Using Phage Display to Select Peptides Binding to Type 8 Capsular Polysaccharide of Staphylococcus Aureus
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Using Phage Display to Select Peptides Binding to Type 8 Capsular Polysaccharide of Staphylococcus aureus by Nina M. Lenkey Submitted in Partial Fulfillment of the Requirements for the Degree of Maters of Science in the Biology Program YOUNGSTOWN STATE UNIVERSITY December, 2016 Using Phage Display to Select Peptides Binding to Type 8 Capsular Polysaccharide of Staphylococcus aureus Nina Lenkey I hereby release this thesis to the public. I understand that this thesis will be made available from the OhioLINK ETD Center and the Maag Library Circulation Desk for public access. I also authorize the University or other individuals to make copies of this thesis as needed for scholarly research. Signature: Nina Lenkey, Student Date Approvals: Dr. Diana Fagan, Thesis Advisor Date Dr. David Asch, Committee Member Date Dr. Gary Walker, Committee Member Date Dr. Salvatore A. Sanders, Dean of Graduate Studies Date Abstract S. aureus aureus (S. aureus) is a major bacterial pathogen causing an array of critical infections. Increasing the problem of S. aureus infections is the fact that forty to sixty percent of S. aureus infections have now become antibiotic resistant, making it difficult to treat. S. aureus is protected by a polysaccharide capsule, which contributes to its virulence. The goal of this project is to identify a peptide that will bind to S. aureus type 8 capsular polysaccharide using phage display technology. Phage display is a technique used to display proteins or peptides on the surface of a phage (a virus that infects a bacterium). If a peptide displayed on the phage surface binds to a molecule of interest, the DNA for that peptide can be sequenced and used to synthesize the pure peptide. In this study, S. aureus capsular carbohydrate was purified and the presence of carbohydrate was confirmed using a red tetrazolium carbohydrate assay and an ELISA using anti- capsular hybridoma antibodies. The sample was determined to be free of contaminants using a Bradford assay for protein, phosphate assay for teichoic acid, and a DNA concentration assay for nucleic acid contamination. Using a phage display library, biopanning was performed to identify peptides that bind to whole bacteria. A series of ELISAs were performed to prove binding of the peptide to type 8 capsular polysaccharide of S. aureus. These ELISAs concluded specific clones 1, 2, 3, and 6 showed the most significant amount of binding to the type 8 capsular polysaccharide of S. aureus. Future studies will include further testing of the specific clones, and DNA sequencing of the most specific clone. The peptide can then be purchased and tested for specificity. The clone DNA may then be transferred to expression vectors and coupled to toxins. iii Acknowledgements First I would like to express my gratitude for my advisor, Dr. Diana Fagan, for the endless support, encouragement and help throughout this process. I would also like to thank the rest of my thesis committee: Dr. Gary Walker and Dr. David Asch. I thank my lab mates and friends. Their support and encouragement meant the world to me. Lastly I want to thank my parents, Calvin and Wilma Swank, this would not have been possible without them. A special thanks to my dad who taught me the value of an education and to never stop pursuing academic achievement. iv Table of Contents Chapter Page 1.0 Introduction …………………………………………………………………………………... 1 1.1 Staphylococcus aureus ………………………………………………………………………..1 1.2 Monoclonal Antibodies ………………………………………………………………….…. 13 1.3 Phage Display ……………………….……………………………………………....……… 23 2.0 Materials……………………..…………………………………………………………….….27 3.0 Methods ………………….…………………………………………………………….….…. 28 3.1 Purification of Type 8 Capsular Polysaccharide of S. aureus ……………………...... 28 3.2 Dialysis of Purified Type 8 Capsule ………………………………….…………............. 29 3.3 Lyophylization …………………..………………………………………………………….. 29 3.4 Carbohydrate Assay: Red Tetrazolium ………………………………………………….. 30 3.5 Tests for Impurities …..…………………………………………………………………….. 30 3.5.A Protein Assay: Bradford Assay ……………………………………………….. 30 3.5.B Phosphate Assay: Test for Teichoic Acid …………………………………..... 31 3.5.C DNA concentration Assay …………………………………………………….…31 3.6 Carbohydrate ELISA ……………………………………….…………………………….… 31 3.7 Surface Panning with Amplification ……………………………………………………... 32 3.7.A Biopanning ………………………………………………………………………. 32 3.7.B Titering ……………………………………………………...………………..….. 33 3.7.C Amplification ……………………….…………………...……………...……….. 34 3.8 Growth of Whole Cell S. aureus …………………………….………………….………… 35 3.9 M13 ELISA ……………………………………..……………………………………..…….. 35 v 4.0 Results ……………………………………………………….……………………………….. 37 4.1 Purification of Type 8 Capsular Polysaccharide of S. aureus……………………….