Progress 10/01/06 to 09/30/10
Outputs
OUTPUTS: Over the timeline of this project, the goals and objectives were presented on a number of occasions both locally and nationally: (i) To the UNH community through seminars delivered each November and December from 2006 through 2010 and in a final Ph.D. thesis defense in May 2010 (a second Ph.D. defense is anticipated in early spring). (ii) Nationally and internationally as two posters at American Society for Microbiology meetings in Philadelphia in May 2009. (iii) Both Ph.D. candidates on this project (Kate Stefani and Lia Jeffery) have gone on to accept positions with Rapid Micro Bio Systems and with Millipore Corp in Massachusetts. (iv) 25 undergraduates were mentored and trained in research ethics and techniques by conducting research on this project. Of these, most have gone on to accept Ph.D., M.P.H. and M.S. fellowships, many have entered Medical School or Physician Assistant programs, while others have accepted research positions in hospitals and biotech companies. (v) 3 manuscripts are in preparation from this work. (vi) 4 posters were presented at the UNH Undergraduate Research Conference on this work in April 2008 and 2009. (vii)Aspects of this research have been incorporated into laboratory exercises in two major UNH courses (MICR 501 and MICR 602). MICR 501 had 140 nursing and nutrition majors while MICR 602 had 60 microbiology, pre-med and medical laboratory science majors per year. All of these students were exposed in detail to information that emerged from the research conducted during this project. PARTICIPANTS: Dr. Frank G. Rodgers, PI; Kate Stefani, Ph.D. Candidate; Lia Jeffery, Ph.D. candidate; (all 3 worked directly on this project); the following undergraduates: Robert Citorik, Jong Yu, Carey Gallini, Erica Sennott, Joshua Collins, Amanda Ramos, Kate Hutchins, Megan Macia, Alexandra Manakos, Fredrick Ta, Afanine Ali, Sarah Bottiglieri, Jennifer Brackett, Christopher Lewis, Robert Tucker, Eric Canzanello, Meredith Cormack, Jennifer Forcina, Emily Godbut, Amy Levesque, Laura Marden, Jennifer Nieves, Jessica Alicea, Saadia Aziz, and Tara Bordalo worked on diverse aspects of this project. TARGET AUDIENCES: Because of the nature of the pathogen investigated, this work targets health care personnel, food industry specialists, veterinarians, molecular biologists, infectious disease professionals and those interested in developing appropriate and safe measures for the production and distribution of food stuffs from "farm gate to dinner plate". The project also serves to increase the knowledge and awareness of the general public about Escherichia coli O157:H7 and the diseases it causes. PROJECT MODIFICATIONS: The generation of a mutant toxin by the pathogen was deemed more likely to generate meaningful data about the binding aspects of stx-2 to neutrophils and therefore shed greater light on the role the toxin plays in arresting neutrophil apoptosis thus leading to greater disease.
Impacts
Enterohemorrhagic Escherichia coli O157:H7 (EHEC) carries the lambda-like phage, 993W. This phage carries potent Shiga-toxins (Stx). In infected individuals the presence of Stx often leads to hemolytic uremic syndrome (HUS), a severe complication of hemorrhagic colitis (HC). Approximately 5 percent of cases with HC progress to HUS, especially in the young and those with impaired immune function. The disease is characterized by renal failure, blood cell damage and system-wide organ shutdown. This condition is often fatal in children under 5 years old. The CDC reports approximately 73,000 cases of food borne illness and over 60 deaths in the US attributed to this pathogen annually. Several major outbreaks of EHEC have occurred in recent years from contaminated foods including unpasteurized apple juice, spinach, lettuce and most commonly ground beef. The production of Stx by the pathogen is a major virulence factor that is responsible for severe disease. These toxins enter cells and shut down host-cell protein synthesis. Toxins pass in the blood stream to the kidneys, brain, and other organs, causing severe damage and result in major organ failure and system collapse. Our research focused on Stx-2, the most virulent toxin produced by EHEC. Our goal was to investigate three Stx-inducing conditions: (i) exposure of the pathogen to antibiotics (antibiotic treatment of HUS patients often makes the condition worse), (ii) impacts of probiotics (do probiotics influence disease outcome by influencing toxin production) and (iii) the type of carbon source used to grow the pathogen (has the switch from grass to corn fed cattle changed the pattern of toxin production). A second facet of this research was to investigate the interactions of Stx with the pro-apoptotic (Bcl-2, Bcl-XL, Bcl-W, Mcl-1, A1) and anti-apoptotic (Bax and Bak) proteins using human neutrophils as a model. Data on toxin production generated by cytotoxicity and ELISA assays indicated that DNA-damaging antibiotics increased Stx-2 production by triggering the DNA SOS response while other antibiotics did not. Likewise, co-cultivation with lactobacilli significantly decreased toxin production. In addition, growth of the organisms in glucose but not glycerol stimulated the production of Stx-2. Having established the techniques for detecting Bcl-2 proteins, Western blots were performed with Caco-2 (human colon carcinoma) and human neutrophil cell lysates to determine which Bcl-2 proteins were present. A mutated Stx-2 with a modified binding site for apoptotic proteins was developed using PCR -SOE. This was produced to determine whether Stx-2 binds to the Bax protein within neutrophils and so inhibits apoptosis, thus offering an explanation as to why Stx is carried in neutrophils to the kidneys and other organs leading to extensive disease.
