BME 236 Microbiology Past Paper 2024 PDF

Microbiology BME 236, HS 2024 http://www.kcl.ac.uk Hubert Hilbi, Institute of Medical Microbiology 1 [email protected]; https://www.imm.uzh.ch Microbiology – Text books Basics: BIO 132 – Microbiology 2  Mic...

Microbiology BME 236, HS 2024 http://www.kcl.ac.uk Hubert Hilbi, Institute of Medical Microbiology 1 [email protected]; https://www.imm.uzh.ch Microbiology – Text books Basics: BIO 132 – Microbiology 2  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 3  I. Bacterial pathogens – Examples and sources John Snow, Cholera outbreak, 1854 Arnold Böcklin, Die Pest, 1898  Bacterial pathogens – History and heroes Cholera: 7 pandemies (first: 1817), London/Florence (1854), Naples (1884); Vibrio cholerae (1854: John Snow, 1883: Robert Koch, 1892: Max v. Pettenkofer) Plague: 3 pandemies (1st: Justinianic plague: 541–549; 2nd: Black Death: 1346-1353); Yersinia pestis (1894: Alexandre Yersin) Tuberculosis: latent TB (chronic disease); White Death; ca. 5’000 years old; Mycobacterium tuberculosis (1882: Robert Koch) Legionnaires’ disease: sporadic/epidemic, fulminant pneumonia; Philadelphia 1976 Legionella pneumophila (1977: Joseph McDade) 5  Bacterial pathogens – Examples Bacterial cell shapes Bacterial pathogens – Gram stain Gram-positive: Staphylococcus aureus Gram-negative: Escherichia coli Hans Christian Gram, 1884 7 Bacterial pathogens – Cell envelope: membrane(s) and cell wall  Bacterial pathogens – Examples Gram-negative rods: Enterobacteriaceae (Escherichia coli, Enterobacter cloacae, Salmonella typhy, Shigella flexneri, Klebsiella pneumoniae, Yersinia pestis), Acineto- bacter baumanii, Pseudomonas aeruginosa, Legionella pneumophila - ESKAPE Gram-positive rods: Clostridium spp. (difficile, perfringens, tetani, botulinum), Bacillus spp. (anthrax, cereus, subtilis) Mycobacterium spp. (tuberculosis, leprae, abscessus) Gram-negative cocci: Neisseria spp. (meningitis, gonorrhoeae) Gram-positive cocci: Streptococcus (pneumoniae, pyogenes) Staphylococcus aureus, Enterococcus faecium - ESKAPE Spirochetes: Treponema pallidum (Syphilis), Borrelia burgdorferi (Lyme disease) 9  Bacterial pathogens – Targets of antibiotics Brock Microbiology 10 Bacterial pathogens – Mechanisms of antibiotics resistance E. Wistrand-Yuen 11 Bacterial pathogens – Sources 12 Sources of respiratory pathogens (aerosols) Alfred Hitchcock, 1960, Psycho Sources of pathogens: Biofilms Biofilm: „city of microbes“ Structured community of bacteria enclosed in a self-produced extracellular polymeric matrix (glycocalix, „slime“), adherent to inert or living surfaces. Within biofilms, bacteria are 100 m protected from - predators (amoebae) - phages - biocides (chemical, physical) - antibiotics - immunophagocytes - antibodies Confocal micrograph of biofilm of P. aeruginosa expressing GFP 14 Sources of pathogens: Biofilms Biofilms (green) might be disseminated around the body, either by single cells or by clumps of protected emboli. Sporadic detachment can lead to cycles of bacteremia. Hall-Stoodley et al. (2004) Nature Rev Microbiol 2: 95. 15 Biofilms and bacterial virulence Dental caries: 500-1000 different species make up plaque: multispecies biofilm; coaggregating bacteria share mutual benefits and form metabolic networks. EPS: extracellular polymeric substances (polysaccharides, proteins, nucleic acids). Colonizers: streptococci (S. gordonii, S. mutans), Actinomyces (Gram-positive rod), Porphyromonas gingivalis (major periodontal pathogen), strict anaerobes (Actinomyces naeslundii, Propionibacterium acnes, Veillonella atypica, Actinobacillus actinomycetemcomitans). 16 Inhibition of biofilms by iron depletion Lactoferrin is a ubiquitous and abundant constituent of human external secretions (humoral innate immunit), which chelates iron and thus stimulates twitching motility causing P. aeruginosa to wander across the surface instead of forming cell clusters and biofilms. GFP-expressing P. aeruginosa in flow cells perfused with lactoferrin-free (a-d) and lactoferrin-containing (e-h) media. Images were obtained 4 h (a, e), 24 h (b, f), 3 days (c, g) and 7 days (d, h) after inoculating the flow cells. Scale bars, 10 m (a, b, e, f), 50 m (c, d, g, h). Singh et al. (2002) Nature 417: 552. 