…37 4.2 Carbohyrate Assay: Red Tetrazolium……………………………………………...………37 4.3 Contaminants in Carbohydrate Sample…………………………………………………...38 4.3A Protein Assay: Bradford Reagent………………………………………………………...38 4.3B Phosphate Assay: Test for Teichoic Acid………………………………………………..42 4.3C DNA concentration Assay…………………………………………………………………42 4.4 Carbohydrate ELISA……………………………………………………………...………….45 4.5 Biopanning……...……………………………………………………………………………..45 4.6 M13 ELISA of Phage to Whole Cell Type 5 and Type 8 S. aureus Bacteria………….47 4.7 M13 ELISA of Phage to Purified Type 8 Capsular Polysaccharide of S. aureus…….47 4.8 M13 ELISA of Phage Clones to Whole Cell Type 8 S. aureus Bacteria………………52 4.9 M13 ELISA of Phage Clones to Purified Type 8 Capsular Polysaccharide of S. aureus……………………………………………………………………………………………...52 5.0 Discussion……………………………………………………………………………………..59 6.0 References……………………………………………………………………………………..67 vi Table of Figures Figure Page Figure 1: Red tetrazolium Carbohydrate Assay of DEAE Column Fractions 42 Figure 2: Red Tetrazolium Carbohydrate Assay of Pooled DEAE Column Fractions 43 Figure 3: Bradford Reagent Protein Assay of pooled carbohydrate fractions 44 Figure 4: Phosphate Assay of Carbohydrate sample 46 Figure 5: Carbohydrate ELISA 46 Figure 6: M13 ELISA of P3T8 binding to whole cell S. aureus bacteria 50 Figure 7: M13ELISA of P3T8 binding to whole cell S. aureus bacteria. 51 Figure 8: ELISA of Type 8 S. aureus carbohydrate to P3 53 Figure 9: Phage clone ELISA to whole cell S. aureus bacteria 54 Figure 10: Phage clone ELISA to Type 8 S. aureus Carbohydrate 57 vii Table of Tables Table Page 1: The Percent Concentration of Nucleic Acids within the Purified Carbohydrate Samples 44 2: Plaque Forming Units before and after each panning round 48 3: Showing PFU/mL of selected titering plaques from before and after amplification 49 4: Fold increases of each plaque with and without S. aureus from the ELISA to whole cell bacteria. P-values of each plaque with and without S. aureus, as determined by a student two-tailed t-test 55 5: Fold increases of each plaque with and without S. aureus from the ELISA to type 8 capsule of S. aureus. P-values of each plaque with and without S. aureus, as determined by a student two-tailed t-test. 58 viii Abbreviations Staphylococcus aureus – S. aureus Methicillin resistant staphylococcal aureus – MRSA Penicillin-binding proteins – PBPs Vancomycin resistant staphylococcal aureus – VRSA Polymerase chain reaction - PCR Capsule polysaccharides - CPs Hypoxanthine-aminopterin-thymidine – HAT Fragment crystallization - Fc Surface plasmon resonance – SPR Complementarity determining region – CDR Very low birth weight - VLBW Iron surface determinant B – IsdB Filamentous phage family - Ff family Bovine serum albumin - BSA Hydrochloric acid – HCl Double stranded DNA - dsDNA Single stranded DNA – ssDNA Single stranded RNA – ssRNA Enzyme – Linked Immunoassay – ELISA Phosphate buffered saline - PBS Optical Density – OD 3,3′, 5,5′-Tetramethylbenzidine - TMB ix Diethylaminoethyl cellulose - DEAE Fetal calf serum – FCS Plaque forming units – PFU Polyethylene Glycol –PEG Too numerous to count - TNC x Chapter 1: Introduction 1.1 Staphylococcus aureus Staphylococcus aureus( S. aureus) is a gram-positive bacterial strain that has become a leading problem among infectious diseases (Holtfreter, 2009). S. aureus infections are seen universally. Newborns usually come into contact with S. aureus soon after birth, and become colonized within the first year of life. The colonization rate decreases with age. Twenty percent of adults are persistent S. aureus carriers; thirty percent of adults carry S. aureus intermittently; while fifty percent of adults are never carriers. Carriers of S. aureus usually have colonization of the nasal cavity. Although it appears that the S. aureus infection of the host is non-threatening, it has been seen that it is a balancing act. When there is a decline in the immune system or the staphylococcal infection invades a new site in the host, the infection can become serious (Holtfreter, 2009). Genome sequencing of 13 S. aureus strains shows a remarkable degree of variability between strains (Holtfreter, 2009). These variations include surface-associated genes, many virulence factors and resistance factors. The diversity of the S. aureus genome allows it to adapt to different microenvironments through the regulation of gene expression and protein synthesis mechanisms. These mechanisms make it possible for S. aureus to attach to the nasal septum, survive inside epithelial cells, grow in hostile environments and survive the bacterial killing conditions within the blood. The diversity of S. aureus, as well as, the diversity of the human host is a complicating factor in research. Some hosts are more susceptible to infection, the mode or location of bacterial 1 entry, the