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Molecular and Functional Characterization of the Long Non-Coding RNA SSR42 in Staphylococcus Aureus

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Molecular and Functional Characterization of the Long Non-Coding RNA SSR42 in Staphylococcus Aureus Molecular and functional characterization of the long non-coding RNA SSR42 in Staphylococcus aureus DISSERTATION zur Erlangung des naturwissenschaftlichen Doktorgrades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Fakultät für Biologie der Julius-Maximilians-Universität Würzburg vorgelegt von M.Sc. Biol. Jessica Horn geboren am 01.11.1989 in Würzburg Würzburg, 2018 Eingereicht am: Mitglieder der Promotionskommission: Vorsitzender: Erstgutachter: Prof. Dr. Thomas Rudel Zweitgutachter: Prof. Dr. Cynthia Sharma Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am: 2 TABLE OF CONTENT TABLE OF CONTENT 1 SUMMARY…………………………………………………………………………………………….…………………………….…...10 1.1 Abstract…………………………………………………………………………………………………..………………………………….10 1.2 Zusammenfassung……………………………………………………………………………………………………………………...11 2 INTRODUCTION……….…..……………………………………………………………………………………………………………13 2.1 Staphylococcus aureus………………………………………………………………………………………………………………..13 2.1.1 General Information………………………………………………………………………………………………………………..13 2.1.2 Host colonization………………………………………………….………………………………………………………………….14 2.1.3 Methicillin-resistant Staphylococcus aureus (MRSA)………………………………………………………………..15 2.1.4 S. aureus infections and diseases……………………………………………………………………………………………..16 2.2 Virulence factors of S. aureus……………………………………………………………………………………………..………18 2.2.1 Surface-associated virulence factors………………………………………………………………………………………..19 2.2.1.1 Teichoic acids…………………………………………………………………………………………………………………….….19 2.2.1.2 Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)………….19 2.2.1.3 Secretable expanded repertoire adhesive molecules (SERAMs) ……………………………………………21 2.2.2 Secreted virulence factors……………………………………………………………………………………………………….22 2.2.2.1 Pore-forming toxins………………………………………………………………………………………………………………22 2.2.2.2 Immune evasion proteins……………………………………………………………………………………………………..25 2.2.3 Iron acquisition………………………………………………………………………………………………………………………..26 2.3 Intracellular virulence and persistence of S. aureus…………………………………………………………………….27 2.3.1 Adhesion………………………………………………………………………………………………………………………………….27 2.3.2 Intracellular fate of S. aureus…………………………………………………………………………………………….…….27 2.4 Regulation of virulence……………………………………………………………………………………………………………….30 2.4.1 Two-component systems…………………………………………………………………………………………………………30 2.4.1.1 The agr quorum-sensing system……………………………………………………………………………………………30 3 TABLE OF CONTENT 2.4.1.2 The SaeRS two-component system……………………………………………………………………………………….32 2.4.1.3 Further two-component systems regulating virulence………………………….………………………………34 2.4.2 SarA-family of transcriptional regulators……………………………………………………………………………….…35 2.4.3 Alternative σ-factors………………………………………………………………………………………………………………..36 2.4.4 Repressor of surface proteins (Rsp) …………………………………………………………………………………………37 2.4.5 Regulatory link between metabolism and virulence regulation………………………………………………..38 2.5 Non-coding RNAs………………………………………………………………………………………………………………………..39 2.5.1 Gene regulation by non-coding RNAs……………….……………………………………………………………………..39 2.5.2 RNA-binding protein Hfq………………………………………………………………………………………………………….41 2.5.3 RNA-turnover…………………………………………………………………………………………………………………………..42 2.5.4 Non-coding RNAs in Staphylococcus aureus…………………………………………………………………………….42 2.6 Aim of the study………………………………………………………………………………………………………………………….45 3 MATERIAL AND METHODS……….…………………………………….……………………………………………………..….46 3.1 Material……………………………………………………………………………………..…………………………………………..….46 3.1.1 Bacterial strains……………………………….…………………………………………………………………………………..….46 3.1.2 Plasmids………………………………………………………………………………………………………………………..……..….56 3.1.3 Cell lines……………………………………………………………………………………….……………………………………..….59 3.1.4 Oligonucleotides and Northern blot probes………………………………………………………………….……..….59 3.1.5 Media and buffers………………………………………………………………………………………….…………….……..….65 3.1.6 Antibiotics and chemicals………………………………………………………………………………….………….……..….70 3.1.7 Enzymes………………………………………………………………………………………………………………………..……..….72 3.1.8 Size standards………………………………………………………………….…………………………………………………..….72 3.1.9 Kits…………………………………………………………….…………………….…………………………………………………..….73 3.1.10 Technical equipment……………………………….…………………….…………………………………………………..….73 3.1.11 Applied Software………………………………………………………………….…………….……………………………..….74 4 TABLE OF CONTENT 3.2 Methods…………………………………………………………………………….…………………………………………………..….75 3.2.1 Cultivation of bacteria……………………………………………….…….…………………………………………………..….75 3.2.1.1 Bacterial culture conditions……………………………………………….……………………………………………..….75 3.2.1.2 Bacterial cryo-stocks for preservation………………………………………..……………………………………..