BME 236 Microbiology Past Paper HS 2024 PDF

Microbiology BME 236, HS 2024 http://www.kcl.ac.uk Hubert Hilbi, Institute of Medical Microbiology 107 [email protected]; https://www.imm.uzh.ch  Microbiology – Table of contents I. Bacterial pathogens – Example...

Microbiology BME 236, HS 2024 http://www.kcl.ac.uk Hubert Hilbi, Institute of Medical Microbiology 107 [email protected]; https://www.imm.uzh.ch  Microbiology – Table of contents I. Bacterial pathogens – Examples and sources II. Evolution of pathogenic bacteria III. Regulation of bacterial virulence IV. Bacterial transport and secretion V. Cellular microbiology / Cell biology of infection 108 V. Cellular microbiology / Cell biology of infection 1. A brief introduction to cell biology 2. Concepts of cellular microbiology 3. Cellular targets - Cell surface and receptors - Cytoskeleton - Small GTPases - Phosphoinositide lipids 4. Legionella spp.: Copycat eukaryotes 109 Cellular vesicle trafficking pathways ER: endoplasmic reticulum ERGIC: ER-Golgi intermediate compartment TGN: trans-Golgi network EE: early endosome LE: late endosome LYS: lysosome RE: recycling endosome M6PR: mannose 6-phosphate receptor TfR: transferrin receptor EEA1: EE antigen-1 LAMP1: LYS-associated membrane protein vATPase: vacuolar ATPase 110 Cellular switches – Small GTPases GEF: guanine nucleotide exchange factors GAP: GTPase activating factors GDI: guanine nucleotide displacement inhibitors Ras, Rho, Rab, Arf/Sar, Ran: Small GTPases (membrane-bound) (host cell) 111 Cellular signposts – Phosphoinositide lipids TEN: tubular endosomal network TGN: trans-Golgi network MVB: multivesicular body 112  Small GTPases, phosphoinositide lipids and SNAREs promote membrane fusion PI(3)K PI(3)K PI(3)K PI(3)K GTPase – PI-kinase/phosphatase – phosphoinositide – adaptor protein - SNARE  Concepts of cellular microbiology Pathogenic bacteria Use secretion systems (type I – VII) and secreted „effector proteins“ to manipulate their interactions with host cells. Interfere with and exploit many processes in eukaryotic host cells. Respond to different environmental (host) conditions by regulating the expression of distinct sets of genes. Constantly evolve and fine-tune their interactions with host cells. 114  Concepts of cellular microbiology Cellular Microbiology 2nd ed., Cossart et al. (2005) 115  Concepts of cellular microbiology Cellular Microbiology 2nd ed., Cossart et al. (2005) 116 Cellular targets of pathogenic bacteria , Legionella spp.) Pathogenic bacteria interfere for their own benefit with normal functions of the host cell in multiple ways. 117 Cellular targets – Cell surface and receptors 118 Cellular targets – Cell surface and receptors Bacterial factors Host cell Cell wall, surface structures Receptors - Lipopolysaccharide, lipoproteins, flagellum Pattern recognition receptors: Toll-like (TLR) receptors, mannose receptor - N-formylmethionine Formylpeptide receptor - C3b-modified bacterial components Complement receptor (binds factor C3b) Exotoxins - AB toxins: Anthrax toxin Integrin Diphteria toxin Growth factor receptor (EGF) Cholera toxin Lipid (ganglioside) Shiga toxin Lipid (ceramide) Pore-forming toxins Target structure - C. perfringens Perfringolysin O Cell membrane (cholesterol) - S. pyogenes Streptolysin O Cell membrane (cholesterol) - S. aureus α-Hemolysin Cell membrane (ADAM metallo-protease) 119  Cellular targets – Cell surface Pathogen induced anti-phagocytosis Macrophage Yersinia YopE: GTPase-activating protein (GAP) for Rho, Rac, Cdc42 YopT: Cys protease specific for RhoA (cleaves prenylated RhoA at C-terminus) Inhibition of actin polymerization und phagocytosis YopH: protein phosphotyrosine phosphatase Knodler, Celli & Finlay (2001) Nat Rev Mol Cell Biol 2: 578. 