Publications
- Stefani, K (2010). Factors regulating the production of Stx-2 in Escherichia coli O157:H7. Ph.D. Thesis University of New Hampshire
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: The goals and objectives of the project were presented on a total of six occasions to the UNH community and nationally and internationally at ASM through seminars and posters delivered in April and May 2009. Two departmental seminars were given and four posters were presented; two at the Undergraduate Research Conference and two at the American Society for Microbiology. See "Publications" for details. In addition, 4 undergraduates: (i) Erica Sennott, (ii) Amanda Ramos, (iii) Kate Hutchins, and (iv) Fredrick Ta continued their mentoring and training in research ethics and techniques by conducting research on this project during this reporting period. These students have gone on to accept positions at a medical school, a graduate school, a biotech company, and at a hospital. Aspects of this research were incorporated into selected laboratory exercises conducted in two major UNH courses (Micr 501 and Micr 602). Micr 501 had 140 nursing and nutrition majors while Micr 602 had 68 microbiology, pre-med and medical laboratory science majors. All of these students were exposed in details and information that emerged from the research conducted on this project. PARTICIPANTS: Dr. Frank G. Rodgers, PI; Kate Stefani, Ph.D. candidate; Lia Jeffrey (nee Nalewak), Ph.D. candidate; (all 3 working directly on this project); Erica Sennott (Graduated 2009), Amanda Ramos (Graduated 2009), Kate Hutchins (Graduated 2009), and Fredrick Ta (Graduated 2009). TARGET AUDIENCES: Because of the nature of the pathogen investigated, this works targets health care personnel, food industry specialists, veterinarians, molecular biologists, infectious disease professionals and those interested in developing appropriate and safe measures for the production and distribution of food stuffs from "farm gate to dinner plate". This project also serves to increase the knowledge and awareness of the general public about Escherichia coli O157:H7 and the diseases it causes. PROJECT MODIFICATIONS: The examination of defining the nature of cell death during different points in the host cell cycle was eliminated and replaced with studies on the interaction of the toxin with human neutrophils; this latter was considered a more appropriate method to define a model pathway for the molecular mechanism of Stx-triggered apoptosis and was more likely to generate meaningful and assessable results on this phenomenon.
