Progress 10/01/04 to 09/30/07
Outputs
OUTPUTS: The major finding of this project was that the leukotoxin of the bovine respiratory pathogen Mannheimia haemolytica does not act solely by creating lesions on the surface of leukocytes. Instead, the leukotoxin triggers changes in these cells that compromises their function and ultimately leads to their death. These findings could be used to develop methods that intervene in the intracellular program that is triggered by the toxin, thus reducing damage and improving the host defense function of bovine leukocytes. These results have been disseminated to the broader animal health community through participation in the USDA NC1027 Regional Committee on Bovine Respiratory Disease, through scientific publications, through seminars at other universities, and attendance at national and international scientific meetings (e.g. Conference of Research Workers in Animal Disease, USDA NRI Project Directors Meetings, International Pasteurellaceae Meeting).
PARTICIPANTS: Accomplishing the goals of this project was facilitated by our participation in USDA NC1027 Regional Committee on Bovine Respiratory Disease. Members of this group have provided insights, bacterial strains and reagents that have assisted us in our Mannheimia research. Collaborators who made particular contributions to the project include Dr. Sri Srikumaran at Washington State, Sam Maheswaran at Minnesota, Bob Briggs at NADC, and Sarah Highlander at Baylor. The novel observations made in this study largely reflect the work of two excellent PhD students. The first was Dr. Fabio Leite, who is now an Assistant Professor at Universidade de Pelotas, Brazil. The second is Dr. Dhammika Atapattu, currently employed as a Scientist at BioSentinel, Madison, WI. Both of these students benefited greatly from the outstanding environment for training graduate students that exists at UW-Madison, and the important contributions of faculty who served on their thesis committees.
TARGET AUDIENCES: The target audience for the information gathered in this study is other investigators of the pathogenesis of bovine respiratory disease. Our participation in the USDA NC-1027 Regional Committee on Bovine Respiratory Disease also makes our work available to those who deal with the problem of bovine respiratory diseases in the field. The meetings of NC-1027 have been held in conjunction with the American Association of Bovine Practitioners meeting the last 2 years. These meetings included open forums with members of the veterinary vaccine industry, feedlot operators, and veterinarians who serve cattle producers. The information we provide will help thse stakeholders gain a better understanding of the underlying principles and mechanisms that result in bovine respiratory disease, and may illuminate novel strategies to prevent or reduce its negative effects.
PROJECT MODIFICATIONS: There were no major changes in the award or project. We made substantial progress towards the goals of the project.
Impacts
Bovine respiratory disease (BRD) is a multi-factorial disease complex reflecting stress, management factors, and both viral and bacterial infectious agents. The principal bacterial agent is Mannheimia (Pasteurella) haemolytica, which can cause a severe fibrinous pleuropneumonia. The most important virulence determinant of M. haemolytica is its leukotoxin (LKT), which is a 104 kD protein that is secreted during logarithmic-phase growth. The LKT is part of a large family of related exotoxins (RTX toxins) produced by a variety of gram negative bacteria. The M. haemolytica LKT, and other members of the RTX family of bacterial exotoxins, bind to the beta2 integrin CD11a/CD18 (i.e. LFA-1), or to CD18 alone, on the surface of leukocytes. There is increasing evidence that primed beta2 integrins transmit a variety of intracellular signals that can lead to cell activation, proliferation, differentiation, or death. These observations pose fascinating questions regarding how LKT
binding to beta2 integrins sets into motion the intracellular events that result in leukocyte activation or death. We found that incubation of the BL-3 bovine lymphoblastoid cell line with small amounts of LKT led to upregulation of pro-apoptotic proteins, and down regulation of anti-apoptotic proteins, that contributed to increased caspase-9 activity in the cells and . We also observe increased mitochondrial permeability, cytochrome c release, and morphological evidence of disruption of the outer membrane in LKT treated mitochondria. More recent evidence suggests that the LKT is internalized in a dynamin-dependent pathway and transported via the cytoskeleton to the mitochondria. Inhibition of the action of dynamin, or of F-actin polymerization, prevents the internalization of the LKT and its ability to cause apoptosis in BL-3 cells. An lktc mutant toxin protein Is not internalized to mitochondria, nor does it cause apoptosis in BL-3 cells. Accomplishing the goals of this research
helps us better understand the pathogenesis of pneumonia caused by Mannheimia haemolytica, and could identify new strategies to prevent or ameliorate the losses it causes.
