EFFECT OF PREBIOTIC OLIGOSACCHARIDES ON ENTEROPATHOGENIC ESCHERICHIA COLI ADHERENCE By Kari Shoaf A DISSERTATION Presented To the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Food Science and Technology Under the Supervision of Professor Robert W. Hutkins Lincoln, Nebraska November, 2006 UMI Number: 3243741 UMI Microform 3243741 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.
ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 EFFECT OF PREBIOTIC OLIGOSACCHARIDES ON ENTEROPATHOGENIC ESCHERICHIA COLI ADHERENCE Kari Shoaf, Ph. University of Nebraska, 2006 Advisor: Robert W. Hutkins Antiadhesion therapy has been found to be a very promising means to prevent and treat infections in the respiratory, urinary, and gastrointestinal tract.
Despite the considerable research interest in antiadhesives, relatively little is known about the ability of commercial dietary oligosaccharides, such as prebiotics, to inhibit pathogen adherence. The goal of this research was to determine if commercially available prebiotic oligosaccharides could function as antiadhesive agents against enteropathogenic E. To investigate the ability of prebiotics to inhibit EPEC adherence, inhibition assays were performed by comparing adherence rates of EPEC on HEp-2 and Caco-2 cells in the presence of galactooligosaccharides (GOS), inulin, fructooligosaccharides, lactulose, or raffinose. These data showed that GOS significantly reduced binding of EPEC more than the other prebiotics tested.
In addition, binding inhibition by GOS was shown to be dose-dependent and saturable at 16 mg/ml. Microscopic analyses indicated that GOS also reduced the number of EPEC per microcolony and the total number of microcolonies per infected cell. Both therapeutic and prophylactic GOS treatments were also examined. GOS was unable to displace previously adhered EPEC, but could prevent EPEC adherence when administered prior to infection.
Additionally, GOS did not affect EPEC autoaggregation. To determine if GOS was inhibiting adherence at a molecular level, the expression of BfpA, a bundle forming pili protein involved in localized adherence, was examined. GOS did not affect BfpA expression indicating that adherence inhibition was not due to the absence of this adhesin. To further examine the role of EPEC adhesins in GOS-mediated adherence inhibition, adherence assays using HEp-2 and Caco-2 cell lines were performed using isogenic EPEC mutants.
In general, GOS did not affect the adherence of strains lacking BfpA, suggesting that GOS may interfere specifically with BfpA- receptor interactions rather than with other potential EPEC adhesins and their receptors. These results show that commercial prebiotics, particularly GOS, directly inhibit the adherence of E. coli, and provides evidence that these agents may be used as antiadhesives against pathogens in both humans and animals. iii Acknowledgement I would like to thank my advisor Dr.
Hutkins for his guidance and patience throughout my Ph. I would also like to express my gratitude to my committee members Dr. Moxley, and Dr. Rupnow for their helpful suggestions.
My special thanks to Joe Nietfeldt and my fellow graduate students Andreia Bianchini, Jennifer Huebner, and Dr. Yong Jun Goh for their help and advice in various aspects of my research. I would also like to thank Dr. Glen Armstrong and his lab at the University of Calgary in Alberta, Canada for their guidance.
Finally, I want to acknowledge my husband, Terry Sweeney, my parents John and Ginny Shoaf, my brother and his wife, Rylee and Crystal Shoaf, and my dear friends for their love, support, encouragement, and understanding during this challenging, yet prolific journey. iv Preface This dissertation consists of three chapters and a conclusions section. Chapter 1 is a literature review on bacterial adherence and the mechanisms by which adherence can be prevented. Chapter 2 describes our published (Shoaf et al.
In press) results focusing on the ability of prebiotic oligosaccharides to inhibit the binding of enteropathogenic Escherichia coli E2348/69 to two types of tissue culture cells. In chapter 3, we report our results on the proposed Escherichia coli adhesins that may be involved in GOS-mediated adherence inhibition and the direct binding of Escherichia coli E2348/69 and other isogenic mutants to immobilized galactooligosaccharides. Lastly, a conclusions section summarizes the major research findings presented within this dissertation and suggests areas for future research.