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

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

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

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

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 Both adhesins and toxins encoded by plasmids easily transferred among different E. coli strains Many different ETEC strains - each usually specific for particular host (reflects adhesion specificity)

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

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 mostly in: Human ETEC Animal ETEC Type Variants LT toxins: LT-I LT-II ST toxins: STa (ST-I) STb (ST-II) ETEC strains may produce LT only (30%), ST only (35%), or both (35%)

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 (binds different gangliosides) facilitates A1 entry LT-I: > 80% homology with CTA & CTB LT-II: A 60% homology with CTA; B - no homology

Cholera usually more severe - Why ? 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

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

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

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 Guanylin-receptor binding activates enzyme production of cGMP in cell STa does exactly same, but is more potent than endogeneous hormone + larger amounts Like excess cAMP, excess cGMP opens CFTR increased Cl secretion net efflux of H2O

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.

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