17  Bacterial pathogens – Sources Water: Vibrio cholerae, Vibrio spp., Legionella spp. Soil: Clostridium spp., Bacillus spp., Legionella longbeachae Biofilms: Pseudomonas aeruginosa, Staphylococcus aureus Protozoa: Legionella spp., Mycobacterium abscessus, Chlamydia-related species Animals: Yersinia pestis, Salmonella enterica, Campylobacter jejunii, Mycobacterium bovis Humans: Shigella spp., Mycobacterium tuberculosis, Chlamydia trachomatis, Helicobacter pylori, Streptococcus pneumoniae, Neisseria gonorrhoeae 18 II. Evolution of pathogenic bacteria 1. Horizontal gene transfer 2. Mechanisms of DNA exchange: - site-specific insertion - homologous recombination 3. Acquisition and loss of genes 4. Genomic pathogenicity islands 5. Virulence plasmids and phages 19  Horizontal gene transfer Transduction (bacteriophages) Conjugation (plasmids) Transformation („naked“ DNA) Horizontal gene transfer: DNA transfer between genomes within the same generation. Requirements: transfer, recombination, expression. 20 Transduction – Temperate phages Temperate phages: lysogenic/lytic phase Lysogeny: Replication in synchrony with host chromosome Phage conversion: Phenotypes caused by lysogenization prophage Horizontal gene transfer – Antibiotic resistance Conjugation Vancomycin resistant Staphylococcus aureus (58 kb multi resistance conjugative plasmid with transposon) Transpostition Weigel et al. (2003) Science 302: 1569. 22  R-plasmids and conjugative transposons R-plasmids - conjugative resistance plasmids - composed of several Tn/IS - example R100 (90 kb): carries several resistance genes, transfers between enteric bacteria Conjugative Transposons - very large (15-100 kb), linear - broad host range (Gram+/-) - spread of antibiotic resistance - example: Tn916 (Enterococcus faecalis) 23  Site-specific recombination – Summary Transposition - Requires transposase on insertion sequence (IS) element or transposon (= IS plus additional gene(s)) - Transfer of DNA element from donor site to target site in the genome - DNA element is inserted at random sites or hot spots - Duplication of integration sequence leads to direct target repeat - Rare event, frequency 10-3 – 10-8 per generation (cell division) Site-specific recombination - Requires recombinase (integrase, resolvase, invertase) - Integration at specific site in the chromosome - Consensus DNA sequences of ca. 15-20 nucleotides required - Integration leads to direct repeat - Involves specific protein-DNA interactions No homologous sequences required for recombination („illegitimate recombination“), independent of RecA (homologous recombination) 24  Site-specific recombination – Integrons Integron: integration of DNA (gene cassette) at specific site, (Integration site) requirements: integrase, att (integration site), promoter to (Gene cassette) express integrated genes Gene cassette: (resistance) gene usually lacking a promoter and containing a short sequence (59-base element) allowing integration; ca. 40 gene cassettes identified, up to 5 per strain (Pseudomonas spp.). Location: chromosome, transposons, plasmids 25 Site-specific recombination – Switch of invertible DNA Phase variation of flagellin (Salmonella enterica Typhimurium) IRR IRL IRL IRR Phase 2 Phase 1 Flagellin synthesis is controlled by the orientation of a promotor relative to the H1 and H2 chromosomal flagellin genes. The Hin invertase binds to the 14 bp inverted repeat sequences and flips the fragment. The „outreading“ promotor drives expression of either H2 flagellin and H1 repressor (phase 2) or none of them. H1 flagellin is transcribed if no repressor is present (phase 1). Frequency of switching: 10-4/generation. 26  Site-specific integration of episomal elements Avirulent Legionella pneumophila Regulation by integration/excision Virulent Legionella pneumophila RecA-independent excision of a 30 kb DNA fragment and high copy episomal replication of the element leads to an altered LPS epitope (phase variation) and a different outer membrane protein pattern. Lüneberg et al. (2001) Mol. Microbiol. 39: 1259. 