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Bacterial Sortase a As a Drug Target UPTEC X 12 009 Examensarbete 30 hp Juni 2012 Bacterial Sortase A as a drug target Caroline Larsson Molecular Biotechnology Programme Uppsala University School of Engineering UPTEC X 12 009 Date of issue 2012-06 Author Caroline Larsson Title (English) Bacterial Sortase A as a drug target Title (Swedish) Abstract Sortase A is a housekeeping enzyme of Gram-positive bacteria that catalyses the anchoring of surface proteins to the bacterial peptidoglycan. The enzyme works to establish an interaction between bacteria and host cells and is essential for pathogenesis. This makes Sortase A a potential suitable target for inhibition, in order to treat bacterial infections. In this degree project Sortase A from Staphylococcus aureus was explored and potential inhibitors were investigated by performing enzyme activity and bacterial binding assays. A robust FRET assay was developed and optimized for a recombinant version of the enzyme and serves as a good starting point for studying inhibition. Keywords Antibiotic resistance, Sortase A, Staphylococcus aureus, Gram-positive bacteria, inhibition, FRET assay, fibronectin-binding Supervisors Ian Henderson Medivir AB Scientific reviewer Helena Danielsson Uppsala University Project name Sponsors Language Security English Classification ISSN 1401-2138 Supplementary bibliographical information Pages 49 Biology Education Centre Biomedical Center Husargatan 3 Uppsala Box 592 S-75124 Uppsala Tel +46 (0)18 4710000 Fax +46 (0)18 471 4687 Bacterial Sortase A as a drug target Caroline Larsson Populärvetenskaplig sammanfattning Antibiotikaresistens är ett aktuellt och växande folkhälsoproblem idag. Ett konstant behov av nya behandlingar för infektioner orsakade av antibiotikaresistenta bakterier innebär starka påfrestningar för sjukvården. Mekanismen bakom antibiotika är att selektivt reducera bakteriernas tillväxt på olika sätt. Detta påverkar det selektiva trycket hos bakterierna, vilket leder till att resistens utvecklas. För att det ska vara möjligt att behandla bakterieinfektioner mer långsiktigt och undvika det selektiva trycket är nya tillvägagångssätt nödvändiga. För att en infektion ska kunna utvecklas krävs en interaktion mellan bakeriecellerna och värdcellerna. Gram-positiva bakterier har en speciell mekanism för att denna interaktion ska kunna ske. Sortas A är ett konserverat enzym hos dessa bakterier som ansvarar för denna mekanism. Enzymet katalyserar en reaktion som fäster ytproteiner till peptidoglykanet hos bakterierna. På så sätt blir interaktionen med andra celler möjlig och genom inhibiering av Sortas A hoppas man att det ska bli möjligt att behandla infektioner som orsakats av Gram-positiva bakterier. Projektet går ut på att nå en ökad förståelse av enzymet och på så sätt möjliggöra inhiberingsstudier. För att kunna studera enzymet har ett aktivitetsprotokoll etablerats samt metoder för inbindning och inmärkning av bakterierna undersökts. Genom utnyttjadet av dessa metoder har potentiella inhibitorer analyserats. Examensarbete 30 hp Civilingenjörsprogrammet Molekylär Bioteknik Uppsala Universitet 2012 Contents 1 Introduction ..................................................................................................................................... 7 1.1 Gram-positive bacteria ............................................................................................................ 7 1.2 Sortase A .................................................................................................................................. 8 1.3 The project .............................................................................................................................. 9 1.3.1 The FRET assay................................................................................................................. 9 1.3.2 The fibronectin-binding assay ....................................................................................... 10 1.3.3 Labeling of bacterial cells .............................................................................................. 10 1.3.4 The aim of the study ...................................................................................................... 10 2 Materials ........................................................................................................................................ 11 2.1 Biological materials ............................................................................................................... 11 2.2 Reagents ................................................................................................................................ 11 2.3 Consumables ......................................................................................................................... 12 2.4 Systems .................................................................................................................................. 12 2.5 Programs ............................................................................................................................... 12 3 Methods ........................................................................................................................................ 13 3.1 The FRET assay ...................................................................................................................... 13 3.2 Mass spectrometry ................................................................................................................ 14 3.3 The fibronectin-binding assay ............................................................................................... 14 3.4 Labeling of bacterial cells ...................................................................................................... 15 3.5 Inhibitors ............................................................................................................................... 15 4 Results ........................................................................................................................................... 18 4.1 The FRET assay ...................................................................................................................... 18 4.1.1 Enzyme .......................................................................................................................... 18 4.1.2 Buffer conditions ........................................................................................................... 18 4.1.3 Mutant ........................................................................................................................... 19 4.1.4 Triglycine substrate ....................................................................................................... 20 4.1.5 Abz/Dnp-substrate ........................................................................................................ 21 4.2 Mass spectrometry ................................................................................................................ 23 4.3 The fibronectin-binding assay ............................................................................................... 25 4.4 Labeling of bacterial cells ...................................................................................................... 27 4.5 Inhibitors ............................................................................................................................... 29 4.5.1 HMB ............................................................................................................................... 29 4.5.2 MV079877, MV079874 ................................................................................................. 30 4.5.3 MV080057 ..................................................................................................................... 32 4.5.4 NEM, MV080100 ........................................................................................................... 33 5 Discussion ...................................................................................................................................... 35 5.1 Assays .................................................................................................................................... 35 5.2 Inhibitors ............................................................................................................................... 36 5.3 Future perspectives ............................................................................................................... 37 6 References ..................................................................................................................................... 38 7 Appendix ........................................................................................................................................ 40 7.1 Constructs .............................................................................................................................. 40 7.2 Michaelis-Menten theory ...................................................................................................... 42 7.3 Fluorescence .......................................................................................................................... 43 7.4 Inner filter effect ................................................................................................................... 43 7.5 Structures .............................................................................................................................. 44 7.6 Mass spectrometry
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