi. Characterization of thistranposoninsertion mutant library shows it to behighly-saturated and randomly dispersed, indicating that this library consists of 10-20 S. Typhi insertion mutants for every gene (except for essential genes) across the genome. 4) Key outcomes or other accomplishments realized: We have generated a genome-wide transposon disruption library in S. Typhi. Transposon sequencing (Tn-seq) will allow us to screen entire bacterial genomes to identify genes involved in its massive growth and survival in plants. Overall, we have successfully completed our research goal initially proposed for the first year, which will allow us to move on the experiments proposed for the second year of this Hatch program.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Chong, A, Lee, S, Yang, YA, Song J. (2017). The role of typhoid toxin in Salmonella Typhi virulence. Yale Journal of Biology & Medicine. 90(2):283-290.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Yang YA, Lee S, Zhao J, Thompson AJ, McBride R, Tsogtbaatar B, Paulson JC, Nussinov R, Deng L, Song J. (2017). In vivo tropism of Salmonella Typhi toxin to cells expressing a multiantennary glycan receptor. Nature Microbiology. Dec 4. doi:10.1038/s41564-017-0076-4. [Epub ahead of print].
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