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Bruce A. McClane, Ph.D.
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Professor
W1147 BSTWR
200 Lothrop Street
Pittsburgh, Pennsylvania 15261
Phone: (412) 648-9022
Fax: (412) 624-1401
E-mail: bamcc@pitt.edu
Lab Phone: (412) 648-9022
Lab Website
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Biography
Dr. McClane is a Professor in the Department of Microbiology and Molecular Genetics at the University of Pittsburgh School of Medicine. He is also a member of the Molecular Virology and Microbiology Graduate Program and Co-Course Director of the Medical Microbiology course for first year medical students. He received his Ph.D. in Microbiology and Cell Biology in 1980 from Penn State University and then performed postdoctoral research at the New York University School of Medicine. His research interests focus on toxin-mediated enteric diseases caused by Clostridium perfringens, Dr. McClane has served on the Editorial Board of Infection and Immunity for the past 13 years, won the Sydney Finegold award for Anaerobic Microbiology research in 1996, and has been elected as a Fellow of the American Academy of Microbiology.
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Research
Our laboratory studies bacterial pathogenesis. Specifically, our research focuses on the enteric diseases caused by Clostridium perfringens type A, including C. perfringens type A food poisoning (the third most common foodborne illness in the USA), several nonfoodborne human GI illnesses (notably antibiotic-associated diarrhea and sporadic diarrhea), and several veterinary enterotoxemias (caused by type B-D isolates). Those type B-D infections are of particular importance because they involve epsilon toxin, a class B CDC/USDA overlap select toxin.
Currently, there are three funded research projects underway in the lab, including:
1. The Mechanism of Action of Clostridium Perfringens Enterotoxin (CPE, funded by NIAID). In the past year, we finished evaluating interactions between CPE and claudins, which are intestinal tight junction proteins. To investigate whether claudin-4 plays a role in CPE action on CaCo-2 cells (a naturally CPE-sensitive polarized human intestinal cell line that forms tight junctions), co-immunoprecipitation and electroelution techniques were used. Those analyses clearly demonstrated interactions between CPE and claudin-1,-3 and -4 in CaCo-2 cells; interactions between CPE and claudin-1 are interesting because this claudin, unlike claudin-3 or-4, cannot serve as a CPE receptor. In addition, we recently showed that rat fibroblast transfectants expressing a C-terminal truncated human claudin-4 are fully CPEsensitive (unlike parent rat fibroblasts); this result indicates that cell signaling cascades, which are mediated by the PDZ domain of the C-terminal region of claudins, are not required for CPE action. Considerable progress has also been achieved on Aim #2 of our NIH grant, which involves elucidating the action of CPE. We have recently shown that neither CPE binding to claudins not formation of the ~155 kDa CPE complex required for CPE action occur in lipid rafts. This finding identifies CPE as an unusual pore-forming toxin whose action does not require lipid rafts. We are also now further exploring the CPE structure:function relationships (grant Aim #3). A recently identified CPE mutant was shown to form the ~155 kDa complex that is responsible for initiating CPE-induced plasma membrane permeability changes; however that mutant does not cause membrane permeability changes. These findings suggest the mutant forms a prepore that cannot progress to a functional pore, i.e., we have putatively identified a new step in CPE action. In addition, we have recently shown that the “~155kDa” CPE complex contains six copies of CPE, i.e., it is a hexameric pore. Since we have also shown in our Aim #1 studies that the “~155 kDa” complex can contain two claudins, it is clear that this CPE complex is larger than previously estimated; we are currently resizing this pore using electrophoretic and chromatography approaches. Finally, substantial progress has been achieved regarding grant Aim #4, which involves dissecting the molecular pathogenesis of CPE-positive C. perfringens isolates. We have completed sequencing of two cpe-encoding plasmid families; this work represents the first completely sequenced C. perfringens virulence plasmids (virulence plasmids encode most toxins produced by this baceterium). A manuscript of this seminal work has been published in J. of Bacteriology. We are now analyzing plasmids in type E isolates that carry silent cpe genes near their iota toxin genes, which should help elucidate evolutionary relationships among cpecarrying plasmids.
2. C. perfringens type B-D virulence plasmids (funded by NIAID). As introduced above, virulence plasmids play a major role in C. perfringens human and veterinary enteric infections. For example, they carry the gene encoding epsilon toxin, a CDC/USDA class B select toxin that is important in veterinary infections caused by type B and D isolates. In the past year we dissected the contributions of various toxins to the pathogenesis of type B and C isolates. Those analyses revealed that beta toxin is essential for the lethal effects of type C supernatants in the mouse i.v., injection model. However, both beta and epsilon toxins are important for the lethal activity of type B supernatants in the mouse i.v., challenge model We have recently developed a new techniques for preparing knock-out mutants in C. perfringens. We have now used that new method to prepare a series of single and double toxin null mutants in a type C background. Those studies have identified beta toxin as being required for both the intestinal and systemic effects of type C infection. In addition, have prepared a type D epsilon toxin null mutant that will soon be tested in animals for virulence.
3. The Molecular Epidemiology of Clostridium perfringens type A Food Poisoning (funded by USDA). Our laboratory is conducting studies on the molecular epidemiology of C. perfringens type A food poisoning. We have recently shown that food poisoning isolates carrying a chromosomal cpe gene are not only highly resistant to heat but also to other food environment stresses (cold, preservatives, etc.). In addition, we are identifying the reservoirs for these food poisoning isolates and how/when they enter the food supply. Finally, we are starting to evaluate the basis behind the resistance phenotype of the food poisoning isolates.
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Selected Publications
- Caserta JA, Hale ML, Popoff MR, Stiles BG, McClane BA. (2008) "Evidence that membrane rafts are not required for the action of Clostridium perfringens enterotoxin" Infect Immun. 76:5677-85. | Abstract
- Miyamoto K, Li J, Sayeed S, Akimoto S, McClane BA. (2008) "Sequencing and diversity analyses reveal extensive similarities between some epsilon-toxin-encoding plasmids and the pCPF5603 Clostridium perfringens enterotoxin plasmid" J Bacteriol. 190:7178-88. | Abstract
- Li J, McClane BA. (2008) "A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates" PLoS Pathog. 4:e1000056. | Abstract
- Carman RJ, Sayeed S, Li J, Genheimer CW, Hiltonsmith MF, Wilkins TD, McClane BA. (2008) "Clostridium perfringens toxin genotypes in the feces of healthy North Americans" Anaerobe. 14:102-8. | Abstract
- Sayeed S, Uzal FA, Fisher DJ, Saputo J, Vidal JE, Chen Y, Gupta P, Rood JI, McClane BA. (2008) "Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model" Mol Microbiol. 67:15-30. | Abstract
- Miyakawa ME, Saputo J, Leger JS, Puschner B, Fisher DJ, McClane BA, Uzal FA. 2007. "Necrotizing enterocolitis and death in a goat kid associated with enterotoxin (CPE)-producing Clostridium perfringens type A" Can Vet J. 48:1266-9. | Abstract
- Sayeed S, Uzal FA, Fisher DJ, Saputo J, Vidal JE, Chen Y, Gupta P, Rood JI, McClane BA. 2008. "Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model" Mol Microbiol. 67:15-30. | Abstract
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