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An Anti-Pyruvate Kinase Monoclonal Antibody and Translocated Intimin Purdue University Purdue e-Pubs Open Access Theses Theses and Dissertations 2013 An Anti-Pyruvate Kinase Monoclonal Antibody And Translocated Intimin Receptor (tir) For Specific etD ection Of Listeria Species And Shiga- Toxigenic Escherichia Coli Titiksha Dikshit Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_theses Recommended Citation Dikshit, Titiksha, "An Anti-Pyruvate Kinase Monoclonal Antibody And Translocated Intimin Receptor (tir) For Specific eD tection Of Listeria Species And Shiga-Toxigenic Escherichia Coli" (2013). Open Access Theses. 3. https://docs.lib.purdue.edu/open_access_theses/3 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By TITIKSHA DIKSHIT Entitled AN ANTI-PYRUVATE KINASE MONCONAL ANTIBODY AND TRANSLOCATED INTIMIN RECEPTOR (TIR) FOR SPECIFIC DETECTION OF LISTERIA SPECIES AND SHIGA-TOXIGENIC ESCHERICHIA COLI Master of Science For the degree of Is approved by the final examining committee: DR. ARUN K. BHUNIA Chair DR. BRUCE M. APPLEGATE DR. JOHN PATTERSON DR. RAMESH VEMULAPALLI To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20) , this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ___________________DR. ARUN K. BHUNIA _________________ ____________________________________ Approved by: DR. BRIAN FARKAS 10/11/2013 Head of the Graduate Program Date AN ANTI-PYRUVATE KINASE MONOCLONAL ANTIBODY AND TRANSLOCATED INTIMIN RECEPTOR FOR SPECIFIC DETECTION OF LISTERIA SPECIES AND SHIGA-TOXIGENIC ESCHERICHIA COLI A Thesis Submitted to the Faculty of Purdue University by Titiksha Dikshit In Partial Fulfillment of the Requirements for the Degree of Master of Science December 2013 Purdue University West Lafayette, Indiana ii To my parents. ii iii ACKNOWLEDGEMENTS I would like to thank: Dr. Arun K. Bhunia, my research advisor, for his guidance, advise and patience. I have learnt much during my research and I sincerely appreciate this opportunity. Thank you for your time and continuous encouragement. Dr. Bruce M. Applegate, Dr. John A. Patterson and Dr. Ramesh Vemulapalli for serving in my graduate committee and for their insights and support. Dr. Mary A. R. Amalaradjou, Dr. Marcelo Mendoça, Dr. Atul K. Singh and Dr. Krishna M. Mishra for helping me learn various techniques. I am grateful to you for your assistance in problem solving, for lending a patient ear and for motivating me to give my best, particularly when the going got tough. My lab mates of Molecular and Microbiology Lab, former and present; and my iii wonderful friends; it has been great working with you and your friendship means a lot to me. I wish you all the best in your future endeavors. David La, Dr. Daisuke Kihara, Dr. Chittaranjan Das, Gwen Shoemaker, Deb Livengood and Linda Webster for their assistance. iv My wonderful friends. Last but not the least, my family. My parents Mumukshu and Nirupama Dikshit, for their unconditional and endless love and support. Thank you for making me the person I am today. And finally, Saattwik Dikshit, for being the best little brother one could wish for. Funding: This research was supported through a cooperative agreement with the Agricultural Research Service of the US Department of Agriculture project number 1935-42000-035 and the Center for Food Safety Engineering at Purdue University. iv v TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................... viii LIST OF FIGURES ...............................................................................................ix ABSTRACT ................................................................................................. xiv CHAPTER 1. INTRODUCTION ........................................................................ 1 1.1 Implications of foodborne illnesses…………………………………..1 1.2 Food diagnostics: Conventional and rapid detection techniques…3 1.3 Research goal and objectives………………………………………...6 CHAPTER 2. LITERATURE REVIEW…………………… ........ …………………9 2.1 Introduction …………………………………………………………… 9 2.1.1 Foodborne pathogens and illnessees .................................... 11 2.1.2 Detection of foodborne pathogens ........................................ 20 2.2 Pathogenic E. coli…………………………………………………… 29 2.2.1 STEC/VTEC/EHEC ............................................................... 33 2.2.2 Infection mechanism ............................................................. 34 2.3 Listeria monocytogenes……………………………………………...40 2.3.1 Infection mechanism ............................................................. 