DOCTORAL THESIS Hijacking of host cellular functions in Staphylococcus aureus pathogenesis Natalia, Bravo-Santano Award date: 2019 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 06. Oct. 2021 Hijacking of host cellular functions in Staphylococcus aureus pathogenesis by Natalia Bravo-Santano, BSc, MRes A thesis submitted in partial fulfilment of the requirements for the degree of PhD Department of Life Sciences University of Roehampton 2019 A mi familia y a Pablo, por estar siempre ahí Abstract Staphylococcus aureus is a facultative intracellular pathogen that invades and replicates within many types of human cells. S. aureus has shown to rapidly overcome traditional antibiotherapy by developing multidrug antibiotic resistance. Furthermore, intracellular S. aureus is protected from the last resort antibiotics – vancomycin, daptomycin and linezolid – since they are unable to achieve intracellular killing. Therefore, there is an urgent need to develop novel anti-infective therapies against S. aureus. Here, I proposed the exploitation of host-directed approaches as a promising strategy. Specifically, I employed three different approaches to unravel those host molecular factors and/or pathways hijacked by MRSA during intracellular infection. By characterizing the host cell metabolism after MRSA infection, I observed several metabolic changes suggesting a starvation-induced autophagic flux and AMPK phosphorylation levels were also increased in MRSA-infected cells. It was hypothesized that intracellular S. aureus induces autophagy for energy generation and nutrient scavenging. Accordingly, host AMPK-inhibition halted S. aureus intracellular proliferation. I further screened 140 host-directed drugs and found three host-directed tyrosine kinase inhibitors – Ibrutinib, Dasatinib and Crizotinib – that substantially impaired intracellular bacterial survival. Particularly, Ibrutinib increased host cell viability after S. aureus infection via inhibition of intracellular bacterial invasion or proliferation and we confirmed the importance of the host receptor EPHA2 for staphylococcal infection. Lastly, I performed a genome-wide shRNA screen to test the importance of 16,000 host genes and identified several host genes important for intracellular MRSA infection. Specifically, I found that silencing the human gene TRAM2 resulted in a significant reduction of intracellular MRSA. TRAM2 is associated with the endoplasmic reticulum SERCA pumps and accordingly, treatment with the SERCA- inhibitor Thapsigargin halted intracellular MRSA proliferation. In summary, I identified three different host-directed drugs – Dorsomorphin, Ibrutinib and Thapsigargin – that impaired intracellular MRSA infection. These findings serve as an important example of feasibility for identifying host-directed therapeutics to tackle S. aureus host cell infection. i Table of contents Abstract Table of contents List of figures List of tables Acknowledgements Author´s declaration Common abbreviations 1. INTRODUCTION .................................................................................................. 1 1.1. Biology of Staphylococcus aureus.............................................................. 1 1.2. S. aureus infections and epidemiology.................................................... 3 1.2.1. Skin and Soft Tissues Infections (SSTI) .......................................... 3 1.2.2. Infective Endocarditis ..................................................................... 3 1.2.3. Osteomyelitis .................................................................................. 4 1.2.4. Toxic shock syndrome (TSS) .......................................................... 4 1.2.5. Pulmonary infections....................................................................... 5 1.2.6. S. aureus bacteraemia ...................................................................... 6 1.3. Emergence of methicillin-resistant S. aureus (MRSA) ............................ 7 1.3.1. Healthcare-associated vs community-associated MRSA .................. 8 1.4. Pathogenesis of S. aureus infections ..................................................... 10 1.4.1. Surface-associated proteins ........................................................... 11 1.4.1.1. Staphylococcal protein A (SpA) ................................................ 12 1.4.1.2. Fibronectin-binding proteins (FnBPs) ........................................ 13 1.4.1.3. Collagen-binding protein (Cna) ................................................. 14 1.4.1.4. Clumping factors (Clfs) ............................................................. 14 ii 1.4.1.5. Elastin binding protein (EbpS) ................................................... 15 1.4.2. Secreted proteins ........................................................................... 15 1.4.2.1. Superantigens ............................................................................ 16 1.4.2.2. Pore-forming toxins ................................................................... 17 1.5. Regulation of S. aureus virulence factors during infection .................... 20 1.6. Biofilm formation................................................................................. 22 1.7. Intracellular fate of Staphylococcus aureus ........................................... 24 1.7.1. S. aureus internalization into non-phagocytic cells ........................ 25 1.7.2. Intracellular persistence, the role of Small Colony Variants (SCVs) ...................................................................................................... 28 1.7.3. S. aureus phagosomal escape ........................................................ 30 1.7.4. Host-cell death induced by S. aureus ............................................. 31 1.8. Clinical management of S. aureus infections ........................................ 33 1.9. Host-directed approaches, a new perspective ........................................ 35 1.10. Justification and aims........................................................................ 43 2. MATERIALS AND METHODS ........................................................................ 46 2.1. Microbiology ....................................................................................... 46 2.1.1. Bacterial strains and culture conditions.......................................... 46 2.1.2. Preparation of bacterial stock ........................................................ 47 2.1.3. Bacterial pre-inoculum preparation for intracellular assays ............ 47 2.1.4. In-vitro growth assays ................................................................... 47 2.2. Cell culture........................................................................................... 50 2.2.1. Cell lines and culture conditions .................................................... 50 2.2.2. Transduction of mammalian cells .................................................. 53 2.2.3. Intracellular infection assays ......................................................... 55 iii 2.2.4. Host cell viability assay ................................................................. 57 2.2.5. Intracellular bacterial estimation.................................................... 58 2.3. Molecular biology ................................................................................ 60 2.3.1. Polymerase chain reaction (PCR) .................................................. 60 2.3.2. Agarose gel electrophoresis ........................................................... 61 2.3.3. DNA extraction ............................................................................. 63 2.3.3.1. Plasmid extraction ..................................................................... 63 2.3.3.2. Bacterial DNA extraction .......................................................... 63 2.3.3.3. Mammalian DNA extraction ...................................................... 64 2.3.4. Molecular cloning and DNA manipulation .................................... 64 2.3.4.1. pCRII-TOPO cloning ................................................................ 65 2.3.4.2. Bacterial transformation ............................................................ 66 2.3.4.3. DNA digestion........................................................................... 66 2.3.4.4. Dephosphorylation of P12-MMP vector..................................... 67 2.3.4.5. DNA ligation ............................................................................. 67 2.3.4.6. Retroviral production and HeLa transduction ............................. 68 2.3.5. ShRNA screening .......................................................................... 70 2.4. Immunoproteomics .............................................................................. 70