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 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 | would like to thank my advisor Dr.
Hutkins for his guidance and patience throughout my Ph. | 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. | would also like to thank Dr. Glen Armstrong and his lab at the University of Calgary in Alberta, Canada for their guidance.
Finally, | 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.
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. Table of Contents page Abstract i Acknowledgements, ————¬IẰ ằ .Ễ00200En xxx scsei ii Preface iv Table of Contents 2u, Vv ListofTables vu viii Listof Figures ees ix Chapter 1 Literature Review 1 Introduction CC UỐỐ 2 Route of Infection 3 Adherence Basics 6 Adherence Kinetics 6 Adherence Specificity ———¬— nh Š re, 9 Adherence Affinity “` ¬nee 10 Specific Pathogen-host Interactions ọỘỒỘỀỘỒỘẼỘẽ.ị-—_ 11 Lectin-carbohydrate Interactions 11 Protein-protein Interactions 12 Hyrdophobin-protein Interactions mọi 12 Intestinal Target Tissues 14 Cell Surface Structures 16 Extracellular Matrix Components = =——— SA 18 Adhesive Components 18 BacterialAdhesins 19 FimbrialAdhesins 19 AfimbrialAdhesins 22 Initial Adherence of Enteropathogenic Escherichia coli = 23 Bundle-forming pili 24 EspA Filaments 25 Flagella 26 Intimin 27 LifA protein aT Anti-adhesives CỐ 28 Adhesin-based Vaccines ỘỘịẹềềỀềỀẽs.ọsọs“Ế_ 29 Host-derived anti-adhesins _ 30 Sublethal concentrations of antibiotics ——_ 3 Probiotics CC 32 Adhesin Analogs 33 Receptor Analogs 35 vii Conclusions 40 References 41 Prebiotic galactooligosaccharides reduce adherence of Chapter 2 Enteropathogenic Escherichia coli to tissue culture cells 88 Abstract.
89 Introduction, — ỐỀỐ uỐ 90 Materials and Methods 92 Results Ốc 98 Discussion — UỐỐỐ 102 References 124 Basis for GOS-mediated adherence inhibition of Chapter 3 Enteropathogenic Escherichia coli 135 Abstract Sun 136 Introduction CS VUỐ 136 Materials and Methods «re 138 Results and Discussion 143 References 158 Chapter 4 Conclusions 165 List of Tables page Chapter 1 1. Pathogen carbohydrate receptors 13 3. Some important ECM and adhesive components used as receptors bypathogens —— hnhnnnnnnhrrrhrrrrreeseeeereeeseeese 20 2. Oligosaccharides that prevent bacterial adherence invivo ---- 37 Chapter 2 1.
Structure and composition of commerical oligosaccharides used 108 2. Effect of oligosaccharides on enteropathogenic E. coli E2348/69 microcolony size and formation on Hep-2cells 109 3. Inhibition of localized adherence by enteropathogenic E.
coli E2348/69 on Caco-2 cells by oligosaccharides ——————¬ Ữ -ằ 110 4. Effect of GOS on autoaggregation of enteropathogenic E. Strains used in this study 147 viii List of Figures page Chapter 1 1. Description of bacterialadherene =———Ặ‹ĂằồÖ--.
Schematic illustration of adherence and anti-adhesiveagens - - 36 Chapter 2 1. Inhibition of enteropathogenic E. coli E2348/69 adherence to HEp-2 tissue culture cells by prebiotic oligosaccharides = 112 2. Effect of galactooligosaccharide concentration on adherence inhibition of enteropathogenic E.
coli E2348/69 to HEp-2 cells = 114 3. Displacement of enteropathogenic E. coli E2348/69 by GOS 116 4. Phase contrast and scanning electron micrographs of cultured cells challenged with enteropathogenic E.
coli E2348/69 in the absence and presenceofGOS 118 5. Inhibition of enteropathogenic E. coli E2348/69 adherence to Caco-2 tissue culture cells by prebiotic oligosaccharides 120 6. Effect of GOS on expression of BpA.
Schematic of Neo-GOS production 148 3. Adherence of enteropathogenic E. coli E2348/69 mutants to HEp-2 tissue culture cells in the presence and absence of GOS 152 4. Adherence of enteropathogenic E.
coli E2348/69 mutants to Caco-2 tissue culture cells in the absence and presence of GOS 154 5. Binding of EPEC E2348/69 to immobilized GOS 156 Chapter 4 4. Antiadhesive model for GOS 168 Chapter 1 Literature Review Introduction Over the last few decades, extensive research and investigation into the mechanisms behind bacterial adherence has established several fundamental principies involved in enteric pathogen interactions with a host. Most importantly, it is now well established that adherence of pathogens to host cell surfaces is an essential step in the infection process (195, 196).