Publications
- Leite, F., C. Kuckleburg, D. Atapattu, R.D. Schultz, and C.J. Czuprynski. 2004. BHV-1 infection and inflammatory cytokines amplify the interaction of Mannheimia haemolytica leukotoxin with bovine leukocytes in vitro. Vet. Immunol. Immunopathol. 99:193-202.
- Leite, F., D. Atapattu, C. Kuckleburg, R. Schultz, and C.J. Czuprynski. 2004. Incubation of bovine PMNs with conditioned medium from BHV-1 infected peripheral blood mononuclear cells increases their susceptibility to Mannheimia haemolytica leukotoxin. Vet. Immunol. Immunopathol. 103:187-193.
- Czuprynski, C.J. and F. Leite, M. Sylte, C. Kuckleburg, R. Schultz, T. Inzana, E. Behling-Kelly and L. Corbeil. 2004. Complexities of the pathogenesis of Mannheimia haemolytica and Haemophilus somnus infections: challenges and potential opportunities for prevention? Anim. Health Res. Rev. 5:277-282.
- Atapattu, D.N. 2006. Cellular mechanisms of Mannheimia haemolytica leukotoxin induced bovine lymphoblastoid cell (BL-3) death. Ph.D. thesis, University of Wisconsin-Madison.
- Atapattu, D. and C.J. Czuprynski. 2005. Mannheimia haemolytica leukotoxin induces apoptosis of bovine lymphoblastoid cells (BL-3) via a caspase-9 dependent mitochondrial pathway. Infect. Immun. 73:5504-5513.
- Atapattu,. D., and C J. Czuprynski. 2007. Mannheimia haemolytica leukotoxin binds to lipid rafts in bovine lymphoblastoid cells (BL-3) and is internalized in a dynamin-2 and clathrin-dependent manner. Infect. Immun. 75:4719-4725.
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Progress 01/01/06 to 12/31/06
Outputs
Bovine respiratory disease (BRD) is a multi-factorial disease complex reflecting stress, management factors, and both viral and bacterial infectious agents. The principal bacterial agent is Mannheimia (Pasteurella) haemolytica, which can cause a severe fibrinous pleuropneumonia. The most important virulence determinant of M. haemolytica is its leukotoxin (LKT), which is a 104 kD protein that is secreted during logarithmic-phase growth. The LKT is part of a large family of related exotoxins (RTX toxins) produced by a variety of gram negative bacteria. Recent reports suggest that the M. haemolytica LKT, and other members of the RTX family of bacterial exotoxins, bind to the beta2 integrin CD11a/CD18 (i.e. LFA-1), or to CD18 alone, on the surface of leukocytes. There is increasing evidence that primed beta2 integrins transmit a variety of intracellular signals that can lead to cell activation, proliferation, differentiation, or death. These observations pose fascinating
questions regarding how LKT binding to beta2 integrins sets into motion the intracellular events that result in leukocyte activation or death. We find that incubation of the BL-3 bovine lymphoblastoid cell line with small amounts of LKT leads to upregulation of pro-apoptotic proteins, and down regulation of anti-apoptotic proteins, that contribute to increased caspase-9 activity in the cells. We also observe increased mitochondrial permeability, cytochrome c release, and morphological evidence of disruption of the outer membrane in LKT treated mitochondria. More recent evidence suggests that the LKT is internalized in a dynamin-dependent pathway and transported via the cytoskeleton to the mitochondria. Inhibition of the action of dynamin, or of F-actin polymerization, prevents the internalization of the LKT and its ability to cause apoptosis in BL-3 cells. An lktc mutant toxin protein Is not internalized to mitochondria, nor does it cause apoptosis in BL-3 cells.
