Luận án tiến sĩ: Effect of prebiotic oligosaccharides on enteropathogenic Escherichia coli adherence

EFFECT OF PREBIOTIC OLIGOSACCHARIDES ON ENTEROPATHOGENIC ESCHERICHIA COLI ADHERENCE Major: Food Science and Technology Under the Supervision of Professor Robert W.. EFFECT OF PREBIOTIC

EFFECT OF PREBIOTIC OLIGOSACCHARIDES ON ENTEROPATHOGENIC

ESCHERICHIA COLI ADHERENCE

Major: Food Science and Technology

Under the Supervision of Professor Robert W Hutkins

Lincoln, Nebraska

November, 2006

UMI Number: 3243741

3243741 2007

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EFFECT OF PREBIOTIC OLIGOSACCHARIDES ON ENTEROPATHOGENIC

ESCHERICHIA COLI ADHERENCE

Kari Shoaf, Ph.D

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 coli (EPEC)

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

Acknowledgement

I would like to thank my advisor Dr Robert W Hutkins for his guidance and patience throughout my Ph.D program I would also like to express my gratitude to

my committee members Dr Andrew K Benson, Dr Rodney A Moxley, and Dr John

H 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

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., 2006 Infect Immun 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