Pathogen Biology The E. coli

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Pathogen BiologyThe E. coli Salmonella
paradigms

• Professor Mark Pallen
• University of Birmingham

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Bacterial Virulence A simplistic view

• Some bacterial exotoxins can elicit the features
  of a bacterial infection when injected as pure
  proteins, e.g.
• tetanus toxin, botulinum toxin
• diphtheria toxin, anthrax toxin
• Vaccination with toxoids led to a spectacular
  decline in the incidence of many bacterial
  infections.
• Leading to the simplistic idea that all bacteria
  need to cause disease is a single toxin.

Diphtheria in England Wales
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Bacterial Virulence A more sophisticated view

• Many different ways to define a virulence factor
• Something
• needed to colonise and/or damage host tissues
• Molecular Kochs postulates
• Biochemical studies
• that distinguishes a pathogen from a commensal
• Comparative genomics
• expressed or essential in vivo
• but not in the lab?
• STM, IVIAT, IVET, expression profiling

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Bacterial Virulence A more sophisticated view

• Virulence as a process is
• MULTIFACTORIAL
• A bacterial army, like a human army, needs more
  than just its firearms to enter and secure enemy
  territory
• An army marches on its stomach Napoleon
• MULTIDIMENSIONAL
• A programme of events organised in time and space

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Steps in successful infection

• Sex comes before disease
• acquire virulence genes
• Sense environment
• and Switch virulence genes on and off
• Swim to site of infection
• Stick to site of infection
• Scavenge nutrients
• especially iron
• Survive stress

• Stealth
• avoid immune system
• Strike-back
• damage host tissues
• Subvert
• host cell cytoskeletal and signalling pathways
• Spread
• through cells and organs
• Scatter

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Bacterial Sex drives bacterial evolution

• Molecular phylogeny ribosomal RNA and other
  sequences allowed realisation of Darwins dream
  of Tree of Life by Woese et al in 1980s
• practical consequence identification of
  non-culturable bacteria, e.g. Trophyerma whippeli
• More recently, genome sequencing suggests
  horizontal gene transfer has played a large role
  in shaping bacterial evolution
• Web or Net of Life
• Genomes as mosaics
• Cores (housekeeping genes) and options
  (niche-specific)

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Bacterial Sex acquiring virulence genes

• Bacteria have three ways of exchanging DNA
• Transformation
• cells take up naked DNA
• Transduction
• phages carry DNA
• Conjugation
• cells mate through specialised appendages

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Bacterial Sex Mobile genetic elements

• Transposons
• ST enterotoxin genes
• Virulence Plasmids
• e.g. TTSSs in Shigella, ST, LT toxins in ETEC
• Phage-encoded virulence
• e.g. Shiga-like toxin, TTSS effectors in
  Salmonella and E. coli.

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Bacterial Sex Pathogenicity Islands

• Concept originated from study of uropathogenic E.
  coli strains
• Hacker and colleagues in early 1990s
• Haemolysin islands, deletable DNA fragments
  encoding alpha-haemolysin
• Also encoded P fimbriae, so renamed
  pathogenicity islands
• Now extended to many bacterial species

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Bacterial Sex Pathogenicity Islands

• Defining Features
• Carriage of (many) virulence genes
• Presence in pathogenic strains versus
  non-pathogenic strains
• Different GC content from host chromosome
• Occupy large chromosomal regions
• (10s to 100s of kilobases)
• Compact distinct genetic units, often flanked by
  DRs, tRNAs, ISs
• Presence of (cryptic) mobility genes
• Unstable, prone to deletion

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Bacterial Sex Pathogenicity Islands

• often encode secretion systems
• LEE region in EPEC
• Spi1, Spi2 in Salmonella
• can also encode adhesins, siderophores, toxins
• Uropathogenic E. coli (Pai I, II, IV, V)

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Sense environment

• Bacteria can sense changes in environment
• e.g. in temperature, nutrient availability,
  osmolarity, cell density (quorum sensing).
• In simplest cases, change in intracellular
  concentration of ion linked directly to gene
  expression
• e.g. fall in intra-cellular iron levels relieves
  Fur-mediated repression of Shiga-like toxin gene
• In more complex cases, sophisticated signal
  transduction cascades allow bacteria to regulate
  gene expression in response to environmental cues
• the pathogen as an information processor

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Switch virulence factors on and off

• Gene expression is regulated
• Inducible versus constitutive genes
• Wasteful if always constitutive
• Artificial constitutive constructs decrease
  fitness
• Co-ordinate gene regulation
• Operon
• Stimulon
• e.g. The oxidative stress response
• Regulon
• e.g. The OxyR regulon
• Co-ordinate regulation of virulence
• in response to in vivo signals