120  Cellular targets – Cell surface Pathogen-induced phagocytosis Epithelial cell Pathogen effectors: Salmonella Sip: Salmonella invasion plasmid SopE, SopE2: Rho GTPase GEF SptP: Rho GTPase GAP SopB: PI phosphatase Shigella GEF GAP Ipa: invasion plasmid antigen IpgD: PI phosphatase Knodler, Celli & Finlay (2001) Activation of GTPases and actin polymerization Nat Rev Mol Cell Biol 2: 578. 121 Cellular targets – Host cytoskeleton Pathogen effectors: Shigella Ipa: invasion plasmid antigen IpgD: PI phosphatase IcsA: actin nucleation VirA: microtubule inhibitor E. coli EspF: actin nucleation Tir: translocated intimin receptor Salmonella Sip: Salmonella invasion plasmid SopE: Rho GTPase GEF SifA: Salmonella-induced filament Legionella LegG1/PieG: RCC1 effectors, microtubule stabilization 122 Cellular targets – Host cytoskeleton (actin „tails/comets“) Shigella flexneri Listeria monocytogenes 123 Cellular targets – Host vesicle transport Pathogen effectors: Legionella SidF: PI phosphatase LepB: PI kinase RalF: Arf GTPase GEF SidM: Rab1 GTPase GEF Shigella Ipa: invasion plasmid antigen IpgD: PI phosphatase Salmonella SopB: PI phosphatase Mycobacterium LAM, PIM: bacterial lipids SapM: PI phosphatase Chlamydia IncV: Vap interactor 124 Cellular targets – Host vesicle transport Pathogen vacuoles: BCV: Brucella-containing vacuole LCV: Legionella-containing vacuole SCV: Salmonella-containing vacuole SIF: Salmonella-induced filaments Inclusion: Chlamydia vacuole 125 Cellular targets – Cytoskeleton and vesicle transport Effectors Cytoskeleton - L. pneumophila LegG1, PpgA Microtubules - S. flexneri IpaA, IpaC, IcsA Actin IpgD Phosphoinositide lipids VirA Microtubules - E. coli EspF, Tir Actin - S. typhimurium SipA, SipC Actin SopE Rho GTPases SifA Microtubules (motor proteins) Effectors Vesicle transport - L. pneumophila SidF, LepB Phosphoinositide lipids RalF, SidM Arf/Rab GTPases - L. monocytogenes Listeriolysin O, PlcA, PlcB Phospholipids - S. flexneri IpgD Phosphoinositide lipids IpaB, IpaC Cholesterol - M. tuberculosis lipids (lipoarabinomannan, LAM; phosphatidylinositolmannoside, PIM) Lipids SapM Phosphoinositide lipids - S. typhimurium SopB Phosphoinositide lipids - C. trachomatis IncV Vap protein (membrane contact sites) 126 Cellular targets – Small GTPases Pathogen effectors: GEFs: RalF (Arf), SidM (Rab1), SopE (Rac, Cdc42) GAPs: LepB (Rab1), YopE/ExoS (Rho, Rac, Cdc42) AMPylase/DeAMPylase: SidM/SidD (Rab1) Phosphocholinase: AnkX (Rab1) Dephosphocholinase: Lem3 (Rab1) (membrane-bound) (host cell) L. pneumophila: At least 7 bacterial effectors target Rab1! (SidM, LepB, SidD, AnkX, Lem3, SidC, LidA) 127 Cellular targets – Phosphoinositide lipids SidC: PtdIns4P binder SidM: PtdIns4P binder LpnE: PtdIns3P binder LidA: PtdIns3P binder MptpB: PI phosphatase SapM: PI phosphatase IpgD: PI phosphatase SopB: PI