Progress 04/01/16 to 06/30/18
Outputs
Target Audience:The target audience for this project includes microbiologists and researchers from agricultural, clinical, industrial, and academic backgrounds.Accordingly, the results of these studies were shared at the 2017 and 2018Microbe conference hosted by the American Society for Microbiology. Additionally, project findings were shared on an ongoing basis with research teams at Cornell University and the University of North Carolina at Charlotte (UNCC). The PD also delivered a guest lecture at UNCC that included findings from this project. Changes/Problems:Early into this project, we encountered difficulty in identifying culture conditions that accomplished the submaximal induction of SPI-1 required to assess the stability of invasion gene expression. The basis of this difficulty lies in the fact that SPI-1 is expressed in vitro only in complex media after bacterial transition from log to lag phase. This condition proved difficult to recreate in our required experimental workflow. Thus, while this line of experiments is ongoing, our research uncovered other aspects of biphasic regulation (i.e. the genetic basis of biphasic expression discussed above) that have been exceedingly fruitful and that we chose to prioritize. A second problem we experienced was in using FACS to sort Salmonella into SPI-1-positive and SPI-1-negative groups using on a GFP reporter of SPI-1. The reason for this difficulty was that standard culture conditions resulted in only a small proportion of GFP+ bacteria, and sorting a sufficient number of these events to enable downstream assays became logistically impractical. A solution to this problem eventually came in the form of our genetic constructs: the mutants we designed to exhibit regulated induction of SPI-1 provide an exogenous method to increase the number of SPI-1-positive bacteria, making isolation of that population by FACS feasible. By the time these mutants were constructed, other aspects of our pursuit had demonstrated more promising results, and so the FACS separation of bacterial population for downstream analysis was triaged. However this line of questioning is of continued interest, and with the appropriate tools now in hand the experimental workflow is feasible. What opportunities for training and professional development has the project provided?For the majority of this project, the PD received mentorship through a unique collaborative arrangement. This arrangement allowed joint guidance from both the primary mentor, Dr. Craig Altier at Cornell University, and supportive mentorship from Dr. James Oliver at UNC Charlotte. An advantage of this arrangement was the depth and diversity in scientific guidance offered by two experienced microbiologist mentors. Furthermore, the networking opportunity offered by this arrangement has been unparalleled, allowing the PD to establish professional partnerships across the East Coast. Indeed, it was because of this arrangement that the PD secured subsequent employment as a faculty member at UNC Charlotte, serving as a Research Assistant Professor in the Department of Chemistry. Here, she is part of an interdisciplinary team working on novel biochemical methods to detect and target pathogenic bacteria. In addition to facilitating this direct career advancement, this project provided several other training and professional development opportunities. One such opportunity was attendance at the annual ASM Microbe conference in 2017 and 2018, where the PD engaged distinguished microbiologists, and delivered oral and poster presentations to disseminate the project findings. Additionally, poster presentations at smaller venues provided an opportunity to refine communication skills and initiate collaborations with colleagues at Cornell and UNCC. Besides these networking opportunities, the PD also acquired experience mentoring students in the theory and profession of scientific research. Two undergraduate students were mentored over the course of the award period. The first student, Savannah Sabbah, developed a multiwell assay to characterize the efficacy of synthetic inhibitors of isoprenoid synthesis in a variety of bacterial species. The second student, M. Seth Flynn, worked with Salmonella to characterize trade-offs between antibiotic resistance and virulence gene expression. How have the results been disseminated to communities of interest?The findings of this project were presented in poster sessions and oral presentations to diverse audiences at both Cornell University and the University of North Carolina at Charlotte. Additionally, an oral presentation based on this work was delivered at the 2017 ASM Microbe conference in New Orleans, and a poster presentation and poster talk based on this work were delivered at the 2018 ASM Microbe conference in Atlanta, GA. In addition to conference presentations, several publications are expected to result from this fellowship award. One portion of this work contributed to a collaborative research project that was recently published in the journal Infection and Immunity. A second collaborative undertaking has been submitted to the same journal, and received favorable review with a request for resubmission. Finally, the major findings from this research project are currently being compiled and finalized prior to manuscript submission. Remaining are data analysis and manuscript composition. The finalized and anticipated publications based on this award are listed in detail below. Manuscripts including work from this award: Salmonella Typhimurium increases functional PD-L1 synergistically with IFNγ in intestinal epithelial cells via Salmonella Pathogenicity Island-2. Sahler J, Eade CR, Altier C, March J. Infect Immun. 