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Switch virulence factors on and offA
multi-layered hierarchy

• Changes in DNA sequence
• Gene amplification
• Genetic rearrangements
• e.g. Hin flip-flop control of flagellar phase
  variation
• Transcriptional Regulation
• Activators and Repressors
• (helix-turn-helix motif)
• mRNA folding and stability

• Translational Regulation
• Trp operon
• Post-translational Regulation
• Stability of protein, controlled cleavage
• Covalent modifications
• e.g. phosphorylation in two-component
  sensor-regulator systems

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Salmonella/E.coli flagellar regulon
Based on Macnab, 1996
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Swim

• Many bacterial pathogens are motile
• Enterics, Campylobacter, Helicobacter,
  spirochaetes
• Motility crucial for virulence in some cases
• Usual organelle of motilityflagellum
• Variants
• Twitching motility
• Swarming

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Stick

• To avoid physical and immunological removal,
  bacteria must adhere to
• cell surfaces and extracellular matrix
• e.g. in respiratory, gastrointestinal and
  genitourinary tracts
• solid surfaces
• e.g. teeth, heart valves, prosthetic material
• other bacteria

• Direct interaction
• Molecular bridging via e.g. fibronectin
• Adherence often combined with manipulation of
  host cell signalling and cytoskeleton
• Invasion
• Intimate adherence

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Stick

• Common adherence mechanisms
• Capsules and slime
• Biofilm formation
• Gram-positive adhesins
• MSCRAMMs (microbial surface components
  recognizing adhesive matrix molecules)
• Fimbriae
• Gram-negative adhesins (CHO and protein
  receptors)
• Fimbriae, Afimbrial adhesins (FHA, Pertactin
  etc.)
• OMPs (e.g. YadA, Opa, Opc, invasin, intimin)
• Types III-IV secretion (e.g. EspA pilus)

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Stick
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Scavenge nutrients

• Free iron levels very low in body fluids
• Acute phase response causes further drop
• Iron overload increases susceptibility to
  infection
• Many different bacterial systems for scavenging
  iron
• Siderophores chelate available iron transport
  it into bacteria
• Iron can be scavenged direct from host
  iron-binding proteins, e.g by lactoferrin-binding
  proteins
• Often co-ordinately regulated e.g. by fur locus
  in E. coli
• Some pathogens avoid the problem by cutting out
  need for iron, e.g. Treponema pallidum
• Iron used to regulate aggressive virulence
  factors
• Diphtheria toxin (DtxR repressor)
• Shiga-like toxin
• Pseudomonas aeruginosa exotoxin A

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Scavenge nutrients

• Urease enables many pathogens to assimilate
  ammonia from urea (major source of nitrogen in
  body fluids)
• Some intracellular pathogens, e.g. the leprosy
  bacillus, scavenge purines pyramidines
• Mutations in genes for aromatic amino acid
  biosynthesis (e.g. aroA) cause attenuation in
  many different pathogens, as body fluids lack
  these amino acids (nutritional defence). Useful
  source of live vaccines (e.g. against typhoid).

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Survive Stress

• In addition to nutrient-limitation stress,
  pathogens face many other stresses
• Acid stress within stomach
• Heat shock during fever
• Oxidative stress within phagocytes
• Stress response proteins, such as chaperonins
  feature as immunodominant antigens
• Detoxification proteins play a role in virulence,
  e.g. periplasmic Cu,Zn-superoxide dismutases
• Infectious dose for enteric pathogens much lower
  in achlorhydria (no need to overcome acid stress)

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Stealthavoid immune system

• IgA proteases
• metalloproteases active against IgA
• Immunoglobulin-binding proteins
• e.g. protein A of S. aureus
• Resist complement, opsonisation
• Capsule (usually polysaccharide)
• Lipopolysaccharide
• Surface proteins and OMPs
• Antigenic mimicry
• e.g. sialic acid capsule of group B meningococcus

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Stealthavoid immune system

• Antigenic or phase variation
• Involves surface structures such as proteins,
  LPS, capsules
• Variety of mechanisms
• slip-strand mispairing
• flip-flop
• cassettes
• Adopt cryptic niche
• inside phagocytes
• in biofilm