phosphatase 128 Cellular targets – Intracellular recognition / host defense PAMP / MAMP: pathogen- / microbe- associated molecular pattern 129 Cellular targets – Intracellular bacterial defense (zinc metallo- protease) 130 Cellular targets – Signal transduction and gene expression 131 Cellular targets – Intracellular recognition and defense Bacterial factors Host cell structures and processes Bacterial components Intracellular recognition Lipopolysaccharide, peptidoglycan, flagellum, Pattern recognition receptors: DNA, RNA Toll-like (TLR) / NOD-like (NLR) receptors C3b-modified bacterial components Complement receptor (factor C3b) Catalases, reductases Intracellular defense - M. tuberculosis ROS, RNS - S. typhimurium ROS, RNS Effectors Intracellular defense - M. tuberculosis Zmp1 Inflammasome - S. flexneri IcsB Autophagy - L. pneumophila RavZ Autophagy Effectors Signal transduction and gene expression - S. typhimurium SteC, SpvC MAP kinase (activation, inhibition) - S. flexneri OspF MAP kinase/ NF-B (inhibition) - L. pneumophila RomA, Lgt1-3 Histone (methylation), ribosome (inhibition) 132 Legionella spp. – the causative agents of Legionnaires’ disease http://www.kcl.ac.uk Legionella: > 65 species, L. pneumophila causes 90% of clinical infections Gram-negative, motile (most species), facultative intracellular, strictly aerobic bacteria Genetic tools: Plasmids, defined mutants, >70 genomes, microarray, RNAseq Therapy: Chinolones (Levofloxacin, Ciprofloxacin), Macrolides (Azithromycin) Community outbreaks of Legionnaires’ disease Year Location Source Cases Fatality rate Cooling tower/ 1976 Philadelphia, USA 221 15% air conditioner 1999 Bovenkarspel, Holland Whirlpool 242 12% 2001 Murcia, Spain Cooling tower 449 1% Cooling tower/ 2004 Pas-de-Calais, France 86 21% petrochemical plant 2005 Toronto, Canada Cooling tower 127 17% 2010 Ulm, Germany Cooling tower 64 8% (105-1010 cfu Lpn per liter) Sewage plant, 2013 Warstein, Germany 165 2% cooling tower Vila Franca de Xira, 2014 Cooling tower 336 3% Portugal Nguyen et al. (2006) J Inf Dis 193: 102. Legionella is an accidental pathogen, which primarily infects elderly and immuno-compromised persons Environmental cycle and pathogenesis of L. pneumophila 135 Interaction of Acanthamoeba with Legionella biofilm Hochstrasser et al. (2022) Environ Microbiol 24: 3672. 136 Interaction of Acanthamoeba with Legionella biofilms WT WT sinR 137 Bacterial clusters on Acanthamoeba in Legionella biofilms 138 Legionella pneumophila: a copycat eukaryote Hilbi & Buchrieser (2022) Microbiology 168: doi: 10.1099/mic.0.001142. 139  Cellular models to study the virulence of Legionella Human or murine macrophages and epithelial cells - Cell lines (RAW264.7, A549) and primary cells - Relevant for Legionella pathogenesis Horwitz & Silverstein (1980) J Clin Invest Acanthamoeba castellanii - Natural host of L. pneumophila - Easy to handle, robust system Gao et al. (1997) Infect Immun Dictyostelium discoideum - Haploid genome, genetically tractable - Genetic, biochemical and cell biological tools Hägele et al. (2000) Cell Microbiol 140 Legionella pneumophila: a copycat eukaryote 141  Legionella RCC1 effectors are encoded by eukaryotic genes Legionella spp. harbor eukaryotic genes Legionella spp. harbor genes encoding eukaryotic proteins