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MIC328: Lecture 19 PowerPoint Presentation
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MIC328: Lecture 19

MIC328: Lecture 19

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MIC328: Lecture 19

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  1. MIC328: Lecture 19 AIMS: To provide • Brief introduction to E. coli: a versatile pathogen • Overview of Enterotoxigenic E. coli (ETEC)

  2. E. coli - a versatile pathogenic species • Single species causes a variety of different diseases • different strains possessing different sets of vir genes • MostE. coli strains - harmless intestinal commensals • Only some strains pathogenic divided into groups based on disease produced

  3. Summary of types of intestinal diseases - enteric pathogens Secretory or watery diarrhea: Large volume watery stools - no blood or pus Site: Small intestine, no invasion - colonization + toxin production Examples: V. cholerae, ETEC, EAEC Bloody watery diarrhea: Large volume, watery + bloody stools (sometimes + pus) Site: Small intestine, tissue invasion Examples: Yersinia enterocolitica, Campylobacter jejuni Colitis or Haemorrhagic colitis: Large volume, watery, bloody stools - no pus Site: Large intestine - invasion absent or limited - A/E lesions Examples: EPEC, EHEC Dysentery: Bloody stools + pus + mucus; scant volume Site: Large intestine, tissue invasion Examples: Shigella, EIEC, Entamoeba histolytica

  4. 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

  5. Both adhesins and toxins encoded by plasmids easily transferred among different E. coli strains ETEC Secretory (watery) diarrhoea: like cholera, but less severe Like V. cholerae, ETEC • do not invade • adhere to + colonize epithelium of small intestine • produce enterotoxins that cause secretory diarrhea Many different ETEC strains - each usually specific for particular host (reflects adhesion specificity)

  6. Human ETEC strains • Often fatal in infants and young children - a major • cause of deaths in many developing countries • Adults less susceptible – acquire natural immunity to • local ETEC strains during previous encounters • (either sub-clinical or disease) • ETEC common cause of traveller's diarrhoea in adults • - little or no immunity to ‘newly’ encountered serotypes

  7. Type Variants LT toxins: LT-I LT-II ST toxins: STa (ST-I) STb (ST-II) mostly in: Human ETEC Animal ETEC Enterotoxins produced by ETEC strains • Two different general types discovered by early ‘70s • Heat-labile enterotoxins (LT) • Heat-stable enterotoxins (ST) • Later, ‘variants’ of each type identified • ETEC strains may produce LT only (30%), ST only (35%), • or both (35%)

  8. E. coli Heat-labile toxin (LT) A-subunit ADP-ribosylates Gs activates A1 A2 adenylate cyclase cAMP B-pentamer B • binds to gangliosides • LT-I: GM1 • LT-II: GD1a or GD1b • facilitates A1 entry LT-I: > 80% homology with CTA & CTB LT-II: A 60% homology with CTA; B - no homology

  9. ETEC versus V. cholerae CT • Both colonize small intestine very effectively • LT & CTx very similar toxins - identical mechanisms Cholera usually more severe - Why ? • Probably combination of reasons, including difference • in the way the toxins are released • Example of importance of protein secretion in bacterial • pathogenicity

  10. Difference in the excretion of LT and CT toxins E. coli V. cholerae Lacks GSP terminal branch LT-A LT-B Sec Sec • LT remains in periplasm • Small quantities ‘leak’ thro’ • OM ( bile salts) CTx actively secreted by the EPS terminal branch of GPS

  11. ‘pro-’ sequence removed outside cell. Role unclear - possibly export thro’ OM ?? N-terminal signal sequence Active toxin consists of only the C-terminal 18 a.a. - these include 6 Cys ETEC: Heat-stable toxins (STa) - structure STa best understood - small peptide toxin, expressed as much larger 72 a.a. ‘prepropeptide’ precursor ‘pre’ ‘pro’ sec In periplasm, protoxin folds + forms 3 disulphide bonds OM ? • Very stable • (resistant to gut proteases) Type V secretion ?

  12. Guanylin-receptor binding activates enzyme production of cGMP in cell Like excess cAMP, excess cGMP opens CFTR increased Cl secretion net efflux of H2O Heat-stable toxins (STa) - mechanism of action ‘mimics’ an intestinal hormone called guanylin ( a 15 a.a. peptide with 2 disulphide bonds) Guanylin regulates guanylate cyclase - ‘receptor cyclase’ located in apical membranes STa does exactly same, but is more potent than endogeneous hormone + larger amounts

  13. The second heat-stable toxin - STb Similar size precursor (71 a.a.) as STa, with N-terminal signal sequence secretion to periplasm by GSP, where it folds + forms intramolecular disulphide bonds (hence stable) However, similarity with STa ends there. • No additional ‘processing’ after secretion to periplasm • Active toxin is a 48 a.a. peptide that does not result in • increased production of either cAMP or cGMP • Mechanism of action still not understood, though shown • to induce fluid-secretion in ligated-ileal loops.