Impacts
Enterohemorrhagic E. coli O157:H7 (EHEC) may cause hemolytic uremic syndrome (HUS), a severe complication of hemorrhagic colitis (HC). Approximately 5 percent of cases with HC progress to HUS, especially in the young and those with impaired immune function. The condition is characterized by renal failure, blood cell damage and system-wide organ shutdown. In children under five years old this condition often proves fatal. The CDC reports approximately 73,000 cases of food borne illness and over 60 deaths attributed to this pathogen annually in the US. Several spectacular outbreaks of EHEC have occurred in recent years from contaminated foods including unpasteurized apple juice, spinach, and most commonly ground beef. The pathogen produces Shiga-toxins (Stx) that are major virulence factors responsible for disease. These enter cells and shut down host-cell protein synthesis. Likewise, toxins pass in the blood stream to the kidneys, brain, and other organs, causing major organ failure and system collapse. Our research focuses on Stx-2, the most virulent toxin produced by EHEC. Our first goal was to investigate three Stx-inducing conditions: (i) exposure of the pathogen to antibiotics (treatment of HUS patients with apparently appropriate antibiotic therapy often makes the condition worse), (ii) impacts of probiotics (do probiotics influence disease outcome by influencing toxin production) and (iii) the type of carbon source used to grow the pathogen (has the switch from grass to corn fed cattle changed the pattern of toxin production and hence disease). Data on toxin production generated by cytotoxicity and ELISA assays indicated that DNA-damaging antibiotics increased Stx-2 production by triggering the DNA SOS response while other antibiotics did not. Likewise, co-cultivation with lactobacilli significantly decreased toxin production. It was also determined that Stx-2 output rose in the presence of increased glucose while varying concentrations of glycerol had no effect on Stx-2 production. Increasing concentrations of cAMP decreased the amount of Stx-2 produced. These studies suggest that glucose but not glycerol stimulated Stx-2 expression in E. coli O157:H7 strains that carried the lambda-like phage 933-W. Our second goal was to investigate the interactions of Stx with the pro-apoptotic (Bcl-2, Bcl-XL, Bcl-W, Mcl-1, A1) and anti-apoptotic proteins (Bax and Bak) using human neutrophils as a model. The effect of Stx-2 on neutrophil apoptosis and necrosis was assessed, and the data on apoptosis rates showed that there was a significant difference in neutrophil apoptosis rates (p<0.05) up to and including 12 hours after the neutrophils were exposed to Stx-2. These data suggest that Stx-2 could delay neutrophil apoptosis by binding to pro-apoptotic Bcl-2 group proteins within the cell. To evaluate whether the delay in apoptosis is due to the interaction of Stx-2 with Bax within neutrophils the generation of a mutated Stx-2 toxin was initiated and this work is ongoing.
Publications
- 1. Jeffrey, L. K., Ramos, A., Hutchins, K.M., Stefani, K.P. and Rodgers, F.G. 2009. Effects of Escherichia coli O157:H7 Stx-2 on Human Neutrophil Apoptosis. Proceeding of the 109th ASM General Meeting, Philadelphia, PA., p 115: May 19, 2009.
- 2. Stefani, K.P., Sennott, E., Jeffrey, L.K. and Rodgers, F.G. 2009. Impacts of glucose, glycerol and cAMP on the Regulation of Stx-2 in Escherichia coli O157:H7. Proceedings of the 109th ASM General Meeting Philadelphia, PA., p 115: May 19, 2009.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The goals and objectives of the project were presented on two separate occasions to the UNH community through seminars delivered in November and in December 2007. In addition, during this reporting period 9 undergraduates: (i) Robert Citorik, (ii) Jong Yu, (iii) Carey Gallini, (iv) Erica Sennott, (v) Josh Collins, (vi) Amanda Ramos, (vii) Kate Hutchins, (viii) Megan Macia and (ix) Fredrick Ta were all mentored and trained in research ethics and techniques by conducting research on this project. Of these, a number has gone on the accept positions at Harvard, UNH, Mass General Hospital and at a biotech company. Aspects of this research were incorporated into selected laboratory exercises conducted in two major UNH courses (Micr 501 and Micr 602). Micr 501 had 140 nursing and nutrition majors while Micr 602 had 60 microbiology, pre-med and medical laboratory science majors. All of these students were exposed in some detail to information that emerged from the research conducted on this project. PARTICIPANTS: Dr. Frank G. Rodgers, PI Kate Stefani, Ph.D. candidate Lia Jeffery (nee Nalewak), Ph.D. candidate Robert Citorik, (Graduated 2008) Jong Yu (Graduated 2008) Carey Gallini (Graduated 2008) Megan Macia (Graduated 2008) Erica Sennott Josh Collins Amanda Ramos Kate Hutchins Fredrick Ta TARGET AUDIENCES: Because of the nature of the pathogen investigated, this works targets health care professionals and serves to increase the knowledge and awareness of the general public about Escherichia coli O157:H7 and the diseases it causes. