Enteropathogenic E. coli (EPEC) is an important cause of serious diarrhea in infants in developing countries and a model organism for studies of Enterohemorrhagic E. coli (EHEC) infections. These pathogens are able to attach intimately to host cells and induce dramatic changes in cytoskeletal organization in a process known as attaching and effacing that is dependent on the Locus of Enterocyte Effacement (LEE) pathogenicity island. Ongoing studies of the genes from one end of the LEE have provided important insights into the pathogenesis of EPEC and EHEC infection. Experiments designed to accomplish four specific aims will further these studies.
The first aim i s to identify the target(s) of and determine the mechanism of action of EspF. Two functions have been identified for EspF. EspF mediates host cell apoptosis and is required for disruption of intestinal barrier function. The mechanism of action of EspF will be revealed by identifying its host cell target(s). These studies will also determine whether these two functions are the result of a single or separate EspF activities.
The second aim i s to test the hypothesis that EspB has a role in attaching and effacing distinct from its role as a component of the translocation apparatus. EspB is required for translocation of EPEC effector molecules into host cells, but EspB is itself translocated to the host cell cytoplasm and EspB causes dramatic changes in epithelial morphology when transfected and expressed in host cells. Mutants with linker-insertion and point mutations in espB will be screened to determine whether EspB proteins that retain translocation activity but lack attaching and effacing activity can be identified. These studies will also yield a detailed analysis of EspB structure-function relationships. The third specific aim is to test the hypothesis that the product of orf23 is a component of the translocation apparatus and the fourth aim is to test the hypothesis that the product of orf27 is a chaperone for an EPEC effector protein that is not required for attaching and effacing activity. These hypotheses are firmly based on data developed during the current funding period and will lead to new insights into the structure of the translocation apparatus and into functions of the LEE other than attaching and effacing. The experiments described in this proposal are essential for improving our understanding of the pathogenesis of EPEC and EHEC infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032074-14
Application #
6878093
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Schmitt, Clare K
Project Start
1992-02-01
Project End
2007-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
14
Fiscal Year
2005
Total Cost
$297,000
Indirect Cost
Name
University of Maryland Baltimore
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Donnenberg, Michael S; Narayanan, Shivakumar (2013) How to diagnose a foodborne illness. Infect Dis Clin North Am 27:535-54
Hazen, Tracy H; Sahl, Jason W; Fraser, Claire M et al. (2013) Refining the pathovar paradigm via phylogenomics of the attaching and effacing Escherichia coli. Proc Natl Acad Sci U S A 110:12810-5
Maddocks, Oliver David Kenneth; Scanlon, Karen Mary; Donnenberg, Michael S (2013) An Escherichia coli effector protein promotes host mutation via depletion of DNA mismatch repair proteins. MBio 4:e00152-13
Aroeti, Benjamin; Friedman, Gil; Zlotkin-Rivkin, Efrat et al. (2012) Retraction of enteropathogenic E. coli type IV pili promotes efficient host cell colonization, effector translocation and tight junction disruption. Gut Microbes 3:267-71
Luo, Wensheng; Donnenberg, Michael S (2011) Interactions and predicted host membrane topology of the enteropathogenic Escherichia coli translocator protein EspB. J Bacteriol 193:2972-80
Dasanayake, Dayal; Richaud, Manon; Cyr, Normand et al. (2011) The N-terminal amphipathic region of the Escherichia coli type III secretion system protein EspD is required for membrane insertion and function. Mol Microbiol 81:734-50
Contreras, C A; Ochoa, T J; Lacher, D W et al. (2010) Allelic variability of critical virulence genes (eae, bfpA and perA) in typical and atypical enteropathogenic Escherichia coli in Peruvian children. J Med Microbiol 59:25-31
Deng, Qiwen; Luo, Wensheng; Donnenberg, Michael S (2007) Rapid site-directed domain scanning mutagenesis of enteropathogenic Escherichia coli espD. Biol Proced Online 9:18-26
Crane, J K; McNamara, B P; Donnenberg, M S (2001) Role of EspF in host cell death induced by enteropathogenic Escherichia coli. Cell Microbiol 3:197-211