and domains Phylogenetic tree of the RCC1 repeat protein PieG from Legionella species and homologous sequences RCC1: Eukaryotic Regulator of Chromosome Condensation-1 (Ran GEF) 142  Legionella RCC1 effectors target the microtubule cytoskeleton RCC1 effectors in Legionella spp. RCC1 effectors: PpgA, PieG, LegG1 Swart et al. (2020) Cell Microbiol 22: e13246. L. pneumophila effectors target small GTPases L. pneumophila effectors: GEFs: RalF (Arf), SidM (Rab1) GAPs: LepB (Rab1) AMPylase/DeAMPylase: SidM/SidD (Rab1) Phosphocholinase: AnkX (Rab1) Dephosphocholinase: Lem3 (Rab1) (membrane-bound) (host cell) L. pneumophila: At least 7 bacterial effectors target Rab1! (SidM, LepB, SidD, AnkX, Lem3, SidC, LidA) 144 Legionella effectors target the ubiquitination system A) SidC, LubX, GobX and RavN effectors are E3 ubiquitin ligases B) SidE effector family (SidE, SdeA, SdeB, SdeC) Komander & Randow (2017) Cell Host Microbe 21:127. 145 Most bacteria are killed in phagolysosomes Endosomes Golgi Arf1 Rab1 Phagosome Phago- lysosome Calnexin ER Legionella spp. – The prey becomes predator http://www.neave.com/webgames/pacman (1980) Legionella replicates within a unique vacuole Endosomes Icm/Dot Golgi Arf1 Icm/Dot Rab1 Calnexin Icm/Dot ER Icm/Dot Arf1 Rab1 Calnexin Icm/Dot Formation of the Legionella-containing vacuole Steiner et al. (2017) EMBO Rep 18: 1817.  Isolation of the Legionella-containing vacuole Legionella-containing vacuole (endoplasmic reticulum-associated) 2 m Urwyler et al. (2009) Traffic 10: 76. Swart et al. (2020) mBio Hoffmann et al. (2014) Cell Microbiol 16: 1034. Rothmeier et al. (2013) PLoS Pathogens Schmölders et al. (2017) Mol Cell Proteomics 16: 622. Vesicle trafficking and phosphoinositide metabolism PI(3,4,5)P 3 Endosomes PI(4,5)P OCRL1 PI4K 2 PI(4)P Golgi PI(3)P Arf1 Rab1 PI3K Calnexin ER  The LCV The LCV is aisPtdIns(4)P-positive a PtdIns(4)P-positive replicative compartment replicative compartment PtdIns(3)P and PtdIns(4)P PtdIns(4)P and calnexin Weber et al. (2014) mBio 5: e00839-13.  Dually labeled D. discoideum: The LCV undergoes phosphoinositide conversion PtdIns(3)P (green) and PtdIns(4)P (red) Dually labeled D. discoideum: PtdIns(3)P (green) and PtdIns(4)P (red) WT, 15 min WT, 45 min icmT, 45 min Weber et al. (2018) mBio 9: e02420-18. 153  Dually labeled D. discoideum: L. pneumophila effectors bind phosphoinositides PtdIns(3)P (green) and PtdIns(4)P (red) Swart & Hilbi (2020) Front Immunol 11: 25. 154  Dually labeled D. discoideum: L. pneumophila effectors metabolize phosphoinositides PtdIns(3)P (green) and PtdIns(4)P (red) MavQ: PtdIns 3-kinase, yields PtdIns(3)P LepB: PtdIns(3)P 4-kinase, yields PtdIns(3,4)P2 SidF: PtdIns(3,4)P2 3-phosphatase, yields PtdIns(4)P Li et al. (2021) EMBO Rep 22: e51163. 155 L. pneumophila – Virulence and communication Dually labeled D. discoideum: PtdIns(3)P (green) and PtdIns(4)P (red) PpgA LppA  Dually labeled D. discoideum: Exit of motile L. pneumophila from LCVs and host cells PtdIns(3)P (green) and PtdIns(4)P (red) Striednig et al. (2021) EMBO Rep 22: e52972. 157 Microbiology – Intricate interactions among the domains of life Dually labeled D. discoideum: PtdIns(3)P (green) and PtdIns(4)P (red) Clinical microbiology Molecular Cellular microbiology microbiology Environmental microbiology 158