….75 3.2.1.3 Bacterial growth curves and determination of minimum inhibitory concentration (MIC)……..75 3.2.1.4 Determination of MIC of antibiotics on agar plates………………………………………………………….…..75 3.2.2 Genetic manipulation of bacteria…………………………………………………………………………….……………...76 3.2.2.1 Preparation of competent E. coli……………..………………………………………………………………………..….76 3.2.2.2 Transformation of E. coli……………………………………………………………………………………………………….76 3.2.2.3 Preparation of electro-competent S. aureus………………………………………………………………….…..…76 3.2.2.4 Electroporation of S. aureus…………………………………………………………………………………….………..….76 3.2.2.5 Transduction of DNA via phage transduction………………………………………………………………..………77 3.2.3 Desoxyribonucleic acid techniques………………………………………………..…………………………………..……77 3.2.3.1 Isolation of Plasmid DNA from E. coli……..…………………………………..…………………………………..……77 3.2.3.2 Isolation of Plasmid DNA from S. aureus……..…………………………………..……………………………..……77 3.2.3.3 Isolation of genomic DNA from S. aureus…………………………………………………………………..………...78 3.2.3.4 Polymerase-Chain-Reaction (PCR)…………………………………………………………………………………………78 3.2.3.5 Quickchange mutagenesis PCR …………….………………………………………………………………………………78 3.2.3.6 Annealing of oligonucleotides……………………………………………………………………………………………….79 3.2.3.7 Phosphorylation of DNA……………………………………………………………………………………………………….79 3.2.3.8 Cloning of DNA into a vector…………………………………………………………………………………………………79 3.2.3.9 In-Fusion cloning …………………………………………..……………………………………………………….…..…..……79 3.2.3.10 Generation of targeted deletion mutants in S. aureus…………..……….………..…………………………79 3.2.3.11 Construction of plasmids used in this study.…………………………………………….…………………………80 3.2.4 Ribonucleic acid techniques……………………………………………………………………..……………………………..83 5 TABLE OF CONTENT 3.2.4.1 RNA isolation……………………………………………………………………………………………..…………………………83 3.2.4.2 Generation of cDNA via reverse transcription……………………………………………………………….………83 3.2.4.3 Quantitative real time PCR (qRT-PCR).…………………………………………………………………..…….…….…84 3.2.4.4 Northern blotting………………………………………………………………………..……………..…………………………84 3.2.4.5 Investigation of transcript stabilities by rifampicin assay…………………….…………………………..……85 3.2.4.6 RNA-Sequencing (RNA-seq)…………………………………………………………………..………………..…………….85 3.2.4.7 Gradient profiling by sequencing (Grad-seq) …………………………………………………………………..……85 3.2.4.8 RNA pulldown approaches……………………………………………………………………………………….………..…85 3.2.5 Investigation of promotor activities…………………………………………………………………..……………….……86 3.2.6 Investigation of haemolytic capacities in S. aureus………………………………….……………………………….87 3.2.7 Protein techniques……………………………………………………………………..……………………………………………87 3.2.7.1 Isolation of bacterial proteins…………………………….………………………………………….……………………..87 3.2.7.2 Bradford assay…………………………………………………………………..……………………………………………….…88 3.2.7.3 SDS PAGE…………………………………………………………………..……………………………………………………….…88 3.2.7.4 Staining of proteins………………………………………………………………………………..………………………….…88 3.2.7.5 Western blotting…………..………………………………………………..…………………………………………….………88 3.2.7.6 Mass spectrometry analysis ………………..…..…………………..…………………………………………….…..……89 3.2.8 Cell culture techniques…………………………………………………………………………………………………….………89 3.2.8.1 Cultivation of cell lines…………………………………………………………………..……………………………..………89 3.2.8.2 Infection of HeLa cells with S. aureus…………………………………………………………………..……….………90 3.2.8.3 CFU enumeration…………………………………………………………………..……………………………….……….……90 3.2.8.4 Cytotoxicity assay (LDH release assay) …………………………………………………………………..…………….90 3.2.8.5 Long-term infection of EA.Hy926 cells……………………………………………….………………………….………90 3.2.9 In vivo infection models…………………………………………………………………..…………………………..….………91 3.2.10 Statistics………………………………………………………………..………..…………………………………………….………91 6 TABLE OF CONTENT 4 RESULTS………….…………………………………….…………..…………………………………………….……………….………92 4.1 Characterization of SSR42, a long ncRNA of S. aureus…………………………………………………………………92 4.1.1 SSR42 sequence conservation…………………………………………………………………..……………………..………92 4.1.2 The genomic locus of SSR42 in S. aureus……………………………………………………………………….…………93 4.1.3 Evidence for full-length SSR42 and procession by RNase Y………………………………………….…………..93 4.1.4 Putative protein-coding sequences in SSR42…………………………………………………………………..……….97 4.1.5 Secondary structure analysis…………………………………………………………………………………………..……….99 4.2 Regulation of haemolysis by Rsp and SSR42………………………………….…………………………………..….….102 4.2.1 SSR42 is required for wild-type haemolysis in S. aureus 6850………………………………………...……..102 4.2.2 Induced transcription of SSR42 is sufficient for hla transcription but not for haemolysis……….105 4.2.3 SSR42 is required for restoring haemolysis in a ΔSSR42-rsp mutant……………………..………..……..106 4.3 ncRNA SSR42 is the main effector of Rsp-mediated virulence in S. aureus…………………….…..……..109 4.3.1 ncRNA SSR42 influences transcription of a plethora of virulence factors………..……………………..110 4.3.2 ncRNA SSR42 influences the protein levels of a multitude of virulence factors………………….…..115 4.3.3 Role of ncRNA in S. aureus-induced cell death……………………….….……….….……….….…………….…..118
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