Progress 10/01/11 to 09/30/14
Outputs
Target Audience: The target audience is the scientific community. They are reached through national scientific conferences including: American Society for Microbiology, Conference of Research Workers on Animal Diseases (CRWAD), Multi-state-NC-1202 meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project helped trained Daniel Shippy a PhD student (now graduated) and Alexis Bennet, a master student. Daniel presented his work at several national conferences. How have the results been disseminated to communities of interest? Findings from this project were presented to scientific communities, including a presentation at the American Society for Microbiology (2011), Conference of Research Workers on Animal Diseases (CRWAD) in 2012, and at the Multi-state-NC-1202 meetings (2013 and 2014). Data from this work was published in several peer-reviewed scientific journals: What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Salmonella is the most frequently reported cause of food-borne illness, causing an estimated 1.4 million cases of food-borne diseases and more than 500 deaths annually in the United States. Controlling Salmonella in food-producing animals is a key strategy for reducing food-borne outbreaks of the pathogen in humans. Salmonella is often associated with gastrointestinal disease outbreaks in humans throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division gene (gidA) rendered Salmonella enterica serovar Typhimurium (STM) unable to cause disease in laboratory animal models of infection. Most importantly, immunization with the gidA mutant protected mice from a lethal dose challenge of WT Salmonella. In this study, we further characterize the gidA mutant STM strain and evaluate its potential use as a live-attenuated vaccine. 1) investigate the mechanistic basis of protection afforded by the gidA mutant. The GidA mutant of Salmonella was unable to cause disease in mice and most importantly, animals infected with the mutant were protected when challenged with the lethal dose of WT Salmonella. Therefore, it will be crucial to examine the immunological basis of protection in animals that have been immunized with the gidA mutant. Such studies are crucial in determining the effectiveness of any attenuated bacterial strain to be used as a vaccine candidate. This information will be important for designing strategies for future animal and human vaccines.The protective efficacy of immunization with the gidA mutant was evaluated by immunization of mice and challenging them with a lethal dose of wild-type (WT) STM. Sera levels of antibodies, passive transfer of sera and cells, and cytokine profiling were performed to study the induction of immune responses induced by immunization with the gidA mutant strain. Mice immunized with the gidA mutant strain were fully protected from a lethal dose challenge of wild-type STM. Naïve mice receiving either immune cells or sera from immunized mice were protected from a lethal dose challenge of wild-type STM. Together, the results demonstrate that immunization with the gidA mutant strain protects mice by inducing humoral and cellular immune responses, with the humoral immune response potentially being the main mechanism of protection. This indicated a potential use of the gidA mutant strain for immunization against Salmonella infections in animals. 2) determine subcellular localization of GidA in Salmonella. GidA is known to complex with another protein known as MnmE, a feature that is important for their function. Therefore, it will be important to determine subcellular localization of GidA and MnmE in Salmonella. This study will help better understand the mechanism and functional characteristics of these proteins. GidA and MnmE were cloned, expressed, and purified. The purified proteins were used to develop polyclonal antibodies to determine the cellular location of GidA and MnmE by fractionation of bacterial cells and immunoblot analysis. Results from objectives showed that the GidA and MnmE proteins of Salmonella are cytoplasmic proteins associated with the membrane of the Salmonella cell. This characteristic is important in regulation of many bacterial activities during infection of animal and human hosts. Additionally, we hypothesized that GidA -MnmE complex modulates ability of Salmonella to cause infections. To test this, and to examine the relative contribution of GidA and MnmE in modulation of Salmonella disease mechanism, we constructed gidA and mnmE single deletion as well as a gidA mnmE double deletion strains of Salmonella. Results from this study showed a marked attenuation of the mutant strains, confirming their role in regulation of Salmonella pathogenesis. 3) identify the method by which GidA contribute to Salmonella filamentous morphology. We have previously shown that deletion of gidA significantly altered the virulence potential of Salmonella in both in vitro and in vivo models of infection. Most importantly, the gidA mutant cells displayed a filamentous morphology compared to the WT Salmonella cells. We investigated the role of GidA in Salmonella cell division using fluorescence and electron microscopy, transcriptional, and proteomic assays. Scanning electron microscopy data indicated a filamentous morphology with few constrictions in the gidA mutant cells. The filamentation of the gidA mutant cells is due to the defect in chromosome segregation. Furthermore, deletion of gidA altered the expression of many genes and proteins responsible for cell division and chromosome segregation, confirming the morphological data. Taken together, this data indicated GidA as a potential regulator of Salmonella cell division genes. In summary, this study: 1) determined the immunization efficacy of animals with gidA mutant strain; 2) determined the mechanisms of protection in animals that have been immunized with the gidA mutant which is crucial in determining the effectiveness of any attenuated bacterial strain to be used as a vaccine candidate; 3) determine the localization and functional characteristics of the GidA and other associated proteins, which is important for understanding of disease modulation in Salmonella. Overall, this work significantly increased our knowledge and understanding of the role of GidA protein during Salmonella infection and the potential use of the gidA mutant in protection against disease outbreaks related to Salmonella and therefore, contribute towards improving health and increase the productivity of domestic livestock and poultry.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Shippy D. C., N. M. Eakley, J. A. Heintz, R. M. Albrecht, and A. A. Fadl. 2011. Deletion of Glucose-Inhibited Division Gene (gidA) Alters the Morphological and Replication Characteristics of Salmonella enterica Serovar Typhimurium. Archives of Microbiology, 194: 405- 412
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Audience:Scientific commnity Effort: General Meeting, American society for microbiology, June 16-19, 2012, San Francisco, CA Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? trained a PhD student Daniel C. Shippy How have the results been disseminated to communities of interest? Data from this project was partly presented at the 112th General Meeting, American society for microbiology, June 16-19, 2012, San Francisco, CA, and will be presented at the Multi-state NC1202 meeting, December 7–8, 2013, Chicago, IL. A manuscript has been published in peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals? Specific aim 2): Determine subcellular localization of GidA and MnmEproteins.
