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SBM 2044: Lecture 10

SBM 2044: Lecture 10. AIMS: To provide. Brief introduction to E. coli: a versatile pathogen Overview of Enterotoxigenic E. coli (ETEC). Types of pathogenic E. coli. Intestinal. Disease. Enterotoxigenic E. coli (ETEC) Enteroaggregative E. coli (EAEC)

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SBM 2044: Lecture 10

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  1. SBM 2044: Lecture 10 AIMS: To provide • Brief introduction to E. coli: a versatile pathogen • Overview of Enterotoxigenic E. coli (ETEC)

  2. Types of pathogenic E. coli Intestinal Disease Enterotoxigenic E. coli (ETEC) Enteroaggregative E. coli (EAEC) Diffusely adhering E. coli (DAEC) Cholera-like watery diarrhoea Enteropathogenic E. coli (EPEC) Enterohaemorrhagic E. coli (EHEC) Colitis or haemorrhagic colitis Enteroinvasive E. coli (EIEC) Dysentery Extraintestinal Urinary tract/ pyelonephritis Uropathogenic E. coli (UPEC) Septicemia/ meningitis Septic E. coli strains

  3. EAEC • Pet – Plasmid-encoded enterotoxin mucus release • First recognised in 1987 as distinct from other ETEC • Characteristic adhesion pattern in vitro reflects bacteria- bacteria (in addition to bacteria-host) adhesion Known virulence factors: • AAF - Aggregative adherence fimbriae • EAST-1 – EAEC heat stable toxin (similar to ETEC Sta) • Pic – role in intestinal colonisation? - various activities (mucinase, serum resistance, haemagglutinin)

  4. EAEC

  5. Strains displaying a distinct adhesion pattern Enteroaggregrative E. coli (EAEC)

  6. Diffusely adhering E. coli (DAEC)

  7. DAEC • Doubts about their importance as pathogens • Like EAEC and ETEC, appear to be heterogeneous • group of strains, sharing certain common factors • Four different adhesins identified – F1845 fimbriae & • 3 non-fimbrial adhesins • No significant information on potential toxins

  8. Enteropathogenic E. coli (EPEC) • First associated with diarrhoea in 1940s • Major cause of watery diarrhoea in infants (< 6 months) • Developing countries - endemic • Developed countries – sporadic outbreaks in nurseries, • paediatric wards, day centres, etc • No detectable enterotoxin • 1970s – first seen by EM to produce unique ‘attaching & effacing’ effects on enterocytes - producing unique lesions, now called A/E lesions

  9. EPEC interactions with epithelial cells 3. Formation of pedestals 1. Initial (non-intimate) attachment 2. Intimate adhesion + effacement Studies on mechanisms very limited until late 1980s

  10. EPEC interactions with epithelial cells 1. Initial (non-intimate) attachment 2. Intimate adhesion + effacement 3. Formation of pedestals Intimin (EaeA) [OM protein] Actin rearrangements Involved: BFP

  11. Knutton et al (1998) EMBO Journal 17: 2166-2176 Anti-EspA • Activation of EPEC • Type III secretion EM Filaments ‘connecting’ EPEC & host cells • Immuno-gold antibodies Filaments composed of EspA

  12. EPEC Type III secretion filament EspB/D pore Host cell membrane Deduced from studies combining: • Mutants • protein-protein binding • Labelling with specific Ab • Electron microscope EPEC OM EscC EPEC IM EscJ IM-associated ‘machinery’ Esc R,S,T,U,V,N,D

  13. Kubori et al (1998) Science 280: 602-605 • Salmonella Type III ‘needles’ • Similar ‘needles’ in EPEC, but Salmonella lack ‘filament’

  14. EPEC: Kenny et al (1997) Cell 91: 511 - 520 Remarkable discovery: • Receptor for intimin (adhesin) is NOT a host cell protein • It is an EPEC protein • Translocated into host cell, phosphorylated, & inserted into the host cell membrane Tir: Translocated intimin receptor