  14. ETEC Enterotoxins - Summary LT-I & LT-II: Very closely related to Cholera toxin - similar structure Identical mechanism of action as CT cAMP Like CT secreted to + assembled in periplasm Unlike CT, not secreted thro’ OM - released by ‘leakage’ STa Small (18 a.a.) peptide, expressed as ‘prepropeptide’ Very stable due to 3 intramolecular disulphide bonds Mimics intestinal peptide hormone guanyline cGMP STb Also relatively small peptide (48 a.a.) - distinct from STa Mechanism of action not elucidated

  15. MIC328: Lecture 21 • ETEC adhesion • EAEC & DAEC • EPEC - introduction

  16. ETEC: Adhesion & colonization • Strong adhesion required to avoid removal by peristalsis • [see Lecture 01] The receptor correspondingly specific molecule on host surface The adhesin specific molecule on bacterial cell surface Carbohydrate components of glycolipids or glycoproteins Protein Usually: Specificity analogous to enzyme-substrate specificity

  17. Adhesin-Receptor specificity Soluble sugar ‘mimicking’ critical part of specific receptor Specific adhesin Specific receptor May bind to the adhesin, blocking the receptor-binding site, Thus inhibiting adhesion • Type I fimbriae: First E. coli adhesin identified (late 1950s) - inhibited by D-mannose - MSHA: mannose-sensitive haemagglutination

  18. Type I fimbriae Originally assumed to mediate ETEC adhesion - BUT • Expressed by > 90% strains, including non-pathogenic Also by some other enterobacteriae – ‘common’ fimbriae • No particular disease-association • 1970s: Discovery of MRHA • ETEC strains from pigs mannose-resistant adhesion Plasmid-encoded K88 fimbriae

  19. Infection experiments in piglets (1971) Result wt E. coli expressing Type I K88 Fimbriae LT toxin Fimbriae Gut colonized Diarrhea • Directin vivo evidence that MRHA adhesin essential for ETEC • Type I fimbriae do not mediate colonization of small intestine • - possibly ‘blocked’ by D-mannose in mucin glycoproteins,

  20. Other E. coli MRHA Adhesins > 40 distinct E. coli fimbrial or afimbrial adhesins - wide variety of different names (e.g. K88, K99, CS1, CS2, CFA/I, CFA/II, F47, Pap, S, AFAI, etc.). - Nomenclature can be very confusing Human ETEC Colonisation factor antigens: CFA/1 - CFA/IV Coli surface antigens: CS1, CS2, CS3, …..etc

  21. Human ETEC: Fimbriae originally designated CFA/II and CFA/IV, later found to include several distinct fimbriae CS3 – thin, flexible CFA/II CS1 CS3 + CS1 or 2 CFA/IV CS6 + CS4 or 5

  22. E. coli MRHA Adhesins Presence/absence of specific receptor determines host or tissue specificity Examples: Adhesin Host Site K88 Pig Small intestine K99 Calves “ CFA/I Humans “ Pap Humans Urinary tract • All probably ‘diverged’ from a common ‘ancestor’

  23. E. coli fimbrial gene clusters – examples K88 (pig ETEC) fae genes A B C D E F G H I J P or Pap (UPEC) pap genes I B A H C D J K E F G Type I (common) fim genes B E A I C D F G H Chaperone Regulators (in cytoplasm) Major subunit ‘Usher’ (OM) Minor subunits

  24. Human ETEC: CFA/I A: Major subunit B: Chaperone C: OM ‘usher’ D: Minor subunit (located at tip) Expression regulated by environment – e.g. temperature (repressed by H-NS at low temp) – expressed at 37ºC

  25. ETEC adhere & form microcolonies Release of LT and/or ST produces diarrhoea

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

  27. 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)

  28. EAEC

  29. Diffusely adhering E. coli (DAEC)

  30. 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

  31. 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

  32. 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

  33. MIC328: Lecture 22 AIMS: • EPEC • Production of A/E lesions • Introduce EHEC

  34. 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

  35. EPEC Two clusters of vir genes involved • Plasmid-encoded • LEE locus in c/s • EAF plasmid (69 kb): encodes - BFP fimbriae • - regulators PerA & PerC • BFP fimbriae: initial, non-intimate, adhesion • PerA: transcriptional activator of bfp operon • PerC: activates ler gene within c/s LEE locus – Ler regulates other LEE locus vir genes Note: Some A/E inducing strains (e.g. EHEC) lack EAF plasmid, but use alternative adhesins for initial step.

  36. Two clusters of vir genes 2. LEE locus: 35.5 kb PI with > 40 ORFs. Encodes • Intimin (EaeA) – ‘intimate’ adhesin • Other proteins required for formation of A/E lesions • Type III secretion system (EPEC secretion components = Esc proteins) • EPEC secreted proteins: EspA, EspB, EspD, EspE, EspF

  37. 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

  38. 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

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

  40. 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, encoded by LEE locus • Translocated into host cell, phosphorylated, & inserted into the host cell membrane Tir: Translocated intimin receptor

  41. 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

  42. MIC328: Lecture 23 AIMS: • To introduce EHEC • E. coli in spesis and meningitis

  43. 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

  44. Shiga-toxin & Vero-toxin producing E. coli (STEC & VTEC) Early ‘80s, found that some E. coli: • produce toxins very like S. dysenteriae Shiga-toxin • – called ‘shiga-like’ toxin (SLT) & strains called STEC • produce a toxin that is particularly active against Vero cells • - called it Vero toxin (VT) & called the strains ‘VTEC’ Soon found that SLT = VT and almost identical to STx Thus: STEC = VTEC

  45. Variants of the E. coli STx STx-1 (=VT1) almost identical to S. dysenteriae ST (differ in only 1 a.a.) STx-2 (=VT2) 55% sequence homology with Stx-1, but essentially same basic structure & mechanism of action STEC strains may contain either one, or both toxins EHEC are a subset of STEC strains

  46. 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

  47. MIC328: Lecture 23 AIMS: • EHEC – virulence factors • UPEC - disease • - toxins

  48. 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

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