BME 236 Microbiology Past Paper 2024 PDF

Microbiology BME 236, HS 2024 http://www.kcl.ac.uk Hubert Hilbi, Institute of Medical Microbiology 57 [email protected]; https://www.imm.uzh.ch  Microbiology – Table of contents I. Bacterial pathogens – Examples...

Microbiology BME 236, HS 2024 http://www.kcl.ac.uk Hubert Hilbi, Institute of Medical Microbiology 57 [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 58  III. Regulation of bacterial virulence 1. Environmental cues - temperature, pH, ions, osmolarity, nutrients 2. Host cells - contact, intracellular conditions 3. Bacterial cell density (quorum sensing) 4. Phenotypic heterogeneity and persistence 5. Inter-kingdom signaling 59 Regulation – Central dogma of molecular biology DNA polymerase RNA polymerase Ribosome 60  Regulation – Transcription Coordinated control by transcription factors and sigma factors Stimulon Regulon Operon RNA polymerase Transcription factor Transcription start Sigma subunits of RNA polymerase bind to different promoters. Transcriptional regulators/factors (RegA, RegB) Sigma-70 (RpoD, house keeping) bind to operator sequences near promoters and Sigma-32 (RpoH, heat shock) act as activators or repressors. Sigma-54 (RpoN, nitrogen limitation) Transcription factors are active as dimers, Sigma-38 (RpoS, stress/stationary phase) DNA-binding motif: helix-turn-helix (HTH). 61 Regulation by temperature – Shigella flexneri 37 °C Transcriptional regulation Regulatory proteins: H-NS: chromosomally encoded temperature sensor (repressor) VirF: master activator (AraC-like) VirB: subordinate activator CpxA/CpxR: two-component system (pH sensor) EnvZ/OmpR: two-component system (osmolarity sensor) Virulence plasmid Target genes grouped in operons: IpaADCB: effector proteins 37 °C, IpgC: chaperone logarithmic growth Mxi-Spa: type III secretion system phase Many Enterobacteriaceae and other pathogens upregulate virulence at 37 °C: Shigella, EIEC, UPEC, Salmonella, Yersinia, Listeria 37 °C 62 Regulation by temperature – Listeria monocytogenes Listeria monocytogenes Translational regulation: Riboswitch Ribosome 37 °C UTR 30 °C PrfA: Positive regulatory factor A S.D.: Shine-Dalgarno ribosome-binding sequence Johansson et al. (2002) Cell 110: 551. 63  (pH, ions, osmolarity) Global control: Two-component systems (TCS) Sensor kinase: His N-terminal signal-sensing domain, C-terminal signal- transmission domain (His). Response regulator: N-terminal signal receiver domain (Asp), C-terminal DNA-binding domain (helix-turn-helix). Asp 64  Regulation by pH, ions, osmolarity Two-component systems Fe 3+ OmpR/EnvZ PmrA/PmrB PhoP/PhoQ Sensor kinase Response regulator PhoP-activated genes OmpR/EnvZ: required for Salmonella infection PhoP-repressed genes and S. flexneri virulence. PhoP/PhoQ: required for survival of Salmonella in macrophages (low Mg2+). 65 Regulation by nutrient availability (starvation) Phenotypic switch in response Stringent response model of virulence regulation to growth conditions (uncharged tRNA) exponential post-exponential tetraphosphate guanosine Legionella pneumophila starvation signal „alarmone“ /“TRANSMISSIVE” Bachman & Swanson (2001) Mol. Microbiol. 40: 1201. 66  Regulation by host cells – Contact, intracellular conditions 1. Adhesion of bacteria, formation of pseudopods 2. Phagocytosis, formation of phagosome 3. Endocytosis, fusion of phagosome with endosome and lysosomes, formation of phagolysosome 4. Degradation of bacteria in phagolysosome 5. Exocytosis, release of bacterial fragments, antigen presentation Elie Metchnikoff, 1908 Nobel Prize © 1997, Duane W. Sears (phagocytosis, macrophages; probiotics, gerontology) 67 Regulation by host cells (contact) – Yersinia Yersinia pseudotuberculosis contacting epithelial cells activates the yopE promotor fused to the luxAB reporter operon. DIC image Merge Light Pettersson et al. (1996) Science 273: 1231. 