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S. aureus is an opportunistic pathogen with an extensive host range, and is the etiologic agent of a wide variety of human diseases, with the ability to promote disease in most human tissues. A repertoire of virulence factors expressed during colonization and infection all contribute to the success of S. aureus as a pathogen. S. aureus is not classically considered as an intracellular pathogen, yet accumulating scientific evidence has demonstrated that this organism has intracellular survival strategies, with the ability to become internalised by, and persist within, a wide range of non-professional eukaryotic phagocytes. This phenomenon has been shown to be linked to the expression of genes under the regulation of the S. aureus quorum sensing system, agr. Specifically, S. aureus has been shown to breach the host-membrane bound endosome within which it is situated upon internalisation, a process which has been hypothesised to rely upon a switch in gene expression due to the accumulation of the agr signalling peptide within the endosome. Many in vitro imaging modalities have been designed to investigate further the mechanisms behind S. aureus internalisation. However, in vivo systems such as bioluminescence and fluorescence reporter genes are limited by various factors including light extinction, restricting investigations to those using small animals. Additionally, as fundamentally two-dimensional modalities, useful anatomical information is minimal in these imaging techniques. At present few imaging modalities exist which enable the non-invasive in vivo detection of bacterial reporter gene expression while offering detailed anatomical information. This study aimed to address the need to develop reporter systems for the non-invasive in vivo tracking systems for the monitoring of infectious disease processes, in the context of using such a technology to investigate further the genes involved in the process of S. aureus internalisation, and more specifically, endosomal escape. Although it is clear that agr regulated genes have a role in endosomal escape of internalised S. aureus, it is not fully clear which genes specifically are important. A number of haemolysin genes are under the control of the agr system, all of which have the ability to disrupt eukaryotic cell membranes. A β-haemolysin mutant was constructed in this study to use in conjunction with existing α-, δ- and γ-haemolysin mutants in in vitro internalisation assays. Initial experiments indicated a role for α-haemolysin in the endosomal escape of internalised S. aureus. The use of an in vivo reporter gene system would allow for further analysis of the role of haemolysins in endosomal escape, in addition to providing detailed anatomical information. MRI bacterioferritin (BFR) reporter genes with inducible or constitutive promoters, the bfr gene and the lux operon, were constructed and evaluated in S. aureus. The reporter gene was shown to be translated in vitro, with the expression of functional BFR which collected iron. However, ICP-MS data revealed relatively low levels of BFR. Pilot in vivo studies were carried out to confirm the potential of the reporter gene for studying specific aspects of staphylococcal disease. Experiments tracking bioluminescence in a mouse tumour model demonstrated expression of the MRI reporter genes, suggesting that bacterioferritin should be successfully synthesised in vivo. However, due to plasmid instability in vivo and the relatively low levels of iron present in S. aureus samples as determined by ICP-MS, the tumours in this study were not scanned by MRI.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 1.0: INTRODUCTION
    • 1.1: Non-Invasive in vivo Molecular Imaging……………………....1 1.1.1: Non-Invasive Optical in vivo Imaging………………….. 2 1.1.1.1: Fluorescence…………………………………….2 1.1.1.2: Bioluminescence……………………………….. 3 1.1.2: Non-Invasive Nuclear in vivo Imaging…………………. 4 1.1.3: Non-Invasive Magnetic Resonance in vivo Imaging….. 5 1.1.3.1: Magnetic Resonance Imaging…………………5 1.1.3.2: Contrast…………………………………………..7 1.1.3.3: Magnetic Resonance Imaging as a Tool……..8 for Non-Invasive in vivo Imaging
    • 1.2: Bacterioferritin as an MRI Reporter…………………………….10 1.2.1: Ferritins…………………………………………………….10 1.2.2: The use of Ferritins as MRI Reporters in vivo…………11
    • 1.3: Staphylococcus aureus………………………………………….. 12 1.3.1: Background………………………………………………..12 1.3.2: Virulence…………………………………………………...14 1.3.3: Quorum Sensing and Virulence Gene Regulation…….15 1.3.3.1: Response to Cell Density………………………16 1.3.3.2: Response to External Stimuli…………………. 17 1.3.4: S. aureus Haemolysins…………………………………..17 1.3.5: S. aureus as an Intracellular Organism………………...19
