Progress 07/01/09 to 09/30/13
Outputs
Target Audience: Research scientists, student researchers and infectious disease clinicians at the University of Arizona. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Multiple laboratory personnel were mentored during this entire project including a Post-Doctoral research associate (Dr. Scott Wilbur), 2 graduate students (Ms. Jennifer Roxas and Ms. Liliana Rounds) and several undergraduate students (Gresa Sylejmani, Hannah Ledvina, Katheryn Larson, Jessica Franco, Jeaninne Kaufer and Asad Mansoor). Under my mentorship, Ms. Sylejmani received a research award from the U. of Arizona Honors program. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impactful discoveries over the course of this project include the finding that phylogenetic relationships between outbreak strains of enteropathogenic and enterohemorrhagic E. coli may be extremely complex, despite the manifestation of similar disease profiles and parameters. Specifically, we evaluated the contributions of sequence differences in the virulence protein EspZ to pathogenesis in both in vitro and in vivo models of infection. We completed and recently published a study focused on the mechanisms by which these pathogenic E. coli strains regulate the survival of infected cells, and another publication on this topic is nearing completion. We also refined and optimized high-throughput proteomic approaches to analyze intricacies in the regulation of survival pathways in host cells infected with pathogenic E. coli, and identified a number of molecules that represent attractive targets for further investigation. We also constructed and preliminarily tested site-directed mutants in EspZ, to define protein domains important for function. Finally, we commenced an in-depth study assessing the impact of thermal stress on E. coli virulence gene expression, and via our optimized high-throughput proteomics, identified molecules whose expression is altered upon transition to elevated temperature. These studies have thus led us to more refined investigations into E. coli pathogenesis, and have yielded valuable enabling information for current and future project on this topic.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Roxas, J.L., Ryan, K., Vedantam, G and Viswanathan, V.K. Enteropathogenic Escherichia coli dynamically regulates EGFR signaling in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2014 Aug 1;307(3):G374-80
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Activities in this period include the mentoring of a post-doctoral research associate (Dr. Scott Wilbur), 2 graduate students (Ms. Jennifer Roxas and Ms. Liliana Rounds) and several undergraduate students (Gresa Sylejmani, Hannah Ledvina, Katheryn Larson, Jessica Franco, Jeaninne Kaufer and Asad Mansoor). Under my mentorship, Ms. Sylejmani received a research award from the U. of Arizona Honors program. PARTICIPANTS: Scott Wilbur, Ph.D. Post Doctoral Research Associate. Jennifer L. Roxas. Graduate Student, Liliana Rounds, Graduate Student. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: Some important discoveries over the past year include the sequencing and comparison of espZ from a variety of outbreak strains of EHEC. This enabled us to deduce phylogenetic relationships between these strains. We are continuing to evaluate the contributions of these sequence differences to pathogenesis in both in vitro and in vivo models of infection. We have also completed a set of studies evaluating the mechanisms by which pathogenic E. coli regulate the survival of infected cells. Continued analysis of our proteomic data have revealed considerable intricacies in the regulation of survival pathways in host cells. Specifically, our studies showed that FAK phosphorylation is involved in the effector-dependent survival pathways in HeLa cells, but not cultured intestinal epithelial cell lines. Further, we were able to show that EPEC effectors protect against intrinsic, but not extrinsic apoptotic signaling. These studies were recently reported in our Infection and Immunity publication. We are now using high-throughput error-prone PCR to generate random mutants in the effector proteins of pathogenic E. coli to define the specific residues required for virulence. Defining these residues will allow us to pinpoint specific interactions with host cells that are required for pathogenesis and, thereby, point to potential targeted intervention approaches in the future.