68 Regulation by host cells (intracellular conditions) – Salmonella Salmonella Typhimurium within macrophages expresses ssaH gene fused to gfp. DIC image Fluorescence Merge Pathogenicity island-2 (SPI-2) components (type III secretion system, effector and regulatory proteins) are preferentially induced within macrophages. Transcription of these genes is dependent on SsrA/SsrB (not PhoP/PhoQ) two-component system located in SPI-2 and an acidic phagosomal environment. Valdivia & Falkow (1997) Science 277: 2007. 69  Regulation by host cells (TCS) – Salmonella Salmonella Typhimurium 1) Regulation of Virulence 2) Adaption to Mg2+ limitation 3) Modification of cell envelope (LPS, PG) 4) Positive feedback loop, crosstalk Groisman (2001) J. Bacteriol. 183: 1835. (phoPQ, pmrAB, pmrD) 70  PhoP/PhoQ TCS-regulated processes (SPI-1 pathogenicity island genes) (SPI-3 pathogenicity island gene) (LPS) (LPS) (Peptidoglycan) (SPI-1 pathogenicity island genes) PhoP-activated genes (pag) are induced at low Mg2+ concentrations of intracellular (phagosomal) compartments. PhoP-repressed genes (prg) are induced at high Mg2+ concentrations (extracellularly). Salmonella Typhimurium phoP or phoQ null mutants are avirulent/ highly attenuated for virulence in mice. 71 PhoP/PhoQ TCS-regulated covalent lipid A modifications (hexaacylated lipid A) L-4-aminoarabinose (pbgPE) Phosphoethanolamine 2-hydroxymyristate Palmitate (pagP) Guo et al. (1997) Science 276: 250. Bishop et al. (2000) EMBO J. 19: 5071. 72 Crosstalk of two-component systems and quorum sensing Legionella pneumophila Hochstrasser & Hilbi (2017) Front. Microbiol 8: 79. 73 Regulation by bacterial cell density (quorum sensing) – Summary Quorum sensing: regulation of gene expression by producing, secreting, detecting, and responding to signaling molecules („autoinducers“, AI) that accumulate in proportion to cell density. Quorum sensing regulated processes: bioluminescence, biofilm formation, virulence, antibiotic production, sporulation, competence. Quorum sensing allows bacteria to collectively control gene expression and thus synchronize group behaviour. 74  Quorum sensing versus diffusion sensing Secretion of exoenzymes Secreted autoinducer senses diffusion Quorum sensing: emphasis on bacterial cell density / concentration Diffusion sensing: emphasis on signaling molecule (autoinducer) concentration Redfield (2002) TIM 10: 365. 75 Quorum sensing – „Autoinducer“ (signaling) molecules AHL or oligopeptide autoinducer: intra-species communication AI-2 autoinducer: inter-species communication, found in > 55 bacterial species (Furanosyl Borate Diester) 76 Quorum sensing – Autoinducer systems: LuxR/LuxI (passive diffusion in and out of the cell) 77 Quorum sensing – Autoinducer systems Vibrio fisheri (LuxRI) Staphylococcus aureus (Agr) Acylated homoserine Oligo - lactone peptide (AHL) Gram-negative bacteria: Gram-positive bacteria: - LuxI (autoinducer synthase) - prepropeptide - LuxR (activator) - peptide autoinducer (AIP) - AHL (autoinducer/ co-activator) - two component system Bassler (2002) Cell 109: 421. 78  Quorum sensing – Autoinducer systems Vibrio harveyi (AHL, AI-2) Vibrio cholerae (AHK, AI-2) HAI-1 AI-2 - autoincucer synthases: - autoincucer synthases: LuxLM (HAI-1 = AHL), LuxS (AI-2) CqsA (CAI-1), LuxS (AI-2), „system 3?