Therefore, research focusing on bacterial-host interactions has led to the identification of numerous bacterial adherence molecules or adhesins and their specific host cell receptor sites. Ina majority of cases, proteins on the surface of bacteria mediate adherence to specific proteins or carbohydrates on the host epithelium. However, hydrophobic interactions and bacterial carbohydrate moieties often play lead roles in adherence. Generally, carbohydrate- or oligosaccharide-bearing moieties are the most common receptors used by bacteria to bind to the intact host cell surface, while protein-type receptors are often associated with bacterial adherence to connective tissues (collagens) or injured tissues that are on the mend (fibronectin, laminin).
The identification and clarification of these detailed bacteria-host interactions has provoked a deeper exploration into the prevention of pathogen-induced disease and infection through the molecular inhibition of adherence. Averting these interactions by physically blocking bacterial contact with intended receptors is the aim of antiadhesion therapy (105, 110, 111, 166, 204, 254). With the rise in antibiotic- resistant bacterial strains, new means of preventing pathogen spread are becoming increasingly necessary. Many novel antiadhesives have already been identified, 3 including adhesin-based vaccines, host-derived antiadhesives, probiotics, adhesin analogs, and receptor analogs.
This review will provide an overview of the architecture of host epithelial cell surfaces, the chemistry behind bacterial interactions with the host cell surface, and types of bacteria-host interactions and how they are mediated. Finally, research on antiadhesion therapies will be reviewed. Route of infection Enteric bacterial pathogens must maneuver through a lengthy stretch of hazardous terrain before they reach their intended adherence or infection site within a host. Initially, they must tolerate salivary enzymes in the mouth, followed by exposure to dead sloughed or shedded epithelial cells in the esophagus, that prevent bacterial adherence (176).
In the stomach, bacteria must endure a potentially devastating environment created by the secretion of digestive enzymes and hydrochloric acid. Once bacteria reach the intestines, they face mechanical, chemical, and cellular barriers including luminal flow, the mucus layer, commensal bacteria, secreted antimicrobial proteins and peptides, and a possible host immune response (176). However, the severity of pathogen resistance within the intestines is dependent upon the physiological dynamics and the prepared mucosal defenses of the target tissues. In the small intestine, the rapid luminal flow, the low pH, and the presence of digestive enzymes ward off many invaders.
The apical surface of the 4 small intestine contains an abundance of intestinal villi ranging from 10 to 40 per mm? of mucosal surface area (127), that, through ciliary action, also help avert some bacterial adherence. These villi contain varying types of epithelial cells, including columnar absorptive enterocytes, goblet cells that secret mucus, and cells that secrete a variety of antimicrobial proteins (57, 170). Additionally, there is a high rate of epithelial cell turnover that assists in preventing adherence (127, 176). The conditions within the large intestine are much less strenuous on invading pathogens.
The luminal flow is less vigorous, there is a neutral pH, and the epithelial cell turnover is around 10 fold slower than that of the small intestine (57, 247). Moreover, the surface of the large intestine does not have villi and is relatively smooth, but still contains absorptive enterocytes, goblet cells, and antimicrobial secreting cells (57). Goblet cells are present in many of the gastrointestinal tissues of humans. They secrete a protective gel-like mucus layer that covers the stomach, small intestine and large intestine (2, 65).
This mucus is chiefly composed of large filamentous gel-forming glycoproteins called mucins (65), that provide both a loosely adherent surface layer and a firmly adherent underlying layer (8). Mucus is thought to act as a medium for protection, lubrication, and transport between the lumen and the epithelial cell surface (65). When considering bacterial adherence in the GIT, the most important role of mucus is as a protective barrier against unwanted bacterial invaders. The mucus layer not only acts as a physical barrier by blocking binding sties at the epithelial cell surface, but also contains other proteins, lipids, and 5 nucleic acids that may deter pathogen adherence, including defensins, lysozyme, antiadherence molecules, secreted lgA and lgM, and other resident microorganisms as reviewed by Pearson and Brownlee (176).
Although the mucus iayer acts as a barrier to some invading pathogens, it also supports the growth and maintenance of a number of commensal bacteria in the GIT.