2018; 86(5). Salmonella Pathogenicity Island One is Expressed in the Chicken Gut and Promotes Bacterial Outgrowth. Eade CR, Bogomolnaya L, Hung C-C, Betteken, MI, Andrews-Polymenis H, Altier C. Submitted; Under Revision Biphasic Expression of Invasion Genes is Critical to Salmonella's Success in the Gut. Eade CR, Hung C-C, Betteken MI, Altier C. In preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Over the duration of this project, we successfully developed a new set of tools to characterize Salmonella virulence, then used these tools to address specific questions about the regulation of invasion gene expression. In addition to addressing some of our original hypotheses, we have also made unexpected discoveries about the nature of SPI-1 expression in Salmonella. The tools we developed include several mutants of Salmonella that genetically isolate individual transcription factors from complex signaling cascades, which permitted us to identify the site of action for environmental repressors of invasion genes. We found that, contrary to prior models, the transcription factors hilD, hilC, and rtsA each responds similarly to repressive environmental signals like short chain fatty acids or bile, though these inhibitors act at different stages of transcription factor gene expression. The mutants we constructed also allowed us to evaluate the mechanism by which invasion gene expression occurs as a biphasic phenotype. We found that while the three transcription factors hilD, hilC, and rtsA together augment SPI-1 induction in a feed-forward loop, it is solely HilD that establishes the biphasic expression of invasion genes. Furthermore, we determined that the open reading frame of hilD is not the essential determinant of biphasic gene expression; rather, it is the hilD promoter that comprises the genetic basis for biphasic SPI-1 expression. Using this information, we devised a technique that accomplishes regulated induction of SPI-1 in either a biphasic or a monophasic fashion. We demonstrated that this tool is useful for both in vitro and in vivo gene induction, and characterized the consequences of converting a SPI-1 expression from a biphasic to a monophasic phenotype. In addition to the immediate insight this tool has provided us, we expect this technique to be widely applicable for studying the consequences of bacterial gene expression both in vitro and in vivo.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
1. Salmonella Typhimurium increases functional PD-L1 synergistically with IFN? in intestinal epithelial cells via Salmonella Pathogenicity Island-2. Sahler J, Eade CR, Altier C, March J. Infect Immun. 2018; 86(5).
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
2. Salmonella Pathogenicity Island One is Expressed in the Chicken Gut and Promotes Bacterial Outgrowth. Eade CR, Bogomolnaya L, Hung C-C, Betteken, MI, Andrews-Polymenis H, Altier C. Submitted; Under Revision
- Type:
Journal Articles
Status:
Other
Year Published:
2018
Citation:
3. Biphasic Expression of Invasion Genes is Critical to Salmonellas Success in the Gut. Eade CR, Hung C-C, Betteken MI, Altier C. In preparation.
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Progress 04/01/17 to 03/31/18
Outputs
Target Audience:The findings from this research were presented at Microbe, the general meeting for the American Society for Microbiology, in June 2017 in New Orleans, LA. Here, the PD delivered an oral presentation of the project findings to a diverse audience of microbiologists from clinical, agricultural, industrial and academic backgrounds. Additionally, the results of these studies were shared on an ongoing basis with research teams at Cornell University and the University of North Carolina at Charlotte (UNCC). The PD also delivered a guest lecture at UNCC that included findings from this project. Changes/Problems:We encountered difficulty in identifying culture conditions that accomplished the submaximal induction of SPI-1 required to assess the stability of invasion gene expression. The basis of this difficulty lies in the fact that SPI-1 is expressed in vitro only in complex media after bacterial transition from log to lag phase. This condition has proven difficult to recreate in our required experimental workflow. Thus, while this line of experiments is ongoing, our research uncovered other aspects of biphasic regulation that have been exceedingly fruitful that we chose to prioritize. Our regulated induction of invasion genes revealed that hilD is the basis of biphasic SPI-1 expression. From this knowledge we developed a system to assess the burden of invasion gene expression in mutants genetically incapable of biphasic regulation of SPI-1, and found that the biphasic nature of SPI-1 expression is essential for bacterial fitness. What opportunities for training and professional development has the project provided?This fellowship has facilitated an intensive training period, with principal mentorship provided by my primary mentor, Dr. Craig Altier, and supportive mentorship through a collaborative arrangement with Dr. James Oliver. Together, Dr. Altier and Dr. Oliver have imparted both scientific and professional guidance. They have also supported professional networking opportunities, including oral and poster presentations at several venues. I have also had the opportunity to mentor two undergraduate students over the course of the award period. The first student, Savannah Sabbah, developed a multiwell assay to characterize the efficacy of synthetic inhibitors of isoprenoid synthesis in a variety of bacterial species. The second student, M. Seth Flynn, is working with Salmonella to characterize trade-offs between antibiotic resistance and virulence gene expression. How have the results been disseminated to communities of interest?The findings of this project were presented in poster sessions and oral presentations to diverse audiences at both Cornell University and the University of North Carolina at Charlotte. Additionally, an oral presentation based on this work was delivered at the 2017 ASM Microbe conference in New Orleans. I plan to attend ASM again this year (in June 2018) and have submitted an abstract based on this project that has been accepted for traditional poster presentation format, along with an oral presentation of the findings. In addition to conference presentations, several publications are expected to result from this fellowship award. One portion of this work contributed to a collaborative research project that was recently accepted for publication in the journal Infection and Immunity. A second collaborative undertaking is expected to be submitted to the same journal this summer. Finally, the major findings from this research project are currently being finalized before manuscript submission. Remaining are experimental repeats, statistical analyses, and manuscript writing prior to submission. What do you plan to do during the next reporting period to accomplish the goals?With the remaining award period, I plan to address the experimental repeats that are necessary to finalize the data set required for publishing the major findings of this project. Remaining are repeats of reporter assays, western blots, and real-time quantitative PCR experiments. The strains and conditions for these experiments are already established, and completing the data set is expected to be straightforward. Additionally, I the data from these experiments will be compiled into a finalized manuscript in the remaining award period.