    • 1.4: MultiSite Gateway® Technology……………………………….. 21
    • 1.5: Aims and Objectives………………………………………………23
    • 2.0: MATERIALS AND METHODS
    • 2.1: Bacterial Strains and Plasmids………………………………….25
    • 2.2: Chemical Reagents…………………………………………......... 32
    • 2.3: Growth Media……………………………………………………….32 2.3.1: Luria-Bertani Medium…………………………………….32 2.3.2: Brain Heart Infusion……………………………………...32 2.3.3: Tryptic Soy Broth………………………………………….33 2.3.4: LK Medium………………………………………………...33 2.3.5: SMMP50…………………………………………………...33
    • 2.4: Supplements………………………………………………………..34
    • 2.5: Growth Conditions…………………………………………………34
    • 2.6: DNA Manipulations……………………………………………….. 35 2.6.1: Genomic DNA Purification……………………………….35 2.6.2: Plasmid Preparation……………………………………...35 2.6.3: Restriction Digests………………………………………..35 2.6.4: Construction of Entry Clones…………………………… 35 2.6.5: LR Recombination Reactions……………………………36 2.6.6: PCR Clean Up…………………………………………….36
    • 2.7: Agarose Gel Electrophoresis…………………………………… 36
    • 2.8: Polymerase Chain Reaction…………………………………….. 37 2.8.1: Primers……………………………………………………. 37 2.8.2: General PCR Parameters………………………………..38 2.8.3: AccuPrime™ Taq DNA Polymerase……………………. 39
    • 2.9: Transfer of Plasmid DNA into Bacterial Cells………………...40 2.9.1: Dialysis of DNA, BP and LR Reactions………………...40 2.9.2: Preparation of Electrocompetent E. coli………………..40 2.9.3: Electroporation of Plasmid DNA into E. coli……………40 2.9.4: Preparation of Electrocompetent S. aureus……………41 2.9.5: Electroporation of Plasmid DNA into S. aureus……….41 2.9.6: Phage Transduction……………………………………...42
    • 2.10: Protein Visualisation using SDS-PAGE Gel………………….43 2.10.1: Preparation of Cell Lysates from E. coli……………….43 2.10.2: Preparation of Cell Lysates from S. aureus………….. 43 2.10.3: SDS-PAGE Gel Electrophoresis………………………. 44 2.10.4: Coomassie Staining…………………………………….. 45
    • 2.11: Western Blotting………………………………………………….45 2.11.1: Electrophoresis………………………………………….. 45 2.11.2: Ponceau Staining……………………………………….. 46 2.11.3: Antibody Staining………………………………………...46 2.11.4: Enhanced Chemiluminescence (ECL) Developing…..46
    • 2.12: Bacterial Mutagenesis…………………………………………...47 2.12.1: Construction of Knockout Mutants……………………..47 2.12.2: Excision of the ErmR Gene……………………………...47
    • 2.13: Iron Quantification………………………………………………. 48 2.13.1: Iron Assay………………………………………………...48 2.13.2: Inductively Coupled Plasma Mass Spectrometry…….48
    • 2.14: Haemolysin Assay………………………………………………..49
    • 2.15: Tissue Culture and Cell Invasion Assay……………………..49 2.15.1: Maintenance of Cell Line………………………………..49 2.15.2: Trypsinisation of Monolayer Cultures………………….50 2.15.3: Cell Invasion Assay……………………………………...50
    • 2.16: Animal Work……………………………………………………….51
    • 3.0: CONSTRUCTION AND IN VITRO EVALUATION OF
    • 3.1: Objectives……………………………………………………………53
    • 3.2: Introduction…………………………………………………………53 3.2.1: Promoters: P3, PS10 and PahpC………………………….. 53 3.2.2: E. coli Bacterioferritin……………………………………. 55 3.2.3: The lux Operon……………………………………………55
    • 3.3: Results……………………………………………………………….56 3.3.1: Construction and Evaluation of Expression Vectors….56 with various Constitutive Promoters 3.3.2: Construction of pUNK1 PS10-bfr-luxterm, pUNK1……..59 P3-bfr-luxterm and pUNK1 PahpC-bfr-luxterm for use as MRI Reporters in S. aureus 3.3.3: Evaluation of pUNK1 PS10-bfr-luxterm, pUNK1………..60 P3-bfr-luxterm and pUNK1 PahpC-bfr-luxterm 3.3.3.1: Growth Curves…………………………..……...61 3.3.3.2: SDS-PAGE of S. aureus Lysates from...……..63 Reporter Constructs 3.3.3.3: Western Blots……………………………………64 3.3.3.4: Sequencing………………………………………66 3.3.3.5: ICPMS…………………………………………… 68
    • 3.3.4: Construction of pUNK1 PS10-bfd/bfr-luxterm and……...69 pUNK1 P3-bfd/bfr-luxterm for use as MRI Reporters in S. aureus
    • 3.3.5: Evaluation of pUNK1 PS10-bfd/bfr-luxterm and………...70 pUNK1 P3-bfd/bfr-luxterm 3.3.5.1: SDS-PAGE of E. coli and S. aureus…..……....70 Lysates from bfr and bfd/bfr Reporter Constructs 3.3.5.2: Iron Assay………………………………..………74 3.3.5.3: ICP-MS…………………………………...………75 3.3.5.4: Evaluation of Plasmid Loss…………….………76 3.3.5.5: Sequencing………………………………………77 3.3.5.6: Bioluminescence Assays to Determine……….78 any Interference of Transcription or Translation due to bfd Gene
    • 6.0: DISCUSSION
    • 6.1: Construction and in vitro Evaluation of an MRI Reporter…. 120 in S. aureus
    • 6.2: Construction and Characterisation of a hlb Mutant and……127 Characterisation of Existing Haemolysin Mutants in S. aureus
    • 6.3: In vivo Evaluation of an S. aureus MRI Reporter in Mice….. 130
    • Abdelnour, A., Arvidson, S., Bremell, T., Ryden, C., and Tarkowski, A. (1993) The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model. Infect Immun 61: 3879- 3885.
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