Progress 03/01/10 to 12/31/12
Outputs
OUTPUTS: Escherichia coli O157:H7 is a major foodborne zoonotic pathogen associated with outbreaks worldwide that pose serious public health concerns. Pathogenesis of E. coli O157:H7 depends on multiple virulence factors. Intimin, tir, the type III secretion system (T3SS) effectors, and other adhesion factors mediate E. coli O157:H7 intestinal colonization. Meanwhile, E. coli O157:H7 produces Shiga toxin (Stx), a major virulence factor responsible for bloody diarrhea and life-threatening hemolytic uremic syndrome (HUS). Cyclic di-GMP is a ubiquitous bacterial second messenger that affects multiple biological functions. In this study, we assessed roles of c-di-GMP signaling in mediating E. coli O157:H7 virulence through deletion c-di-GMP specific phosphodiesterase (PDE), YhjH or overexpression of c-di-GMP specific PDE in E. coli O157:H7 to alter intracellular c-di-GMP level. Consistent with c-di-GMP's role in other proteobacteria, overexpression of PDE decreased, while deletion of PDE increased biofilm formation in E. coli O157:H7. Data further showed that c-di-GMP signaling is necessary for proper E. coli O157:H7 colonization to epithelial host cells. Overexpression of c-di-GMP specific PDE reduced E. coli O157:H7 adherence to HT-29 epithelial cells and cattle colon explants, which was at least partially through alteration of multiple adhesion factors. In addition, the deletion of the c-di-GMP specific PDE, YhjH, affected E. coli O157:H7 virulence via multiple pathways. On the one hand, the yhjH deletion mutant was more adhesive to epithelial cells via increased T3SS production. On the other hand, yhjH deletion reduced Stx production and cytotoxicity to host cells. Furthermore, the yhjH deletion mutant was less invasive and/or resilient to macrophages, likely due to its increased susceptibility to oxidative stress. Our results highlight the complex manner in which c-di-GMP signaling affects the virulence of E. coli O157:H7. We further explored roles of polynucleotide phosphorylase (PNPase) in E. coli O157:H7 virulence. Deletion of pnp in E. coli O157:H7 not only impaired its motility, cold adaptation, and adhesion to intestinal epithelial, but also decreased the activation of spontaneous lambdoid prophages and Stx prophage, and dramatically decreased Stx2 mRNA expression and protein production. These data show that PNPase is critical in E. coli O157:H7 pathogenesis, in addition to its contribution to the growth of E. coli O157:H7 at low temperatures. PARTICIPANTS: Mei-Jun Zhu, assistant professor, Animal science; Mark Gomelsky, professor, Molecular biology; Baolin Wang, Research Scientist, Animal science; Warrie J. Means, associate professor, Animal science; Wanfu, Yue, Research Scientist, Animal science; Jia Hu, PhD student, Molecular and Cellular Life Science/Animal science; Harries, Shaun, MS student, Animal science; Sarena Olsen, MS student, Animal science. TARGET AUDIENCES: Scientific community and food producers through presentations and peer-reviewed publications. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Shiga toxin producing E. coli O157:H7 is a major food safety threat that results in significant economic losses, and poses serious public health concerns. Shiga toxin (Stx) is the major virulence factor in E. coli O157:H7, which causes bloody diarrhea and life threatening Hemolytic-uremic syndrome. Fecal shedding of E. coli O157:H7 from beef cattle is the major contamination source of this pathogen and biofilms on facility surfaces in processing plants provide a constant contamination source. Our studies demonstrated that c-di-GMP signaling plays a critical role in E. coli O157:H7 biofilm formation and gut colonization. The current study further showed that PNPase had regulatory roles in E. coli O157:H7 motility, biofilm formation, gut epithelial cell adhesion and its Shiga toxin production, as well as growth at cold temperature. These studies provide insight into methods to reduce virulence and intestinal adhesion, biofilm formation and growth of E. coli O157:H7 in meat during cold storage, improving the safety of beef.
Publications
- Olsen, S., J. Hu, Y. Xue, M. J. Zhu. (2013). Effects of grape seed extract on the growth of top six non-O157 Shiga toxin producing Escherichia coli. 2013 International Association for Food Protection, Charlotte, North Carolina, July 28-31, 2013.