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Enterohemorrhagic E. coli O157:H7 (EHEC) is a cause of hemolytic uremic syndrome (HUS), a severe complication of hemorrhagic colitis (HC). Approximately 5 percent of cases with HC progress to HUS. The condition is characterized by renal failure, blood cell damage and system-wide organ shutdown. In children under five years old this condition is often fatal. The CDC reports that in the US there are approximately 73,000 reported cases of food borne illness and over 60 deaths attributed to this pathogen annually. Several spectacular outbreaks of EHEC have occurred in recent years from contaminated foods including spinach, lettuce and ground beef. The pathogen produces Shiga-toxins (Stx) that are major virulence factors responsible for disease by entering cells and shutting down protein synthesis. Likewise, toxins pass in the blood stream to the kidneys, brain, and other organs, causing major organ failure and system collapse. Our research focuses on Stx-2, the most virulent toxin produced by EHEC. The goal was to investigate three Stx-inducing conditions: (i) exposure of the pathogen to antibiotics (treatment of HUS patients with antibiotics often makes the condition worse), (ii) impacts of probiotics (do probiotics influence disease outcome) and (iii) the type of carbon source used to grow the pathogen (has the switch from grass to corn fed cattle changed the pattern of disease). Data on toxin production generated by MTT cytotoxicity and ELISA assays indicated that DNA-damaging antibiotics increased Stx-2 production by triggering the DNA SOS response while other antibiotics did not. Likewise, co-cultivation with lactobacilli decreased toxin production. Investigations are in progress to determine the precise cause of these changes. A second facet of this research was to create a model for the interactions of Stx with the pro-apoptotic (Bcl-2, Bcl-XL, Bcl-W, Mcl-1, A1) and anti-apoptotic proteins (Bax and Bak) using human neutrophils. Having established the techniques for detecting Bcl-2 proteins, Western blots were performed with Caco-2 (human colon carcinoma) and human neutrophils cell lysates to determine which Bcl-2 proteins were present. A mutated Stx-2 with a modified binding site for apoptotic proteins is in development. This will determine whether Stx-2 binds to the Bax protein within neutrophils and so inhibits apoptosis, if so this may explain why Stx is carried in neutrophils to the kidneys and other organs.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: 1. Presentations: (i) Departmental Seminar: "Interactions of Escherichia coli O157:H7 Shiga-like Toxins Stx-1 & Stx-2, and the Bcl-2 Family of Proteins" November 14, 2006. (ii) Departmental Seminar: "Stx-2 Production in Escherichia coli O157:H7" November 21, 2006. (iii) Doctoral Committee Seminar: "Interactions of Escherichia coli O157:H7 Shiga-like Toxins Stx-1 & Stx-2, and the Bcl-2 Family of Proteins" May 17, 2007. (iv) Doctoral Committee Seminar: "Factors Regulating Stx-2 Production in Escherichia coli O157:H7" July, 24, 2007. 2. Publications: Two Posters (see details in "Publications") 3. Graduated Students: (i) Sarah Bottiglieri, B.S., Biology (i) Jennifer Forcina, B.S., Medical Laboratory Science (ii) Jennifer Brackett, B.S., Microbiology (iii) Meghan Macia, B.S., Microbiology (iv) Christopher Lewis, B.S., Biology 4. Courses: (i) Pathogenic Microbiology (Micr 602); 58 students (ii) Microbes in Human Disease (Micr 501); 140 students (iii) Germs 101 (Micr 407); 401
students
PARTICIPANTS: Individuals: Frank G. Rodgers Principal Investigator. Directed all aspects of this project. Partner organizations, collaborators, and contacts: None. Training or professional development: Graduate students: (i) Kate Stefani, responsible for day-to-day functions of facet 1 and (ii) Lia Nalewak responsible for day-to-day functions of facet 2. Undergraduate students: (i) Sarah Bottiglieri, (ii) Robert Citorik, (iii) Jong Yu, (iv) Carey Gallini, (v) Meghan Macia, (vi) Jennifer Brackett, (vii) Jennifer Forcina, (viii) Joshua Collins, (ix) Erica Sennott, (x) Ammanda Hurley, (xi) Kate Hutchins, (xii)Alex Manachos, (xiii) Christopher Lewis, (xiv) Afanine Ali. These students worked on different aspects of the project over the year under the direct supervision of the PI and the two graduate students.