Impacts
Accomplishing the goals of this research will help us better understand the pathogenesis of pneumonia caused by Mannheimia haemolytica, and could identify new strategies to prevent or ameliorate the losses it causes.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Bovine respiratory disease (BRD) is a multi-factorial disease complex reflecting stress, management factors, and both viral and bacterial infectious agents. The principal bacterial agent is Mannheimia (Pasteurella) haemolytica, which can cause a severe fibrinous pleuropneumonia. The most important virulence determinant of M. haemolytica is its leukotoxin (LKT), which is a 104 kD protein that is secreted during logarithmic-phase growth. The LKT is part of a large family of related exotoxins (RTX toxins) produced by a variety of gram negative bacteria. Recent reports suggest that the M. haemolytica LKT, and other members of the RTX family of bacterial exotoxins, bind to the beta2 integrin CD11a/CD18 (i.e. LFA-1), or to CD18 alone, on the surface of leukocytes. There is increasing evidence that primed beta2 integrins transmit a variety of intracellular signals that can lead to cell activation, proliferation, differentiation, or death. These observations pose fascinating
questions regarding how LKT binding to beta2 integrins sets into motion the intracellular events that result in leukocyte activation or death. We find that incubation of the BL-3 bovine lymphoblastoid cell line with small amounts of LKT leads to upregulation of pro-apoptotic proteins, and down regulation of anti-apoptotic proteins, that contribute to increased caspase-9 activity in the cells. We also observe increased mitochondrial permeability, cytochrome c release, and morphological evidence of disruption of the outer membrane in LKT treated mitochondria. More recent evidence suggests that the LKT is internalized in a dynamin-dependent pathway and transported via the cytoskeleton to the mitochondria.
Impacts
Accomplishing the goals of this research will help us better understand the pathogenesis of pneumonia caused by Mannheimia haemolytica, and could identify new strategies to prevent or ameliorate the losses it causes.
Publications
- Atapattu, D. and C.J. Czuprynski. 2005. Mannheimia haemolytica leukotoxin induces apoptosis of bovine lymphoblastoid cells (BL-3) via a caspase-9 dependent mitochondrial pathway. Infect. Immun. 73:5504-5513.
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Progress 01/01/04 to 12/31/04
Outputs
Bovine respiratory disease (BRD) is a multi-factorial disease complex reflecting stress, management factors, and both viral and bacterial infectious agents. The principal bacterial agent is Mannheimia (Pasteurella) haemolytica, which can cause a severe fibrinous pleuropneumonia. The most important virulence determinant of M. haemolytica is its leukotoxin (LKT), which is a 104 kD protein that is secreted during logarithmic-phase growth. The LKT is part of a large family of related exotoxins (RTX toxins) produced by a variety of gram negative bacteria. Recent reports suggest that the M. haemolytica LKT, and other members of the RTX family of bacterial exotoxins, bind to the beta2 integrin CD11a/CD18 (i.e. LFA-1), or to CD18 alone, on the surface of leukocytes. There is increasing evidence that primed beta2 integrins transmit a variety of intracellular signals that can lead to cell activation, proliferation, differentiation, or death. These observations pose fascinating
questions regarding how LKT binding to beta2 integrins sets into motion the intracellular events that result in leukocyte activation or death. We find that incubation of the BL-3 bovine lymphoblastoid cell line with small amounts of LKT leads to upregulation of pro-apoptotic proteins, and down regulation of anti-apoptotic proteins, that contribute to increased caspase-9 activity in the cells. We also observe increased mitochondrial permeability, cytochrome c release, and morphological evidence of disruption of the outer membrane in LKT treated mitochondria. These events suggest that exposure to LKT initiates a series of events, either via a signal transduction pathway or intracellular transport of LKT into the cell, that results in activation of the mitochondria-dependent caspase 9 pathway of apoptosis.
Impacts
This research will help us better understand the pathogenesis of pneumonia caused by Mannheimia haemolytica, and could identify new strategies to prevent or ameliorate the losses it causes.
Publications
- Atapattu, D., Albrecht, R., Oshel, P., Czuprynski, C.J. 2004. Cellular trafficking of M. haemolytica leukotoxin. 84th Conf. Res. Work. Anim. Dis.
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