- Hu, J., B. Wang, X. Fang, W. J. Means, R. J. McCormick, M. Gomelsky, and M. J. Zhu. (2013). c-di-GMP signaling regulates E. coli O157:H7 adhesion to colonic epithelium. Veterinary Microbiology, in press.
- Wang, B., W. J. Means, M. Gomelsky, and M. J. Zhu. (2013). C-di-GMP phosphodiesterase YhjH affects virulence of Escherichia coli O157:H7 via multiple pathways. Microbes and Infection, submitted.
- Harries, S. M., W. F. Yue, S. A. Olsen, J. Hu, W. J. Means, R. J. McCormick, M. Du, and M. J. Zhu. (2012). Salt at concentrations relevant to meat processing enhances Shiga toxin 2 production in Escherichia coli O157:H7. International Journal of Food Microbiology, 159:186-192.
- Yue, W. F., M. Du, and M. J. Zhu. (2012). High temperature in combination with UV irradiation enhances horizontal transfer of stx2 gene from E. coli O157:H7 to non-pathogenic E. coli. PLoS ONE, 7: e31308 (1-7)
- Hu, J., W. J. Means, R. J. McCormick, M. J. Zhu. (2013). Polynucleotide phosphorylase is critical for E. coli O157:H7 low temperature growth in milk. Food Microbiology, submitted.
- Hu, J., X, Fang, M. Gomelsky, M. J. Zhu. (2013). Polynucleotide phosphorylase regulates Shiga toxin 2 production in Escherichia coli O157:H7. 113th General Meeting of the American Society for Microbiology, Denver, Colorado, May 18-21, 2013.
- Xue, Y., J. Hu, J., H. Wang, H. Zhang, M. Du, and M. J. Zhu. (2013). Host inflammatory response inhibits Escherichia coli O157:H7 adhesion to gut epithelium through augmenting mucin expression. 113th General Meeting of the American Society for Microbiology, Denver, Colorado, May 18-21, 2013.
- Hu, J., W. J. Means, R. J. McCormick, M. J. Zhu. (2013). Polynucleotide phosphorylase is required for E. coli O157:H7 growth at low temperature. 2013 International Association for Food Protection, Charlotte, North Carolina, July 28-31, 2013.
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Progress 03/01/11 to 02/28/12
Outputs
OUTPUTS: The gastrointestinal tract of beef cattle is the primary reservoir of E. coli O157:H7 and fecal shedding is the main source of cattle-related contamination. Therefore, it is critical to eliminate or reduce E. coli O157:H7 colonization in the gastrointestinal (GI) tract. Up to now, however, mechanisms regulating E. coli O157:H7 gut colonization are poorly defined. We previously showed that c-di-GMP specific phosphodiesterase over-expression in E. coli O157:H7 reduced its adhesion to gut epithelial cells and decreased biofilm formation. To further define underlying mechanisms regulating colonization and virulence, we explored roles of polynucleotide phosphorylase (PNPase) in E. coli O157:H7 gut adherence and associated virulence. Our results indicated that PNPase knockout (deltaPNP) pronouncedly inhibited E. coli O157:H7 motility, and reduced E. coli O157:H7 adhesion to cattle gut epithelial cells and explants. In addition, deletion of PNPase in E. coli O157:H7 dramatically impairs its growth at 10 C as well as its Shiga toxin production, which will provide insights for the development of preventive measures to the meat industry to improve E. coli O157:H7 meat safety. Currently, we are continuing to define the underlying mechanisms linking c-di-GMP and PNPase to their roles in E. coli O157:H7 gut colonization. PARTICIPANTS: Mei-Jun Zhu, assistant professor, Animal science; Mark Gomelsky, professor, Molecular biology; Baolin Wang, research scientist, Animal science; Warri Means, associate professor, Animal science; Wanfu, Yue, research scientist, Animal science; Jia Hu, PhD student, MCLS/Animal science. TARGET AUDIENCES: Scientific community and meat industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
E. coli O157:H7 is the most common bacterial etiologic agent in outbreaks associated with beef products. The GI tract of cattle is the major source of contamination. Once contaminated, it grows in meat during transportation and cold storage. Therefore, it is critical to reduce E. coli O157:H7 gut colonization and its growth in the meat. Our data show that c-di-GMP signaling and PNPase have regulatory roles in E. coli O157:H7 motility, biofilm formation, gut epithelial cell adhesion and its Shiga toxin production, the main virulence factor, production. In addition, we observed that the deletion of PNPase impaired E. coli O157:H7 growth at cold temperature. These studies will impact the beef industry, by providing insight into methods to reduce or eliminate E. coli O157:H7 gut colonization, biofilm formation and its growth in the meat during storage, improving the safety of beef.