“ - two-component systems - two-component systems - phospho-LuxO binds RpoN and - LuxU (histidine phosphotransferase protein) activates expression of repressor - LuxO and HapR are required for expression of toxin coregulated pili (TCP), cholera toxin (CT) and virulence in mice. Bassler (2002) Cell 110: 303. 79  Quorum sensing – Signaling molecules Autoinducer molecules of Gram-negative bacteria Autoinducer-2 (AI-2; furanosyl borate diester) N-acyl-homoserine lactone (AHL), 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS), 3,5-dimethylpyrazin-2-ol (DPO), cis-11-methyl-2-dodecenoic acid (DSF), 3-hydroxypalmitic acid methyl ester (3-OH-PAME), L. pneumophila 3-hydroxypentadecane-4-one (Legionella autoinducer-1, LAI-1; -hydroxyketone, AHK) 80 Inter-species communication via LuxS/AI-2 (Gram-positive and Gram-negative) 81 No luxS gene: V. fisheri, P. aeruginosa, L. pneumophila  Quorum sensing and c-di-GMP signaling 82 Hochstrasser & Hilbi (2020) Curr Opin Microbiol 55: 9. Quorum sensing and c-di-GMP signaling – L. pneumophila Diguanylate cyclase 83  Processes regulated by quorum sensing Natural interference with quorum sensing: - AHL-antagonizing inhibitors (halogenated furanones, Delisea pulchra red algae) - AHL-lactonase (AiiA, Bacillus spp.) - AHL-acylase (AiiD, Ralstonia spp.) - Inhibition of autoinducer peptide-mediated signaling (S. aureus) 84 Bioluminescence – Symbiotic Vibrio fisheri Pinecone fish (12 cm), Bobtail squid (2 cm), red organ in lower jaw, light organ close to ink sac, 1010 V. fisheri per ml 1011 V. fisheri per ml Symbiosis: exchange nutrients for light (attract prey, camouflage), recycling of reducing equivalents, provision of photoreactivating wavelengths for DNA repair. 85  Bioluminescence – lux operon luxRI luxCDE luxAB 86  Biofilms – Interference with quorum sensing Development of antipathogenic (not bacteriocidal or P. aeruginosa AHLs Synthetic QS inhibitors bacteriostatic) drugs based on quorum sensing inhibition. Advantages: Desia pulchra brominated furanones Breakdown of biofilm structures. No selective pressure for the development of resistance. Inhibition of - AHL signal generation - Signal reception - Bacterial dissemination Hentzer & Givskov (2003) J Clin Invest 112: 1300. 87  Biofilms – Interference with quorum sensing P. aeruginosa biofilms (a) treated with the synthetic furanone C30 (b) are flat and undifferentiated and less tolerant to the antibiotic tobramycin. (Live/dead stain of bacteria; live bacteria: green, dead bacteria: red). Hentzer & Givskov (2003) J Clin Invest 112: 1300. 88  Biofilms – Drug resistance and drug persistance Periplasmic glucans, Physical barriers (EPS) (Starvation, stress-response via RpoS) Physiological heterogeneity 89 Biofilms – Phenotypic/physiological heterogeneity Mixed-species biofilm K. pneumoniae biofilm (monochloramine) Bulk medium K. pneumoniae biofilm (acridine orange, RNA/DNA ratio) Bulk medium Mah & O’Toole (2001) Substratum Substratum TIM 9: 34. red-orange: high RNA/DNA ratio, rapid growth red-orange: high respiratory activity yellow: low RNA/DNA ratio: slow growth green: no respiratory activity 90 Phenotypic heterogeneity – Cues and consequences Signals Bet-hedging Nutrients Survival strategy of a clonal bacterial population, metabolites comprising the expressing of different traits, which pH, O2 allow adaptation to specific conditions in fluctuating ions, osmolarity environments. temperature Division of labor Survival strategy of a clonal bacterial population, comprising the co-existence of different traits, which Striednig & Hilbi (2022) allow concurrent complementary interactions. Trends Microbiol 30: 379. 