Publications
- 1. Roxas JL, Wilbur JS, Zhang X, Martinez G, Viswanathan VK. 2012. The enteropathogenic Escherichia coli secreted protein EspZ inhibits host cell apoptosis. Infection & Immunity. 80(11):3850-7. 2. McQuade RM, Roxas BAP, Viswanathan VK, Vedantam G. 2012. Clostridium difficile clinical isolates exhibit variable susceptibility and proteome alterations upon exposure to mammalian cationic antimicrobial peptides. Anaerobe, Anaerobe, 18(6):614-20. 3. Vedantam G, Clark A, Chu M, McQuade R, Mallozzi M, Viswanathan VK. 2012. Clostridium difficile infection: Toxins and non-toxin virulence factors, and their contributions to disease establishment and host response. Gut Microbes. 2012. 3(2):121-34.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Activities in this period include the mentoring of a post-doctoral research associate (Dr. Scott Wilbur), a graduate student (Ms. Jennifer Roxas) and several undergraduate students (Farhan Anwar, Giovanna Martinez, Gresa Sylejmani and Erik Anderson). Under my mentorship, Mr. Anwar received the Stuart Brotman Summer Research Fellowship from the American Gastroenterological Association, and Ms. Martinez received the Minority Health Disparities Award from the University of Arizona (for Summer 2011). PARTICIPANTS: Scott Wilbur, Ph.D. Post Doctoral Research Associate. Jennifer L. Roxas. Graduate Student TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: Over the past year, we have made some key discoveries on the pathogenesis of Escherichia coli pathotypes, and the way in which it manipulates signaling in host intestinal epithelial cells. Specifically, we have performed proteomic analysis of infected epithelial cells to monitor signaling alterations. Our studies demonstrate the phosphorylation/activation of key signaling molecules in these cells, including those that have been individually/independently identified by other investigators. This validates our approach, and gives us confidence in pursuing some of the novel molecules identified in our screen as putative molecules that play an important role in pathogenesis/disease. We have initiated new studies to investigate some of these key proteins, and will have additional information within the coming year. In addition, we have completed and compiled our studies on host cell survival in the context of infection with pathogenic Escherichia coli. A manuscript has been submitted to Cellular Microbiology outlining our discoveries.
Publications
- Vedantam G, Viswanathan VK. Unlocking the gates to inflammatory bowel disease: the role of Enterococcus faecalis gelatinase. Gastroenterology. 2011. 141(3):795-8.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Activities: Conducting and analyzing experiments: We have made considerable progress in determining the mechanisms by which specific virulence factors in the intestinal pathogens Enterohemorrhagic and Enteropathogenic Escherichia coli contribute to pathogenesis. Teaching & mentoring: In the past year, I have recruited and mentored a post-doctoral research associate, Dr. Scott Wilbur. Dr. Wilbur has been instrumental in deriving new strains of EHEC and EPEC, and initiating animal experiments. In addition, I have trained a graduate (Rebecca McQuade) and two undergraduate students (Kevin Cho & Steriance Tchemy) to conduct laboratory research. Training includes theoretical and practical instructions in conducting experiments, and involvement in laboratory meetings (including presentations). Events: Attended Digestive Diseases Week Conference. PARTICIPANTS: V.K. Viswanathan, Ph.D. - principal investigator, directed the project John Scott Wilbur, Ph.D. - Post doctoral research associate. Helped design several of the experiments. Coordinated preliminary mouse studies, and assisted with the training of undergraduate students. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our studies over the last year revealed two key pieces of information: First, the virulence factor EspZ is essential for these pathogens to cause infection and disease in a rabbit model of infection. Studies done in collaboration with Dr. Edgar Boedeker revealed that rabbit EPEC lacking espZ failed to colonize animals, and did not cause any signs of disease. Secondly, we have been able to show that EspZ interacts with host cell proteins, including those involved in the survival of intestinal epithelial cells. We are currently investigating the contribution of these interactions to pathogenesis in the in vivo situation.