Impacts
What was accomplished under these goals?
Since the project start date, we have completed construction of several complex mutants of Salmonella that have been used to assess SPI-1 expression. Experimentally, we have found that the SPI-1 regulator HilD is essential to the biphasic nature of SPI-1 expression, and that while other transcription factors augment SPI-1 induction in a feed-forward loop, it is solely HilD that establishes the biphasic expression of invasion genes. We have also found that, contrary to prior models, each transcription factor in this loop responds similar to repressive environmental signals like short chain fatty acids and bile, though these inhibitors act at different stages of gene expression. We have also pioneered a technique that accomplishes regulated induction of SPI-1 in either a monophasic fashion, and used this to assess the importance of biphasic SPI-1 expression to bacterial fitness and colonization of animals.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Sahler JM, Eade CR, Altier C, March JC. Salmonella enterica Serovar Typhimurium Increases Functional PD-L1 Synergistically with Gamma Interferon in Intestinal Epithelial Cells via Salmonella Pathogenicity Island 2. Infect Immun. 2018 Apr 23;86(5). pii: e00674-17.
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Progress 04/01/16 to 03/31/17
Outputs
Target Audience:The work from this project was disseminated to diverse scientific research groups at Cornell University and the University of North Carolina at Charlotte. Changes/Problems:One experimental difficulty we have encountered is in defining conditions of submaximal induction that permit us to monitor biphasic stability of SPI-1 expression. In reculturing sorted Salmonella into various (conditioned) media formulations, we find that SPI-1 expression is invariably repressed. We are currently using our collection of reporter assays to identify appropriate conditions for maintaining SPI-1 expression, yet this pursuit has led us to ask more basic questions about the signaling that initiates SPI-1 induction. We have begun to evaluate nutrient availability and metabolic signaling as iniators of SPI-1 induction, and the physiological implications for such control. What opportunities for training and professional development has the project provided?The key to my training has been the mentorship provided by my mentor, Dr. Craig Altier. I continue to learn from him about the scientific underpinnings of Salmonella biology, while he challenges me with new facets of bacteriology, host-pathogen dynamics, and experimental design. Dr. Altier has also facilitated my professional development by fostering a collaboration with the University of North Carolina at Charlotte, where I have been able to participate in a cooperative research experience. This stimulating environment has provided a unique perspective to my own work. How have the results been disseminated to communities of interest?The findings of this project were presented in poster sessions and oral presentations to diverse audiences at both Cornell University and the University of North Carolina at Charlotte. Additionally, an oral presentation based on this work has been accepted for delivery at this year's ASM Microbe conference in New Orleans. A portion of this work is also in final stages of manuscript preparation as part of a collaborative publication, while other findings from this project are being formed into a separate independent publication that we hope to publish later this year. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will employ our completed strain collection to perform the proposed experiments. Using our optimized FACS sorting protocol, we will determine the amount of HilD protein expressed in SPI-1 positive populations in the presence or absence of repressive treatments. We will also build on our preliminary results from SPI-1 inductions experiments to evaluate the stability of SPI-1 positive versus SPI-1 negative phenotypes. These results are expected to yield two manuscripts in the coming reporting period.
Impacts
What was accomplished under these goals?
In this year, we have nearly completed the complex strain construction that is necessary to address our project goals. Using those strains that are already complete, we have optimized experimental systems, and performed important preliminary experiments that address our hypotheses. Regarding optimization, we have established conditions that permit FACS sorting of SPI-1-positive and SPI-1 negative populations, and established our western blot protocol for our tagged HilD protein. Experimentally, we have found that the SPI-1 regulator HilD is essential to the biphasic nature of SPI-1 expression, and that while other transcription factors augment SPI-1 induction in a feed-forward loop, it is solely HilD that establishes the biphasic expression of invasion genes. We have also found that, contrary to prior models, each transcription factor in this loop responds similar to repressive environmental signals like short chain fatty acids and bile. We have also made progress in establishing the conditions that induce and maintain biphasic SPI-1 expression, which will facilitate our upcoming phenotypic stability studies.
Publications
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