67700 67710 67720 GAAGTGCATTTAACTTGGGGGG
GGGGGTAAT GAAGTGCATTTAACTTGGGGGGGGGGGGTAAT GAAGTG
CATTTAACTTGGGGGGGGGGGGGTAAT GAAGTGCATTTAACTTGGG
GGGGGGGTAAT GAAGTGCATTTAACTTGGGGGGGGGGGTAAT GAAG
TGCATTTAACTTGGGGGGGGGTAAT GAAGTGCATTTAACTTGGG
GGGGGGGGGTAAT GAAGTGCATTTAACTTGGGGGGGGGGGTAAT GA
AGTGCATTTAACTTGGGGGGGGGGTAAT GAAGTGCATTTAACTT
GGGGGGGGGGTAAT GAAGTGCATTTAACTTGGGGGGGGGGTAAT
GAAGTGCATTTAACTTGGGGGGGGGGGTAAT GAAGTGCATTTAACTT
GGGGGGGGGGGGTAAT
Homopolymeric tract in Campylobacter jejuni
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Strike-back Damage host tissues

• Endotoxin
• Exotoxins
• Toxins acting on cell membranes
• Toxins active inside cells
• Superantigens

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Endotoxin of Gram-negatives
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Strike-back Endotoxin

• Actions of Endotoxin
• Pyrogenicity
• Leucopenia then leucocytosis
• Hypotension
• Gram-negative Shock
• Life-threatening complication of septicaemia
• e.g. in meningococcal infection, in ITU or
  oncology patients
• Endotoxic shock seen with dirty intravenous
  equipment
• Most of the effects of endotoxin are mediated by
  tumour necrosis factor
• Attempts at therapy using anti-endotoxin or
  anti-TNF antibodies

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Strike-back Membrane-Damaging Exotoxins

• Many bacterial toxins form pores in eukaryotic
  cell membranes, producing oligomeric rings, e.g.
• streptolysin O of Streptococcus pyogenes
• listeriolysin of Listeria monocytogenes
• alpha-toxin of S. aureus
• Other toxins, such as phospholipases, degrade
  components of the membrane
• e.g. Clostridium perfringens alpha toxin

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Strike-back Toxins active inside cells

• Toxins often consist of translocation and binding
  B subunit(s) that deliver(s) the active A subunit
  into the host cell cytoplasm
• Shiga-like toxin
• Enzymatic A chain in red
• Cell binding B chains (Pentamer)
• The A-subunits of Shiga toxin, the Shiga-like
  toxins (SLTs), and ricin inactivate eukaryotic
  ribosomes by enzymatically depurinating 28S rRNA

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Heat-Labile Enterotoxin (LT)

• Lunar lander modules
• single hexameric protein assembly as seen in the
  crystal structure of the LT AB5 holotoxin.
• Lunar surface
• the outer membrane of an intestinal epithelial
  cell.
• Protrusions from membrane under toxin
• 5 copies of saccharide component of ganglioside
  GM1 to which toxin binds.
• GM1 is a normal membrane component which the
  toxin co-opts as a receptor

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Heat-Labile Enterotoxin (LT)

• A (enzymantic) subunit
• ADP-ribosylating toxin
• not enzymatically active until nicked to A1, A2
• ADP-ribosylates membrane GTPase Gs
• regulates host cell adenylate cyclase, determines
  level of cAMP
• active (GTP-bound) Gs increases activity of
  adenylate cyclase
• GDP-bound form renders adenylate cyclase inactive

• active Gs normally produced following hormone
  stimulation, converted to inactive form after a
  short time
• ADP-ribosylation of Gs short-circuits off-on
  control by locking Gs in "on" form
• alters the activities of Na and Cl- transporters
  
• ion imbalance leads to water loss, i.e. diarrhoea

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Subvert

• inject proteins into host cells to subvert the
  cytoskeleton and signal-transduction pathways
• manipulating e.g. Rho GTPases and the
  cytoskeleton to induce membrane ruffling and
  bacterial invasion
• Salmonella has evolved a GAP (SptP) and a GEF
  (SopE) by convergent evolution
• remaining within a vacuole by manipulating host
  cell vesicular transport and endocytosis

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Subvert
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Subvert
From Nature 412, 701
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Spread

• through cells and organs
• within macrophages, e.g. in typhoid
• through blood (need to be complement-resistant)
• within cells
• actin-based motility of Listeria monocyogenes,
  depends on ActA protein.

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Spread Transmission, virulence and evolution

• Established dogmas
• balanced pathogenicity
• being too virulent is no good
• high virulence is a sign of recent emergence of a
  pathogen
• pathogens evolve towards symbiosis

• Counter-arguments
• Where pathogens rely on spread through biting
  arthopods, high bacteraemias advantageous
• Where pathogens rely on shedding into water,
  highest possible shedding rates good for pathogen
• Where pathogens cause incidental disease (e.g.
  Legionella) no selective pressure towards low
  virulence
• Virulence as a local adaptation (why meningitis?)
• Bad vaccines and effect on virulence