A Thesis entitled Identification and Description of Burkholderia pseudomallei Proteins that Bind Host Complement-Regulatory Proteins via in silico and in vitro Analyses by Caroline Lambert Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master’s Degree in Biomedical Sciences ________________________________________ R. Mark Wooten, Ph.D., Committee Chair ________________________________________ Robert M. Blumenthal, Ph.D., Committee Member _________________________________________ Jyl Matson, Ph.D., Committee Member _________________________________________ Amanda Bryant-Friedrich, Dr. rer Nat., Dean College of Graduate Studies The University of Toledo August 2018 Copyright 2018, Caroline Lambert This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Identification and Description of Burkholderia pseudomallei Proteins that Bind Host Complement-Regulatory Proteins via in silico and in vitro Analyses by Caroline Lambert Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master’s Degree in Biomedical Sciences The University of Toledo August 2018 Burkholderia pseudomallei (Bp) is a motile Gram-negative bacillus and causative agent of the febrile disease melioidosis. Bp is an opportunistic bacterium, with diabetes mellitus as a major risk factor. Bp can evade clearance by the alternative pathway of the complement cascade enabling it to invade cells and persist intracellularly. Factor H (fH) is a negative regulator of the alternative pathway, which protects host surfaces from complement-mediated damage. Several microbial species are known to mimic host surfaces or deceive fH self-recognition domains by producing a fH binding protein (fHbp). Bacteria with the ability to bind fH to their surface include Yersinia enterocolitica, via adhesin YadA, Neisseria meningitidis via fHbp, and Haemophilus influenzae via P5. This study used in silico and in vitro methods to investigate the ability of Bp to bind to host complement regulatory protein factor H. In vitro studies found that Bp can bind host complement regulatory protein fH on its surface via one or more proteins with a iii molecular weight of approximately 37 kDa. Candidate fHbps OmpA and Omp38 were recognized by mass spectrometry analysis. BLAST database searches identified OmpA and BpaC as candidate fHbps. Topological algorithms predicted BpaC and OmpA are partially extracellularly exposed on the bacterial surface. Rigid-body docking methods characterized conformations in which OmpA and BpaC would interact with fH domains 19-20. Binding affinities between BpaC and OmpA bound to fH domains 19-20 were predicted to be stronger than the interaction between known fHbp Borrelia burgdorferi OspE and fH domains 19-20. A direct interaction between fH and the recombinant versions of candidate fHbps Omp38 and OmpA has not yet been confirmed using molecular biology methods. In vitro methods to investigate the BpaC and fH interaction are still to be explored. The identification of Bp proteins that bind to fH will provide a therapeutic target, which may have potential as vaccine candidates to be used towards reducing the global burden of melioidosis. iv Acknowledgements I would like to thank Dr Laura Stanbery and Irum Syed for all their time and effort spent trying to make me into a scientist, as well as John Presloid and Muhammed Saad Moledina for their support in the lab. I would like to thank Dr R. Mark Wooten for taking a bioinformaticist into his lab for the first time, Dr Robert Blumenthal for telling me from the first moment I contacted him and throughout my studies that it was possible for me to obtain a masters in bioinformatics and Dr Jyl Matson for her advice and guidance in my research. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Tables .....................................................................................................................x List of Figures .................................................................................................................... xi List of Abbreviations ....................................................................................................... xiv 1 Introduction 1.1 Burkholderia pseudomallei is causative agent of Melioidosis .....................1 1.2 Melioidosis ...................................................................................................3 1.3 Melioidosis Distribution ..............................................................................7 1.4 Melioidosis Risk Factors............................................................................11 1.5 Burkholderia pseudomallei as a possible Biological Weapon ...................12 1.6 Burkholderia pseudomallei virulence ........................................................13 1.7 Burkholderia pseudomallei and thailandensis ...........................................16 1.8 Complement ................................................................................................18 1.8.1 Classical pathway ...........................................................................18 1.8.2 Lectin pathway ...............................................................................19 1.8.3 Alternative pathway .......................................................................19 vi 1.9 Importance of Complement in the control of Burkholderia pseudomallei ..............................................................................................21 1.10 Factor H .....................................................................................................23 1.11 Bacteria that bind Factor H and their Factor H binding proteins ...............26 1.12 Vaccine Potential .......................................................................................32 1.13 Protein Structural Modeling .......................................................................34 1.14 Proteomic Software Assignment of Protein Characteristics .......................36 1.15 Use of Bioinformatics to Identify Bacterial Proteins .................................37 1.16 Thesis goals .................................................................................................38 2 Materials and Methods ....................................................................................................41 2.1 In silico methods ........................................................................................41 2.1.1 Strategies of Multiple Sequence Alignment ..................................41 2.1.2 Identifying putative fHbps in silico ...............................................42 2.1.3 Logo Analysis ................................................................................44 2.1.4 Protein Localization and Topological Assessment ........................44 2.1.5 Protein Structure Prediction ...........................................................46 2.1.6 Predicting Interacting Regions between Factor H and Factor H binding proteins .............................................................................47 2.2 In vitro methods .........................................................................................48 2.2.1 Bacterial strains, Media and Growth Conditions ...........................48 2.2.2 Whole bacteria Factor H Binding assay .........................................48 2.2.3 Outer membrane protein extraction ...............................................49 2.2.4 Far-Western analysis of Factor H binding .....................................50 vii 2.2.5 Cloning of putative Factor H binding proteins into Escherichia coli..................................................................................................50 2.2.6 Protein Expression .........................................................................52 2.2.7 Protein Extraction ..........................................................................53 2.2.7.1 Soluble Protein Extraction ................................................53 2.2.7.2 Inclusion-Body Protein Extraction ...................................54 2.2.8 Affinity Purification of Recombinant protein ................................54 2.2.9 Protein Immunoblot assay ..............................................................55 3 Results ………….. .......................................................................................................57 3.1 In vitro results ............................................................................................57 3.1.1 Burkholderia pseudomallei binds Factor H ...................................57 3.1.2 Identification of Burkholderia pseudomallei protein facilitating Factor H binding via far western blot analysis ..............................58 3.1.3 Putative Factor H binding proteins identified via mass spectrometry analysis .....................................................................59 3.1.4 Expression of putative Factor H binding proteins OmpA and Omp38.............................................................................................60 3.1.5 Protein Purification