91 Drug resistance versus drug tolerance MIC: minimum inhibitory concentration MDK: minimum duration for killing 92 Inter-kingdom signaling by a bacterial signaling molecule L. pneumophila LAI-1 Hochstrasser & Hilbi (2017) Front Microbiol 93  Inter-kingdom signaling by host adrenergic compounds Enterohemorrhagic E. coli (EHEC) Adrenergic signaling in EHEC - TCS: QseBC, QseFE - FlhDC: motility - StxAB: Shiga toxin AB - LEE: locus of enterocyte enfacement (T3SS) Hughes & Sperandio (2008) Nature 6: 111. 94 IV. Bacterial transport and secretion 1. Bacterial transport - porins - transporters - siderophores 2. Bacterial secretion - toxins - secretion systems (type I-VII) 95 Bacterial transport – Outer membrane porins Porins: - Allow diffusion of small (5 different porins Many porins provide specific docking sites for bacteriophages (phage : LamB) 96 Bacterial transport – plasma membrane transporters - Active transport against concentration gradients - Transport consumes energy (ion gradient, ATP) Saturation effect a) transporter b) group translocation c) ABC-transporter 97 Transport across three membranes: Intra-vacuolar bacteria 98  Bacterial transport – Siderophores ABC transporter IrtAB (Mycobacterium spp.) Mycobactin, MBT (membrane-bound) Carboxymycobactin, cMBT (soluble) Arnold et al. & Seeger IrtAB imports and reduces iron-mycobactin (2020) Nature 580: 413. 99 Bacterial secretion – Toxins 100  Protein secretion systems of Gram-negative bacteria Type VI secretion Type VII secretion Autotransporter Fronzes et al. (2009) General secretion pathway/ Nat Rev Microbiol 7: 703. type IV pilus assembly machinery Conjugation machinery Injectisome/flagellum 101  Protein secretion systems – Type III secretion systems T3SS (Salmonella typhimurium Galan & Waksman (2018) Cell 172: 1306. 102  Protein secretion systems – Type IV secretion systems T4BSS T4ASS (Legionella (conjugative pneumophila): plasmids) A, G T4ASS (Helicobacter pylori): E, F H Ghosal et al. (2017) Galan & Waksman (2018) EMBO Rep 18: 726. Cell 172: 1306. 103  Protein secretion systems – Type IV secretion system T4BSS (Legionella pneumophila) Böck et al. (2021) mBio 12: e02180-21. 104  Protein secretion systems – Type VI secretion systems V. cholerae Amoebophilus asiaticus Actin crosslinking Cell wall degradation Böck et al. & Pilhofer (2017) Science 357: 713. Galan & Waksman (2018) Cell 172: 1306. 105 Protein secretion systems (Gram-negative bacteria) – Summary Secretion Representative bacteria Distinctive features, examples system Type I Many, Enterobacteriaceae Haemolysin transporter Type II Many, Enterobacteriaceae General secretory pathway Type III Enterobacteriaceae, Vibrio, Homologous to flagellar apparatus; Pseudomonas, Chlamydia, pathogen-host protein transport; Bordetella pertussis target for anti-virulence compounds Type IV Coxiella, Legionella, Homologous to conjugation systems; Rickettsia, Helicobacter, pathogen-host protein (DNA) transport; Brucella, Bartonella, target for anti-virulence compounds Bordetella pertussis Type V Many, Neisseria Autotransporter Type VI Pseudomonas, Vibrio, Homologous to bacteriophages; contact- Serrratia, Yersinia pestis dependent interbacterial transport Type VII Mycobacterium tuberculosis, ESX-1 – ESX-5; pathogen-host transport; Mycobacterium spp. Mutants: vaccine candidates (BCG) 106