27  Site-specific recombination – Phage  Lytic state Phage : ds DNA virus; lytic state or lysogenic state (prophage) Phage DNA (POP’) attP O: core sequence (15-mer) common to attP and attB attachment sites Bacterial DNA (BOB’) attB Excision Integration (Excisionase) (Integrase) Lysogenic state attL attR Direct repeats (2x) Integrase and IHF bind to attP (240 bp), the Int-IHF-attP-complex binds to attB and cleaves within the 15 bp O-sequence. Staggered cleavages in the common core sequence of attP and attB lead to crosswise recombination. Xis, formed after induction of the lytic cycle, binds to the P region of POB‘ (attR) and excises the phage dependent on Int and IHF. Integration (attP X attB) and excision (attL X attR) require recognition of different pairs of reacting sequences allowing to control the direction of the reactions. 28  Transposition – Phage Mu Temperate phage Mu(tator): large ds DNA virus (37 kb), icosahedrical head, helical tail, six tail fibers - Lysogenic phase: Mu integrates randomly by non-replicative transposition (pro-phage) - Lytic cycle: upon induction, Mu amplifies by replicative transposition IR IR DR DR (37 kb) 29  Transposition – Transposon mutagenesis Advantages: random mutagenesis, mutation is not repaired, selectable markers (antibiotic resistance), mutated gene can be cloned („rescue cloning“) Disadvantages: not useful for essential genes, continued transposition leads to unstable mutations Mini-transposons: small transposon, transposase is encoded by suicide plasmid, generates stable transpositions (e.g. Tn903dIIlacZ) Frequently used: Tn5 (NeoR, KmR), Tn10 (TetR), Bacteriophage Mu 30  Restriction/Mutagenesis by CRISPR/Cas9 CRISPR/Cas: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR-associated (Cas) Cas9: Dual RNA–guided DNA endonuclease (tracrRNA, crRNA); sgRNA (single guide RNA, engineered) Introduction of site-specific double- stranded breaks in target DNA sgRNA Doudna & Charpentier (2014) Science 346: 1077. PAM: protospacer adjacent motif 31  Homologous recombination – Summary Identical or very similar sequences in the crossover region - sequence homologies extend over > 50 (often many more) nucleotides - takes place between homologous IS elements (-> F- plasmid), or after horizontal gene transfer Strand breaks, complementary base pairing between double-stranded DNA molecules, heteroduplex formation Formation of Holliday junction, branch migration Recombination enzymes (> 25 proteins), dependent on RecA (present in all prokaryotes, yeast, higher eukaryotes) In vitro reconstitution: purified RecA, RecBCD, DNA and ATP => Holliday junctions Non-essential process (bacterial cloning strains often lack recA) 32  Homologous recombination – F-Plasmid The tra genes encode a type IV secretion system (T4SS) tra oriT IS3 oriV , transposon Tn1000 IS3 Regulation by integration/excision F‘ 33  Homologous recombination - Mechanism RecBCD: nuclease + helicase Holliday helicase junction RecA DNA-polymerase, nucleases, ligase ligases Pre-Synapsis Synapsis Post-Synapsis 34 Homologous recombination – Holliday model Holliday model: single strand breaks (2x), double strand invasion. More recent model: double strand break (1x), single strand invasion, repair (= double strand break-repair model). Holliday junction „patch“-product „splice“-product (crossover, recombination) 35 Homologous recombination – Mutagenesis Marker exchange mutagenesis (a) Insertion mutagenesis (b) 36 Evolution of bacteria – „Gene pools“ Essential physiological Physiological processes processes required for required for survival in specific survival in general environments (niches) 37 Evolution of bacteria - Acquisition and loss of genes Reductive evolution by gene decay (Mycobacterium leprae, Yersinia pestis, Mycoplasma spp., Chlamydia spp.) 38 Evolution of bacteria – Horizontal gene transfer Genomic islands Fitness: reproductive success of a genotype in a given environment (survival, spread, transmission). Islands are defined by specific niches rather than by genetic composition. 