Date of Award

Summer 8-15-2013

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Uropathogenic Escherichia coli (UPEC) are the primary etiology of urinary tract infections (UTIs), one of the most common bacterial infections afflicting the human population. While UPEC cause disease throughout the urinary tract, bladder infection, or cystitis, is most prevalent. A key aspect of UPEC pathogenesis in the bladder is the modulation of the host inflammatory response. At acute time points, UPEC delay the arrival of immune cells, such as neutrophils, to the bladder. The lack of neutrophils in the bladder lumen enables UPEC to replicate freely in the urine and invade the bladder epithelium, a requirement for bacterial persistence, in the absence of immune pressure. The UPEC products responsible for delaying the arrival of immune cells to the bladder had not been identified.

This thesis work identified a bacterial protein, YbcL, that was modestly up-regulated upon UPEC exposure to either cultured bladder epithelial cells or human neutrophils. We demonstrated that YbcL suppressed the migration of neutrophils across bladder epithelia in an in vitro model of transuroepithelial neutrophil migration and an in vivo murine model of cystitis. Suppression of PMN migration by YbcL was dependent upon the presence of threonine at position 78 (T78). In fact, T78 in YbcL is highly conserved in clinical UPEC isolates, suggesting that inhibition of neutrophil migration across epithelial barriers by YbcL is a conserved mechanism of immune modulation among UPEC.

Using a number of complementary approaches, we demonstrated that liberation of YbcL from the bacterial periplasm was required for suppression of neutrophil migration across a bladder epithelium. YbcL was detected in the supernatant and in association with bladder epithelial cells and neutrophils. Release of YbcL from the periplasm occurred in a manner that was dependent upon the concentration of YbcL in the periplasm, the duration of the infection and the presence of bladder epithelial cells. Although YbcL was soluble in the supernatant, we demonstrated that YbcL was not secreted from the periplasm by a canonical secretion system. Despite the apparent absence of a dedicated secretion system, these findings demonstrate that YbcL functions as an exoprotein.

Investigations into the mechanism underlying suppression of neutrophil migration by YbcL revealed that YbcL did not influence the production of chemoattractant molecules by bladder epithelial cells or bacteria or the ability of neutrophils to chemotax in response to stimuli, requirements for neutrophils to traverse epithelial barriers. This work identified and began the characterization of a bacterial protein, YbcL, that contributes to modulation of the innate immune response by UPEC. Additional experimentation is required to elucidate the importance of T78, the mode of delivery of YbcL from the periplasm, and the mechanism of action of YbcL. By delaying the arrival of immune cells, the activity of YbcL likely facilitates formation of the acute intracellular niche occupied by UPEC and required for persistence in the urinary tract.

Language

English (en)

Chair and Committee

David A. Hunstad

Committee Members

Douglas E. Berg, Michael G. Caparon, Daniel E. Goldberg, David B. Haslam, Jeffrey P. Henderson, Scott J. Hultgren

Included in

Biology Commons

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