42 2.3.2 Listeria surface proteins ........................................................ 46 2.3.3 SecA2 based Listeria surface protein transport ..................... 47 2.3.4 Pyruvate kinase ..................................................................... 49 v 2.3.5 Immunologic techniques for Listeria detection ....................... 50 CHAPTER 3. PYRUVATE KINASE, A SECA2-DEPENDENT SURFACE ASSOCIATED PROTEIN, IN LISTERIA SPECIES ............................................. 52 3.1 Introduction……………………………………………………………52 3.2 Materials and methods……………………………………………… 55 3.2.1 Bacterial cultures, plasmids and primers ............................... 55 3.2.2 Purification of anti-PyK antibody, EM-7H10........................... 57 3.2.3 Identification of PyK in Listeria by MALDI-TOF MS/MS ......... 57 vi Page 3.2.4 Surface localization of PyK .................................................... 58 3.2.5 PyK cloning and expression .................................................. 59 3.2.6 Analysis of enzyme activity of rPyK ....................................... 60 3.2.7 Analysis of PyK localization in SecA2 mutants ...................... 60 3.2.8 Reverse transcriptase PCR (RT-PCR) for pyk ...................... 62 3.2.9 Effect of growth media on PyK expression ............................ 63 3.2.10 Fiber optics biosensor ........................................................... 63 3.2.11 Adhesion assay ..................................................................... 64 3.2.12 Statistical analysis ................................................................. 64 3.3 Results…………………………………...……………………………65 3.3.1 MALDI-TOF MS/MS revealed MAb EM-7H10 reactive protein to be PyK…………………………………………… ................................. ……………65 3.3.2 PyK is located on the cell surface of all Listeria species........ 67 3.3.3 PyK translocation to cell wall is SecA2 dependent ................ 70 3.3.4 Pyk transcript reduced in ΔsecA2 strain ................................ 71 3.3.5 Enzyme activity of PyK .......................................................... 72 3.3.6 Differential PyK expression in selective enrichment broths ... 73 3.3.7 Listeria detection with fiber optics .......................................... 74 3.3.8 Role of PyK as a bacterial adhesion ...................................... 76 3.4 Discussion…………………………………………………………… .76 CHAPTER 4. TIR (TRANSLOCATED INTIMIN RECEPTOR) FOR CAPTURE AND DETECTION OF STEC………………………………………………………...82 4.1 Introduction……………………………………………………………82 4.2 Materials and methods……………………………………………… 85 4.2.1 Bacterial cultures and growth conditions ............................... 85 vi 4.2.2 Cloning and expression of TIR, TIR-IBD, CesT & TIR-CesT . 87 4.2.3 Recombinant protein expression ........................................... 89 4.2.4 TIR antibody development ..................................................... 90 4.2.5 Reverse transcriptase (RT) PCR ........................................... 91 4.2.6 Effect of growth media on TIR expression ............................. 91 4.2.7 ELISA .................................................................................... 92 4.2.8 Statistical analysis ................................................................. 93 4.3 Results and discussion………………………………………………94 4.3.1 TIR antibody ......................................................................... 94 4.3.2 TIR protein cloning and expression ....................................... 95 4.3.3 TIR-IBD cloning and expression ............................................ 98 4.3.4 TIR and TIR-IBD RT-PCR ..................................................... 99 4.3.5 TIR-CesT coexpression ......................................................... 99 4.3.6 TIR based STEC detection .................................................. 101 4.3.7 TIR-CesT protein separation ............................................... 102 4.3.8 Next steps……………………………………………………….103 vii Page REFERENCES ............................................................................................... 105 APPENDICES Appendix A: Identifying specific amino-acids involved in LAP-Hsp60 interaction…………………………………………………………………………..128 Materials and methods ..................................................................................... 129 Results & Discussion ........................................................................................ 132 Appendix B: Identifying natural Lactobacillus spp. isolates from cow rumen……………………………………………………………………………….137
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