39  Genomic islands – Summary Characteristics: - Linked to tRNA genes - Insertion sequence (IS) elements - Flanked by direct repeats (DR) - Mobility elements (int, tnp) - G+C content differs from core genome - Genes abc, def, ghi kb confer “fitness” 40 Genomic islands – Features 41  Pathogenicity islands (PAI) – Summary Presence of genomic island in pathogenic strains, and absence (or sporadic distribution) in less pathogenic strains of the same or a related species Carriage of (often many) virulence genes (toxins, adhesins, invasins, secretion systems (type III, type IV) and their substrates (effectors) Occupation of large chromosomal regions (often > 30 – 200 kb) with a different G+C content in comparison to DNA of host bacteria Compact, distinct genetic units, often flanked by direct repeats (DR) and/or harboring insertion sequence elements (IS), associated with tRNA genes Presence of (often cryptic) „mobility elements“ (IS elements, integrases, transposases, origins of plasmid replication, recombination hot spots). Stabilization of PAI by mutation of „mobility“ genes (int, ori, IS). „Unstabilized“ PAI delete (duplicate) with a frequency of 10-4-10-5/generation. Expression of acquired genes is under the control of chromosomal or PAI-encoded regulators; interaction of the PAI with the genome is required. E.g., expression of invasion genes within the Salmonella SPI-1 is governed by the PhoP/PhoQ two-component system outside of SPI-1. 42 Pathogenicity islands – Location Attachment/insertion sites: tRNA genes Insertion at the 3‘ end of tRNA genes by non-homologous (site- specific) recombination. tRNA genes (att sites) are conserved across species, expressed constitutively and may occur in multiple copies with the same anticodon (redundancy). phage attachment sites (att) Insertion sequence (IS) elements 61 43  Evolution of pathogens – Virulence phages Conversion = change of phenotype due to incorporation of temperate bacteriophage 44  Evolution of pathogens – Enterobacteria SPI-2 (systemic disease) SPI-1 (diarrhea) Chromosomal organization of Shigella and E. coli shares more than 90% homology (branched ca. 35‘000-270‘000 years ago); Shigella and E. coli could be considered members of the same genus. E. coli and Salmonella separated from a common ancestor ca. 140 million years ago. Yersinia spp. are more distantly related (Y. enterocolitica and Y. pseudotuberculosis branched ca. 100 million years ago), Y. pestis emerged only ca. 1‘500-20‘000 years ago. 45  Mobile DNA elements in E. coli EHEC: pO157 (enterohemolysin, katalase, type II secretion, large clostridial toxin) EPEC: pEae (bfp: bundle forming pilus, Per) EIEC: pInv (Shigella virulence plasmid) EAEC: (aaf1: aggregative adherence fimbriae 1) ETEC: (heat-labile, heat-stable enterotoxins) EHEC: Shiga toxin (Stx) EPEC/EHEC: LEE (locus of enterocyte effacement) EAEC: SHI-1 (Shigella) EIEC: SHI-2 (Shigella) All E. coli pathotypes: HPI (high pathogenicity island, iron siderophore; Yersinia) Intestinal E. coli pathotypes: Extraintestinal E. coli pathotypes: EPEC – enteropathogenic E. coli UPEC – uropathogenic E. coli EHEC – enterohemorrhagic E. coli Sepsis E. coli ETEC – enterotoxigenic E. coli Meningitis E. coli EAEC – enteroaggregative E. coli 46 EIEC – enteroinvasive E. coli  E. coli pathogenicity islands UPEC Sepsis E. coli UPEC UPEC EHEC UPEC Salmonella (SPI-5) EPEC/EHEC Shigella (SHI-2) EHEC Salmonella (SPI-3) Salmonella (SPI-2) UPEC UPEC Sepsis E. coli EHEC Meningitis E. coli UPEC EHEC EHEC 47  Salmonella pathogenicity islands Fimbriae (virulence, growth within macrophages) (Putative T1SS) Fimbriae (90 kb virulence plasmid) Mg2+-uptake Fimbriae (growth within macrophages, Pathogenicity islet (SPI-1 effector protein) PhoPQ-regulated) (SopB) Systemic infection (growth within macrophages) (SPI-2 T3SS, effector proteins, regulatory proteins) Fimbriae Early infection, cell invasion (epithelial cells) Pathogenicity islet (SPI-1 T3SS, effector proteins, regulatory proteins) Prophage Prophage (SPI-1 effector protein) Sop proteins: SPI-1 effector proteins Sif proteins: SPI-2 effector proteins 48  Evolution of pathogens – Shigella selC (90 kb deletion) Outer membrane protease T Lysine decarboxylase (cleaves IcsA) (produces cadaverine, which inhibits Shigella enterotoxin) Shigella pathogenicity requires the acquisition of a virulence plasmid and two chromosomally encoded regions as well as the absence of two genes detected in E. coli (“black holes”). SHI-1: Shigella IgA protease autotransporter Sig, Shigella enterotoxin SetAB (AB toxin, IS) SHI-2: aerobactin iron siderophore, colicin immunity protein, integrase Ochman, Lawrence, & Groisman (2000) Nature 405: 299. 