Enteropathogenic E. coli (EPEC), a cause infantile diarrhea, is associated with significant morbidity and mortality. EPEC is not invasive and does not produce toxins, but expresses a type III secretory system (TTSS) that allows translocation of bacterial proteins into host cells. EPEC disrupts intestinal epithelial tight junctions (TJ) in a TTSS-dependent manner which likely contributes to diarrhea. The overarching hypothesis of this proposal is that EPEC effector molecules work in concert to alter TJs in vitro and in vivo. The overall goal of this proposal is to define the mechanisms by which EPEC alters TJs. Several EPEC effectors appear to contribute to TJ disruption including the translocated intimin receptor (Tir) and E. coli secreted protein F (EspF). Tir is inserted into the apical host cell membrane triggering """"""""pedestal"""""""" formation through recruitment of actin, ezrin and other cytoskeletal proteins. Tir may allow for effective translocation of effectors into host cells;alternatively, activation and recruitment of ezrin may be key. EspF binds cytokeratin 18 and 14-3-3 and is crucial for TJ disruption. EspF has also been shown to localize to mitochondria and induce apoptosis;the contribution to TJ disruption has not been examined. Our data suggest that TJ disruption can be dissected into 2 events mediated by different effectors: cytoskeletal contraction via myosin light chain (MLC) phosphorylation and endocytosis of TJ proteins. While in vitro studies have yielded important information regarding EPEC pathogenesis, corroboration of these findings in vivo has been delayed by the lack of a small animal model. We have reported the C57BL/6 mouse as an in vivo model of EPEC infection and our preliminary data suggest that intestinal TJs are disrupted. This model will allow us to determine if data from in vitro and in vivo models of EPEC infection correlate. We hypothesize that Tir-mediated intimate attachment and pedestal formation promote translocation of effectors into host cells wherein they activate signaling pathways or interact with proteins resulting in altered TJ structure and function. The following specific aims will address this hypothesis: 1. To determine the role of Tir in EPEC-induced disruption of tight junctions (TJ). 2. To investigate the relationship between EspF/14-3-3 interactions, mitochondrial targeting, and apoptosis to EPEC-induced alterations in TJ structure and barrier function. 3. To dissect the mechanisms underlying EPEC-induced disruption of TJs and define the effector molecules responsible for each component. 4. To evaluate the effect of EPEC infection on TJ structure and function in vivo using a mouse model and investigate the effector molecules and mechanisms involved.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK050694-14
Application #
7806501
Study Section
Special Emphasis Panel (ZRG1-DIG-C (02))
Program Officer
Grey, Michael J
Project Start
1997-06-01
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2012-04-30
Support Year
14
Fiscal Year
2010
Total Cost
$299,341
Indirect Cost
Name
University of Illinois at Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Nguyen, Mai; Rizvi, Jason; Hecht, Gail (2015) Expression of enteropathogenic Escherichia coli map is significantly different than that of other type III secreted effectors in vivo. Infect Immun 83:130-7
Battle, Scott E; Brady, Michael J; Vanaja, Sivapriya Kailasan et al. (2014) Actin pedestal formation by enterohemorrhagic Escherichia coli enhances bacterial host cell attachment and concomitant type III translocation. Infect Immun 82:3713-22
Glotfelty, Lila G; Zahs, Anita; Hodges, Kimberley et al. (2014) Enteropathogenic E. coli effectors EspG1/G2 disrupt microtubules, contribute to tight junction perturbation and inhibit restoration. Cell Microbiol 16:1767-83
Glotfelty, Lila G; Zahs, Anita; Iancu, Catalin et al. (2014) Microtubules are required for efficient epithelial tight junction homeostasis and restoration. Am J Physiol Cell Physiol 307:C245-54
Hodges, Kim; Hecht, Gail (2013) Bacterial infections of the small intestine. Curr Opin Gastroenterol 29:159-63
Annaba, Fadi; Sarwar, Zaheer; Gill, Ravinder K et al. (2012) Enteropathogenic Escherichia coli inhibits ileal sodium-dependent bile acid transporter ASBT. Am J Physiol Gastrointest Liver Physiol 302:G1216-22
Glotfelty, Lila G; Hecht, Gail A (2012) Enteropathogenic E. coli effectors EspG1/G2 disrupt tight junctions: new roles and mechanisms. Ann N Y Acad Sci 1258:149-58
Rhee, Ki-Jong; Cheng, Hao; Harris, Antoneicka et al. (2011) Determination of spatial and temporal colonization of enteropathogenic E. coli and enterohemorrhagic E. coli in mice using bioluminescent in vivo imaging. Gut Microbes 2:34-41
Vingadassalom, Didier; Campellone, Kenneth G; Brady, Michael J et al. (2010) Enterohemorrhagic E. coli requires N-WASP for efficient type III translocation but not for EspFU-mediated actin pedestal formation. PLoS Pathog 6:e1001056
Thanabalasuriar, Ajitha; Koutsouris, Athanasia; Weflen, Andrew et al. (2010) The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli. Cell Microbiol 12:31-41

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