Impacts
What was accomplished under these goals?
1) Examine the relative contribution of GidA and the MnmE in attenuationof Salmonella. 2) initiated last specific aim: "Determine subcellular localization of GidA protein".
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
1. Shippy, D. C., N. M. Eakley, C. T. Lauhon, P. N. Bochsler and A. A. Fadl. 2013. Virulence characteristics of Salmonella following deletion of genes encoding for the tRNA modification enzymes GidA and MnmE. Microbial Pathogenesis. 57: 1-9.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: A finding from this project was partly presented at the Conference of Research Worker in Animal Diseases, 12/05/2011, Chicago, IL and will be presented at the Multi-state NC1202 meeting, 12/01/2012, Chicago, IL. Two manuscripts have been published in peer-reviewed journals. PARTICIPANTS: Amin Fadl: project director, Department of Animal Sciences; Daniel C.Shippy: graduate student, Department of Animal Sciences; Nicholas M. Eakley: collaborator, Department of Animal Sciences; Joseph A. Heintz: Collaborator, Department of Animal Sciences; Ralph M. Albrecht: Collaborator, Department of Animal Sciences. TARGET AUDIENCES: Scientific community PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our previous studies have shown that deletion of glucose-inhibited division gene (gidA) significantly attenuated Salmonella enterica serovar Typhimurium (STM) virulence in both in vitro and in vivo models of infection. Most importantly, immunization with the gidA mutant protected mice from a lethal dose challenge of wild-type STM. In this study, we further characterize the gidA mutant STM strain for potential use in a live-attenuated vaccine. The protective efficacy of immunization with the gidA mutant was evaluated by challenging immunized mice with a lethal dose of wild-type STM. Sera levels of IgG2a and IgG1, passive transfer of sera and cells, and cytokine profiling were performed to study the induction of humoral and cellular immune responses induced by immunization with the gidA mutant strain. Additionally, a lymphocyte proliferation assay was performed to gauge the splenocyte survival in response to treatment with STM cell lysate. Mice immunized with the gidA mutant were fully protected from a lethal dose challenge of wild-type STM. Mice passively immunized with splenocytes or sera from immunized mice were protected from a lethal dose challenge of wild-type STM. The lymphocyte proliferation assay displayed a significant response of splenocytes from immunized mice when compared to splenocytes from non-immunized control mice. Furthermore, the immunized mice displayed significantly higher levels of IgG1 and IgG2a with a marked increase in IgG1. Additionally, immunization with the gidA mutant strain evoked higher levels of IL-2, IFN-g, and IL-10 cytokines in splenocytes induced with STM cell lysate. Together, the results demonstrate that immunization with the gidA mutant strain protects mice by inducing humoral and cellular immune responses with the humoral immune response potentially being the main mechanism of protection.
Publications
- Shippy D. C., N. M. Eakley, J. A. Heintz, R. M. Albrecht, and A. A. Fadl. 2011. Deletion of Glucose-Inhibited Division Gene (gidA) Alters the Morphological and Replication Characteristics of Salmonella enterica Serovar Typhimurium. Archives of Microbiology, 194: 405- 412.
- Shippy, D. C. and A. A. Fadl. 2012. Immunological characterization of a gidA mutant strain of Salmonella for potential use in a live-attenuated vaccine. BMC Microbiology (Accepted for publication)
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Results from this study was presented in a national conference and submitted for publication to a peer-reviewed journal. PARTICIPANTS: Daniel Shippy, Department of Animal Sciences, University of Wisconsin-Madison. Joseph Heintz, Department of Animal Sciences, University of Wisconsin-Madison. Ralph Albrecht, Department of Animal Sciences, University of Wisconsin-Madison. Nicholas Eakley, Department of Animal Sciences, University of Wisconsin-Madison. Amin Fadl, Department of Animal Sciences, University of Wisconsin-Madison. This project provided opportunity for professional development for Daniel Shippy TARGET AUDIENCES: Scientific community PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In this study, we investigated the role of GidA in Salmonella cell division using fluorescence and electron microscopy, transcriptional, and proteomic assays. Scanning electron microscopy data indicated a filamentous morphology with few constrictions in the gidA mutant cells. The filamentation of the gidA mutant cells is most likely due to the defect in chromosome segregation, with little to no sign of septa formation observed using fluorescence and transmission electron microscopy. Furthermore, deletion of gidA altered the expression of many genes and proteins responsible for cell division and chromosome segregation as indicated by global transcriptional profiling and semi-quantitative Western blot analysis. Taken together, our data indicate GidA as a potential regulator of Salmonella cell division genes.
Publications
- No publications reported this period
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