49  Shigella SHI-O pathogenicity island LPS is an important virulence factor in Shigella. The host antibody response is specific to the O antigen of LPS (serotype-specific). Thus, serotype conversion is a mechanism of immune evasion. The enzymes that modify the basic O antigen are encoded on the SHI-O PAI (serotype 1a) or by lysogenic phages SfII, SfV, SfX (serotypes 2a, 5a). serotype-converting enzymes G+C (SHI-O) = 40%, Shigella = 49-53% attL and attR are about 6.5 kb apart 50 Shigella flexneri bacteriophages O-antigen glycosylation due to gtr gene cluster on SfII, SfV, SfX bacteriophages. The “sword and shield” strategy: interaction between LPS and type III secretion system (TTSS). Hyperinvasive strains that have truncated LPS molecules are susceptible to being killed by the innate immune response in vivo. Bacteria expressing non-glycosylated LPS are compromised for invasion and therefore attenuated. Glycosylation of the O antigen halves the length of the LPS molecule, which allows efficient function of the TTSS while it retains resistance to antimicrobial factors in vivo. West N.P. et al. (2005) Science 307: 1313. 51 Shigella flexneri virulence plasmid Genetic map of the 213 kb virulence plasmid pWR100 of Shigella flexneri serotype 5a. 31 kb region - 31 kb region sufficient for entry into epithelial cells - Mxi-Spa type III secretion system - ca. 25 secreted effector proteins, including IpaA-D - actin nucleating protein IcsA - transcriptional requlatory proteins VirF, VirB - 93 (!) IS elements (58 kb), mosaic structure Buchrieser et al. (2000) Mol. Microbiol. 34: 760. 52  Evolution of pathogens – Yersinia Ysc T3SS, „High Yop effectors, pathogenicity adhesion, island“ antiphagocytosis, immune evasion -- severe/lethal infections Conjugative resistance plasmids -- broad host range 53  Yersinia „high pathogenicity island“ (HPI) - Canonical PAI (large chromosomal fragment, virulence locus (yersiniabactin iron siderophore (phenolate): biosynthesis, transport, regulation), IS, integrase, G+C (yersiniabactin locus) = 60%, Y. pestis = 46-50%, flanked by a tRNA gene). - Excision is mediated by the HPI-encoded P4-like integrase and occurs at a frequency of 10-4, generating an att-like sequence at the junction site and restoring intact asn tRNA locus (Y. pseudotuberculosis, Y. pestis). In Y. enterocolitica HPI is stable (int not functional). - HPI is present in different bacterial genera (pathogenic E. coli, Salmonella enterica, Enterobacter, Klebsiella, Citrobacter). 54 Evolution of pathogens – Vibrio cholerae avirulent V. cholerae O1 a. Acquisition of VPI phage, including toxin co-regulated type IV pili (TcpA) a avirulent V. cholerae O1 b. Phage conversion by CTX phage encoding cholera toxin (AB toxin) aa b virulent V. cholerae O1 c. Acquisition of rfb genes a b virulent V. cholerae O139 “El Tor” c 55  Evolution of (pathogenic) bacteria – Summary Bacteria constantly encounter selective pressure from the environment: nutrients, predators (amoebae, nematodes), arthropods or warm-blooded animals (host defenses), or antibiotics. Survival of and adaptation to these challenges allows the colonization of new niches. Local sequence changes (point mutations), genomic rearrangements and horizontal (lateral) gene transfer are driving forces in microbial evolution. Horizontal DNA transfer between genomes is mediated by transmissible genetic elements (plasmids, bacteriophages) or by natural competence and causes the rapid dissemination of traits (virulence, symbiosis, antibiotic resistance, metabolic capacities, etc.). Horizontal gene transfer may lead to „evolution in quantum leaps“. Integration (deletion) of genetic elements into (from) the bacterial chromosome can generate „fitness islands“ („black holes“). Fitness is defined as a set of properties that enhance survival, spread, and/or transmission in a specific niche. 56