Publications
- Royan SV, Jones RM, Koutsouris A, Roxas JL, Falzari K, Weflen AW, Kim A, Bellmeyer A, Turner JR, Neish AS, Rhee KJ, Viswanathan VK, Hecht GA. Enteropathogenic E. coli non-LEE encoded effectors NleH1 and NleH2 attenuate NF-κB activation. Mol Microbiol. 2010 Dec;78(5):1232-45.
- Viswanathan V.K. Tossed salads. Invited Opinion. Gut Microbes. 2010 Sep;1(5):291-292.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The objective of this study is to examine the effects of the intestinal pathogens enteropathogenic and enterohemorrhagic E. coli on epithelial cells. We have made substantial progress in evaluating the role of the secreted effector proteins EspZ and EspF in the pathogenesis of EPEC and EHEC. The results have been communicated as publications (2 primary articles and 1 review article), and in invited talks at the following venues: Invited lecture, Digestive Diseases Week, Chicago, IL, May 2009; Co-chair, Intestinal absorption and malabsorption, Digestive Diseases Week, Chicago, IL, May 2009; Invited lecture at Microlunch, University of Arizona, May 2009 PARTICIPANTS: Edgar C. Boedeker, M.D., Professor of Medicine(Gastroenterology), University of New Mexico, Albuquerque, NM Gail Hecht, M.D., Professor of Medicine, University of Illinois, Chicago. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
EPEC and EHEC inject effector proteins into intestinal epithelial cells (IECs) via the type 3 secretion system. Secreted effectors can interfere with host cell signaling pathways via specific protein-protein interactions. The secreted protein EspF binds sorting nexin 9 (SNX9), an endocytic regulator, causing tubulation of the plasma membrane. We demonstrated that point mutation of the SNX9 lipid binding domains or truncation of the EspF SNX9 binding domains inhibited tubulation, as did inhibition of clathrin coated pit (CCP) assembly. While EPEC is considered to be non-invasive, invasion has been observed both in vitro and in vivo. Invasion of Caco-2 cells, like tubulation, was blocked by inhibitors of CCPs. Curiously, dynamin inhibitors did not prevent tubulation or bacterial invasion. This data shows that EPEC invasion requires EspF interaction with SNX9. In another set of studies, we examined the role of inflammation in EHEC infection. EHEC causes a range of symptoms from non-bloody diarrhea to more severe outcomes including hemorrhagic colitis, and, sometimes, severe complications such as hemolytic uremic syndrome. The more severe outcomes of EHEC infection are due to the effects of Shiga toxin (Stx) on vasculature. Curiously, while intestinal epithelial cells do not express the Stx receptor Gb3, they translocate Stx. Our studies show that Stx is a potent inducer of inflammation in intestinal epithelial cells. We have also been able to show that EHEC attenuates the cytokine-induced IL-8 production. Moreover, EHEC is able to dampen Stx-dependent inflammatory responses, and this effect requires the type III secretion system. Finally, we have extended our studies on the role of EspZ in pathogenesis. Specifically, we have extended our investigations on the ability of this protein to suppress host cell death and thereby promote colonization. We have collaborated with Dr. Ed. Boedeker at the University of New Mexico and demonstrated that strains lacking EspZ are unable to colonize the rabbit intestine. Current studies are evaluating the precise mechanism by which EspZ supports the virulence of EPEC, EHEC and related pathogens.
Publications
- Weflen AW, Alto NM, Viswanathan VK, Hecht G. 2010. E. coli secreted protein F promotes EPEC invasion of intestinal epithelial cells via an SNX9-dependent mechanism. In press.
- Bellmeyer AA, Cotton C, Kanteti R, Koutsouris A, Viswanathan V.K., Hecht G. 2009. Enterohemorrhagic Escherichia coli suppresses inflammatory response to cytokines and its own toxin. Am J Physiol Gastrointest Liver Physiol. 297(3):G576-81.
- Viswanathan, V.K., K. Hodges and G. Hecht. 2009. Mechanisms of infection-induced diarrhea. Invited Review, Nature Microbiology Reviews 7, 110-119
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