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Tiong Okstate 0664D 14712.Pdf (2.865Mb) IDENTIFICATION OF PUTATIVE SURFACE ADHESINS BY COMPARISON OF SURFACE-ADHERENCE VARIANTS OF LISTERIA MONOCYTOGENES USING LC-MS/MS AND RT-QPCR By HUNG KING TIONG Bachelor of Science in Plant and Soil Sciences (Option: Biotechnology) Oklahoma State University Stillwater, Oklahoma 2004 Master of Science in Microbiology and Molecular Genetics (Microbiology/Cell and Molecular Biology) Oklahoma State University Stillwater, Oklahoma 2009 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY July, 2016 IDENTIFICATION OF PUTATIVE SURFACE ADHESINS BY COMPARISON OF SURFACE-ADHERENCE VARIANTS OF LISTERIA MONOCYTOGENES USING LC-MS/MS AND RT-QPCR Dissertation Approved: Dr. Peter M. Muriana Dissertation Adviser Dr. Glenn Zhang Dr. Udaya DeSilva Dr. Babu Fathepure ii Name: HUNG KING TIONG Date of Degree: JULY, 2016 Title of Study: IDENTIFICATION OF PUTATIVE SURFACE ADHESINS BY COMPARISON OF SURFACE-ADHERENCE VARIANTS OF LISTERIA MONOCYTOGENES USING LC-MS/MS AND RT-QPCR Major Field: FOOD SCIENCE (MICROBIOLOGY) Listeria monocytogenes is a serious foodborne human pathogen that is known for biofilm formation, but the association of Listeria adherence in food environments and the resulting contamination of RTE products is not well characterized. A total of five methods for extraction of surface proteins from a strain of L. monocytogenes was evaluated for cytoplasmic protein availabilities in protein extracts using a LC-MS/MS (orbitrap) mass spectrometer. The surface protein profiles of weakly and strongly adherent strains of L. monocytogenes from food environments were examined to identify potential surface adhesins. Different subcellular localization tools were utilized for prediction of cytoplasmic protein association with cell envelop. The expression levels of the select genes that possessed higher expression in strongly than weakly adherent L. monocytogenes determined in this study were quantitated using real-time RT-PCR (RT- qPCR). LC-MS/MS analysis of five surface extracts revealed that one of them showed the least cytoplasmic proteins using Tris-buffered urea extraction of hypotonic-stressed cells (UB- Ghost) among five extraction methods. Protein subcellular localization prediction revealed that many of the isolated cytoplasmic proteins may be ‘moonlighting’ proteins, suggesting that some cytoplasmic proteins may also moolight as surface proteins and adhesins. Comparative analysis of surface proteins recovered from strains of weakly and strongly- adherent L. monocytogenes planktonic and sessile cells using these techniques revealed higher differential protein expression (i.e. >5-fold) in the strongly than in the weakly- adherent strain studied, hence suggesting the presence of other surface proteins acting as adhesins. Relative RT-qPCR analysis of 14 transcripts recovered from L. monocytogenes pre-incubated under planktonic or sessile condition at different temperatures revealed higher gene expression primarily for the strongly than the weakly adherent strain of L. monocytogenes. The analysis also showed higher gene expression for transcript extracts of both cells pre-incubated under conditions of sessile growth and higher temperatures. Some surface adhesins in L. monocytogenes may be present as cytoplasmic proteins whereby expression is strongly influenced by growth as planktonic or adhered cells. Further studies may identify conditions to better eliminate L. monocytogenes from plant facilities where they can remain adhered and possibly contaminate manufactured foods. iii TABLE OF CONTENTS Chapter Page I. INTRODUCTION ................................................................................................1 II. REVIEW OF LITERATURE ...............................................................................4 Listeria monocytogenes - a foodborne pathogen of serious human health concern ..................................................................................................................4 L. monocytogenes as an emerging foodborne pathogen .......................................6 CDC’s systematic documentation of L. monocytogenes as a pathogenic foodborne infectious agent: FoodNet, PulseNet ...................................................7 Incidence of listeriosis ..........................................................................................8 Prevalence of listeriosis L. monocytogenes .........................................................9 Transmission of listeriosis ..................................................................................10 Virulence factors of L. monocytogenes pathogenesis .........................................12 Model of pathogenesis for intracellular infection of L. monocytogenes .............16 Immune response against L. monocytogenes ......................................................19 Interaction between Listeria and intestinal epithelial cells: mechanisms and implications of adhesion and invasion ................................................................22 Interaction between Listeria and food processing plants: mechanism and implication of adhesion and cross-contamination...............................................32 Method for high performance identification of surface proteins ........................35 Methods for extraction of L. monocytogenes surface proteins ...........................40 High performance quantification of mRNAs ......................................................43 Conclusions and research phases ........................................................................45 References ...........................................................................................................47 iv III. Comparison of five methods for direct extraction of surface proteins from Listeria monocytogenes for proteomic analysis by orbitrap mass spectrometry..............75 Introduction .........................................................................................................76 Materials and Methods ........................................................................................79 Bacterial culture and growth conditions .........................................................79 Reagents and chemicals ..................................................................................79 Extraction protocols .......................................................................................80 Lithium chloride extraction (LiCl) .................................................................80 Tris-buffered urea (8M) extraction (UB) .......................................................81 Hypotonic extraction buffer (UB-Ghost) .......................................................81 Trypsin extraction containing BICAM (Tryp-B+S) .......................................81 Trypsin extraction containing Tris (Tryp-T+S) ..............................................82 Protein concentration assays ..........................................................................82 SDS-PAGE analysis .......................................................................................82 Digestion ........................................................................................................82 Liquid chromatography and tandem mass spectrometry (LC-MS/MS) .........83 MS data analysis .............................................................................................83 Results .................................................................................................................85 Discussion ...........................................................................................................88 Conclusion ..........................................................................................................92 Acknowledgements .............................................................................................93 References .........................................................................................................100 IV. Comparison of surface proteomes of adherence variants of Listeria monocytogenes using LC-MS/MS for identification of potential surface adhesins .............................................................................................................108 Introduction .......................................................................................................109 Materials and Methods ......................................................................................111 Bacterial cultures and growth conditions .......................................................111 Strain characterization: Adherence assays, electron microscopy, molecular typing, serotyping, and cellular hydrophobicity ............................................111 Microplate fluorescence assay and quantification of adhered cells ...............111 Scanning electron microscopy .......................................................................112 Molecular typing ............................................................................................112 Serotyping ......................................................................................................112 v Microbial adherence to solvent (MATS) assay for cell surface hydrophobicity characterization of Listeria strains .................................................................113 Extraction of surface proteins from Listeria monocytogenes ........................113 Cells grown in broth.......................................................................................113
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