  15. filaments EspB/D pore Tir + ? signals translocated into host cell Tir- P Ca++ Intimate adhesion by EaeA to Tir- P EPEC: Model of A/E Lesion Formation Initial adhesion (via Bfp ) Quorum sensing ? Activation of Type III secretion Details of signalling still unclear Actin polymerization pedestal formation

  16. Enterohaemorrhagic E. coli (EHEC) • 1982: two cases of severe food-poisoning (hamburgers) in USA associated with rare serotype of E. coli - O157:H7 • 1983: E. coli O157:H7 produces a Shiga-like toxin • 1986: Like EPEC, O157:H7 produce A/E lesions ‘Enterohaemorrhagic E. coli’ (EHEC) first used in 1987 to describe what seemed to be a new type of pathogenic E. coli Main difference with EPEC is production of Shiga-toxin

  17. Origin of E. coli O157:H7 ? First isolated in US in 1982 - now worldwide • Quantitative population genetic studies (MLEE) O157:H7 isolates throughout world are single clone that emerged relatively recently - closely related to a much less virulent EPEC clone • Both SLT-I & SLT-II genes encoded by bacteriophages, • - could facilitate their transmission into new strains O157:H7 probably emerged when an EPEC strain (already capable of producing A/E lesions & diarrhoea) acquired slt phages, resulting in a dramatic increase in virulence

  18. Shiga-toxins haemorrhage diarrhoea renal failure Typical A-B subunit toxins A1 subunit - N-glycosidase A1 27kDa • Hydrolyses eucaryotic cell 28S rRNA S S 4kDa A2 Pentameric B subunit (5 x 7kDa) B • Receptor: glycolipid called Gb-3 • Gb3-rich cells (particularly sensitive to STx) include • vascular endothelial cells • absorptive enterocytes • kidney endothelial cells

  19. STx: Entry via RME retrograde transport via Golgi & ER From: Groisman

  20. haemorrhage diarrhoea Renal failure (10% - 20% cases) S. dysenteriae E. coli producing high levels of STx Strongly associated: E. coli Shiga-toxins (STx) Role in disease • Local effects of toxin in colon • Absorption – systemic effects Haemolytic uraemic syndrome (HUS) Very serious – often fatal.

  21. E. coli O157:H7 DNA damage SOS response • Clinical reports that therapy with certain antibiotics • increased severity of O157 infections WHY ? Some antibiotics (e.g. quinolones) inhibit DNA replication. Induces lysogenic phage higher levels of STx in gut

  22. Urinary tract infections (community acquired)

  23. septicemia pyelonephritis ureteritis cystitis urethritis Uropathogenic E. coli(UPEC) Bloodstream Kidneys Urine Flushing Ureter Bladder Removes non-adherent bacteria Urethra Ascending infection source: UPEC in colon - harmless commensals

  24. Toxicity inflammation Uropathogenic E. coli - virulence mechanisms • Adhesion - essential to avoid removal by urine • Survival - studies limited to some aspects only • iron-acquisition (siderophores) • capsules (evasion of host defences) - more important in kidney than lower UT - critical if organism enters bloodstream • LPS (endotoxin) • a-haemolysin (membrane damaging toxin) • cytotoxic necrotizing factor 1(CNF-1)

  25. high concentrations ion leakage osmotic lysis • lower concentrations more subtle cytotoxic effects E. colia-haemolysin (HlyA) • Membrane-damaging toxin: • Produces small hydrophilic pores in mammalian cells • Produced by about: • 50% strains from upper UTI – most tissue damage • 30% strains from lower UTI • 10% GI strains Suggests strong association with UTI

  26. E. colia-haemolysin (HlyA) • Role in kidney damage supported by studies in mouse • models: Parent strain expressing P fimbriae + HlyA colonized bladder + kidneys 66% mice died Isogenic mutant expressing P fimbriae, but not HlyA colonized as above, but no apparent damage or deaths

  27. Cytotoxic necrotizing factor 1 (CNF-1) • Large (110,000Da) protein, produced by ca 30% UPEC • Studies in vitro: • epithelial cells: rearrangements of actin filaments & • membrane ruffling • bladder cells: reduced migration & proliferation – • - impede repair of damage bladder ?? • No direct evidence for role in UTI

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