MIC328: Lecture 19

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

4. 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

5. 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

6. 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)

7. 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

8. 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%)

9. 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

10. 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

11. 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 CTx actively secreted by the EPS terminal branch of GPS

12. ‘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 ?

13. 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

14. 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.

15. 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