Philip R. Hardwidge, Ph.D.

Assistant Professor

Postdoctoral Fellow, University of British Columbia, Vancouver, Canada
Ph.D., Biomedical Sciences, Mayo Clinic, Rochester, MN (2002)
B.S., Microbiology, University of Illinois, Urbana, IL (1997)

Office: 4031A Wahl Hall West | Lab: 4001 & 4031 Wahl Hall West
913-588-7085 | email

Research Interests

I am currently accepting highly motivated graduate students and postdoctoral fellows to join a productive, multidisciplinary research group focused on the understanding, treatment, and prevention of diarrheagenic E. coli disease.

Our laboratory studies several types of Escherichia coli that cause diarrhea and malnutrition in humans and livestock. Enterotoxigenic E. coli, (ETEC) is the leading cause of travelers' diarrhea and a major cause of diarrheal disease in underdeveloped nations. Enteropathogenic E. coli (EPEC) is the leading cause of bacterial mediated diarrhea in young children and, like ETEC, poses a major endemic health threat in developing countries. Enterohemorrhagic E. coli (EHEC) is an important problem health problem in the United States and is commonly transmitted through undercooked ground meat products (Hamburger Disease) or vegetables. Infection may develop, especially in young children and the elderly, into a fatal kidney disease known as hemolytic uremic syndrome (HUS). Specific projects include:

The role of ETEC enterotoxins in enhancing bacterial colonization. We hypothesized that the heat-labile enterotoxin (LT) preconditions the host intestinal epithelium for ETEC adherence. To test this hypothesis, we used an in vitro model of ETEC adherence to examine the role of LT in promoting bacterial-host interactions. We demonstrated that elaboration of LT promotes a significant increase in E. coli adherence. This phenotype is primarily dependent on the inherent ADP-ribosylation activity of this toxin, with a secondary role observed for the receptor-binding LT-B subunit. Rp-cAMP, an inhibitor of protein kinase A, abrogates LT’s ability to promote subsequent bacterial adherence. We also documented a role for bacterial sensing of host-derived 3’,5’-cyclic adenosine monophosphate in promoting adherence to host cells.

Mechanisms of Type III secreted EHEC/EPEC effector proteins. We presented the first large-scale proteomic analysis of a human cellular response to a pathogen. We analyzed the host response to T3S-delivered EPEC effector proteins by infecting polarized intestinal epithelial monolayers with either wild-type or T3S-deficient EPEC. Host proteins were isolated and subjected to quantitative profiling using isotope-coded affinity tagging (ICAT) combined with electrospray ionization tandem mass spectrometry. We identified over 2000 unique proteins from infected Caco-2 monolayers, of which ~13% were expressed differentially in the presence of TTSS-delivered EPEC effector proteins. These data provide a framework for future biochemical analyses of host-pathogen interactions.

Bacterial GTPases and outer membrane vesicles. ETEC produce outer-membrane vesicles (OMVs) containing LT that are endocytosed into host cells. The LeoA protein plays a role in secreting LT from the bacterial periplasm. To begin to understand the function of LeoA and its role in ETEC H10407 pathogenesis, a site-directed mutant lacking the putative GTP-binding domain was constructed. This domain was important to LeoA’s function in LT secretion, and may play a modest role in the formation and protein content of OMVs. Deletion of leoA reduced the abundance of OmpX in outer-membrane protein preparations and of LT in OMVs. Immunoprecipitation experiments revealed that LeoA interacts directly with OmpA, but that the GTP-binding domain is nonessential for this interaction. Deletion of leoA rendered ETEC H10407 non-motile, through apparent periplasmic accumulation of FliC.