Publications
- Hu, J., B.L. Wang, W.F. Yue, X. Fang, W.J. Means, K.W. Christensen, R.J. McCormick, M. Gomelsky, and M. J. Zhu. (2012). Cyclic-di-GMP specific phosphodiesterase regulates E. coli O157:H7 colonization. Food Microbiology, In preparation.
- Harries, S.M., W.F. Yue, S.A. Olsen, J. Hu, W.J. Means, R.J. McCormick, M. Du, and M. J. Zhu. (2012). Salt at concentrations relevant to meat processing enhances Shiga toxin 2 production in Escherichia coli O157:H7. International Journal of Food Microbiology, Accepted.
- Yue, W.F., M. Du, and M. J. Zhu. (2012). High temperature in combination with UV irradiation enhances horizontal transfer of stx2 gene from E. coli O157:H7 to non-pathogenic E. coli. PLoS ONE,7(2): e31308.
- Hu, J., B.L. Wang, W.F. Yue, X. Fang, W.J. Means, K.W. Christensen, R.J. McCormick, M. Gomelsky, M. J. Zhu. (2012). Role of cyclic di-GMP-specific phosphodiesterase in E. coli O157:H7 gut colonization. 112th General Meeting of the American Society for Microbiology, San Francisco, California, June 16-19, 2012
- Wang, B.L., X. Fang, W.J. Means, M. Gomelsky, M. J. Zhu. (2012). The EAL domain protein YdiV regulates motility, biofilms and virulence in enterohemorrhagic Escherichia coli. 112th General Meeting of the American Society for Microbiology, San Francisco, California, June 16-19, 2012
- Olsen, S.A., J. Hu, W.J. Means, R.J. McCormick, M. J. Zhu. (2012). Effects of grape seed extract on the growth and Shiga toxin production of Escherichia coli O157:H7. 112th General Meeting of the American Society for Microbiology, San Francisco, California, June 16-19, 2012
- Yue, W., M. Du, W. J. Warrie, and M. J. Zhu. (2011), Horizontal stx2 gene transfer from E. coli O157:H7 to non-pathogenic E. coli occurred in feedlot conditions. 2011 ADSA-CSAS-ASAS Joint Annual Meeting, New Orleans, Louisiana, July 10-14.