TARGET AUDIENCES: Two graduate and fourteen undergraduate students trained through this project. Techniques developed and resulting data from this project were introduced into the formal classes (i) Micro 602 Pathogenic Microbiology (80 students), (ii) Micro 501 Microbes in Human Disease (140 students) and (iii) Micro 407 Germs 101 (854 students).
PROJECT MODIFICATIONS: None
Impacts
Enterohemorrhagic E. coli O157:H7 (EHEC) is the cause of "hamburger disease", or hemorrhagic colitis (HC). Complications beyond gastrointestinal disturbances are reported in approximately five percent of cases and often include hemolytic uremic syndrome (HUS). HUS is characterized by renal failure and system-wide organ shutdown. In children under five years old this syndrome is often fatal. The CDC reports that in the US there are approximately 73,000 reported cases of food borne illness and over 60 deaths attributed to this pathogen annually. Several outbreaks of EHEC have occurred in the last year from contaminated foods including spinach, lettuce and ground beef. Shiga-toxins (Stx) are the major virulence factors responsible for disease. These have the ability to enter cells and shut down protein synthesis. Likewise, the toxins pass in the blood stream to the kidneys, brain, and other organs, causing major organ failure and system collapse. We focus on Stx-2, the
most virulent toxin produced by EHEC. Three toxin-inducing conditions are investigated: (i) exposure of the pathogen to antibiotics (treatment of HUS patients with antibiotics often makes the condition worse), (ii) impacts of probiotics (do probiotics affect disease outcome) and (iii) the type of carbon source used to grow the pathogen (has the switch from grass to corn fed cattle changed the pattern of disease). Our goal was to assess the action of these factors on the production of Stx-2 by EHEC. Preliminary research included growing the pathogen under conditions (i) to (iii) above and quantifying the resultant toxin. Toxin levels measured by MTT cytotoxicity assay have indicated that our hypotheses were correct. DNA-affecting antibiotics increased Stx-2 production, presumably by triggering the SOS DNA repair systems. The presence of lactobacilli decreased toxin production when grown in co-culture with EHEC. Investigations are in progress to determine the precise cause of these
changes. Optimization of more sensitive assays (ELISA and Real Time RT-PCR) will provide quantitative results of both Stx-2 release and stx2 gene transcription. A second facet of this research was to ascertain binding affinities of intracellular Stx with the pro-apoptotic (Bcl-2, Bcl-XL, Bcl-W, Mcl-1, A1) and anti-apoptotic proteins (Bax and Bak) to create a model pathway for the molecular mechanism of Stx-triggered apoptosis. Western blots were performed with Caco-2 (human colon carcinoma) cell lysates to determine which Bcl-2 proteins were present. Far-western blots were performed by probing membranes containing Caco-2 lysates with the A-subunit (protein synthesis inhibiting component) of Stx-2 to define direct binding between the Stx-2 and Caco-2 cell lysates. In addition, co-immunoprecipatation assays have been performed to demonstrate binding of the toxin to the Bcl-2 proteins within Caco-2 cells. A cell binding approach is currently in progress to determine whether Stx-2 binds
to the Bax protein within neutrophils and so inhibits apoptosis, if so this may explain why Stx is carried in neutrophils to the kidneys and other organs.
Publications
- Forcina, J., Nalewak, L., Rodgers, F., 2007. "Interactions Between the Bcl-2 Family of Proteins and Stx-2 Produced by Escherichia coli O157:H7 in Neutrophils" 16th COLSA Undergraduate Research Conference Proceedings p 19: Poster presentation April 28, 2007.
- Citorick, R., Yu, J., Stefani, K., Rodgers, F., 2007. "Effects of Probiotics and Organic Acids on Toxin Production in Escherichia coli O157:H7" 16th COLSA Undergraduate Research Conference Proceedings p 12: Poster presentation April 28, 2007.
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