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Progress 03/01/10 to 02/28/11
Outputs
OUTPUTS: E. coli O157:H7 is a major food safety threat that results in huge losses, especially in the beef industry. Cyclic dimeric guanosine monophosphate, c-di-GMP, is a bacterial second messenger that regulates motility, biofilm formation and virulence in various proteobacteria. To test the role of c-di-GMP in E. coli O157:H7 virulence, we deleted the yhjH gene, which encodes one of the potent c-di-GMP phosphodiesterases in E. coli. We found that disruption of yhjH decreased motility but had no effect on biofilm formation. qRT-PCR analysis indicated that deletion of yhjH decreased (p < 0.05) expression of Shiga toxin I (stx1) and Shiga toxin II (stx2A and 2B) genes. However, expression of genes involved in protein translocation and host cell adhesion, i.e. intimin (eae), EspA (espA) and EspB (espB), increased (p < 0.05). Deletion of yhjH had no effect on hemolysin A (hlyA) and translocated intimin receptor (tir) expression. C-di-GMP is synthesized by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We also electroporated wild type E. coli O157: H7 with an empty vector, or a vector overexpressing a heterologous DGC (slr1143) or PDE (blrP1). Intracellular c-di-GMP levels were analyzed using LC-MS/MS. Swimming motility was assayed using 0.3% agar plates. Biofilm formation was tested using a crystal violet stain. Shiga toxin II mRNA and protein content were measured by qRT-PCR and Western blotting. The virulence was assayed using Vero cells. Our data show that overexpression of a DGC dramatically increased, while overexpression of a PDE decreased intracellular c-di-GMP levels. In agreement with the altered c-di-GMP levels, biofilm formation was increased by the DGC expression and drastically reduced by the PDE expression. Motility of E. coli O157:H7 overexpressing a DGC was also reduced, whereas it was unaffected in E. coli O157:H7 overexpressing PDE. qRT-PCR and Western blotting analyses indicated that overexpressed PDE, but not DGC, greatly increased both mRNA and protein contents of Shiga toxin II. Consistently, the strain overexpressing PDE was more virulent than the wild type or the strain overexpressing DGC. In summary, our data show that c-di-GMP regulates motility, biofilm formation and virulence of E. coli O157:H7. PARTICIPANTS: Mei-Jun Zhu, assistant professor, animal science; Mark Gomelsky, professor, molecular biology; Warrie J. Means, associate professor, animal science; Min Du, associate professor, animal science; Baolin Wang, laboratory technician, animal science; Jia Hu, PhD student, MCLS, animal science. TARGET AUDIENCES: Scientific community through presentations and peer-reviewed publications. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
E. coli O157:H7 induces huge losses to the meat industry and is a threat to consumer safety. Further, the runoffs from cattle farms can contaminate vegetables. Since fecal shedding of E. coli O157:H7 from beef is the major source of this pathogen, it is important to reduce E. coli O157:H7 shedding in beef cattle, which depends on the reduction in E. coli O157:H7 gut colonization. On the other hand, biofilms on facility surfaces in processing plants provide a constant source of pathogens, including E. coli O157:H7. However, up to now, our knowledge about the mechanisms regulating biofilm formation and gut colonization is very limited. The overall objective of this proposal is to evaluate the role of c-di-GMP in E. coli O157:H7 biofilm formation and gut colonization. These studies will impact the beef industry, by providing insight into methods to reduce or eliminate E. coli O157:H7 biofilm formation, as well as its colonization in the gastrointestinal tract of beef cattle, improving the safety of beef.
Publications
- Zhu, M. J. 2011. Local inflammation, Escherichia coli (E. coli) O157: H7 colonization and shedding in beef cattle. U S Meat Animal Research Center, Clay Center, NA, Feb 25, 2011.
- Hu, J., B.L. Wang, X. Fang, W.J. Means, R.J. McCormick, M. Du, M. Gomelsky, M.J. Zhu. 2011. Overexpression of a cyclic di-GMP-specific phosphodiesterase increases Shiga toxin II production and virulence of E. coli O157:H7. 111th General Meeting of the American Society for Microbiology, New Orleans, Louisiana, May 21-24, 2011
- Zhu, M.J., B.L. Wang, W. Yue, V.K. Koseoglu, H. Wang, X. Fang, W.J. Means, R.J. McCormick, and M. Gomelsky. 2010. C-di-GMP signaling pathways are critical for acid resistance of E. coli O157:H7. 2010 ADSA-CSAS-ASAS Joint Annual Meeting, Denver, CO, July 11-15.
- Wang,B.L., X. Fang, M. Gomelsky, M. Du, W.J. Means, R.J. McCormick, M.J. Zhu. 2010. A cyclic di-GMP-specific phosphodiesterase (PDE) controls virulence gene expression of E. coli O157:H7. 110th Genearl Meeting of the American Society for Microbiology. San Diego, California, May 23-27, 2010
- Koseoglu,V.K., B.L. Wang, N. Ertas, M. Gomelsky, M. Du, W.J. Means, R.J. McCormick1, M.J. Zhu. 2010. Identification of genes expressed by E. coli O157:H7 during bovine gut colonization. 110th General Meeting of the American Society for Microbiology. San Diego, California, May 23-27, 2010
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