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SBM 2044 Lecture 9

SBM 2044 Lecture 9. Vibrio cholerae . Diarrhoea . Secretory or watery diarrhoea is caused by the loss of electrolytes and fluids from the small intestine Infections of the intestinal tract can lead to local tissue damage or inflammation and cause bloody diarrhoea or dysentery.

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SBM 2044 Lecture 9

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  1. SBM 2044 Lecture 9 Vibrio cholerae

  2. Diarrhoea • Secretory or watery diarrhoea is caused by the loss of electrolytes and fluids from the small intestine • Infections of the intestinal tract can lead to local tissue damage or inflammation and cause bloody diarrhoea or dysentery

  3. Enteric pathogens • Enterobacteriacea are short G– rods • E. coli and most enteric bacteria form circular convex, smooth colonies with distinct edges • Klebsiella and some other enterobacter colonies are very mucoid in structure • Some strains of E. coli produce haemolysis on blood agar. • Lactose fermentation: • lactose fermenters : E. coli, K. pneumoniae • non-lactose fermenters : Shigella, Salmonella

  4. London, 1854 John Snow “with a notebook, a map and my five senses... traced source of the most terrible outbreak of the cholera which has ever occurred in this kingdom”

  5. Cholera • The connection between cholera and faecal bacteria in drinking water was discovered in London by John Snow in 1854. • Not an invasive infection. Organisms remain in intestinal tract. V. cholerae attach to the microvilli of epith cells whereby they multiply and liberate cholera toxin. • Incubation period 1-4 days, presented with sudden onset of nausea, vomiting and profuse diarrhoea with abdominal cramps. Stools (“rice water”) contain mucus, epith cells and a lot of vibrios.

  6. Vibrio cholerae • Gram-negative • Comma-shaped rods • Highly motile • Single polar flagella

  7. Vibrio cholerae • Survives well in environment (fresh & salt water) • Majority of strains do NOT cause cholera • For >70 years, combination of biotyping & serotyping (O-antigen) used to distinguish V. cholerae isolates • Before 1992, all identified disease isolates were O1

  8. O139 Bengal • Began in the Indian subcontinent in 1992-1993, and spread to Asia • Very similar to V. cholerae O1 El Tor biotype • V. cholerae O139 does not produce the O1 lipopolysaccharide and does not have all the genes necessary to make this antigen • O139 makes a polysaccharide capsule like other non-O1 V cholerae strains, while V. cholerae O1 does not make a capsule.

  9. Cholera toxin • Heat-labile enterotoxin (Mr 84k) • Ganglioside GM1 serves as the mucosal receptor for subunit B, and promotes entry of subunit A. • Diarrhoea – 20-30 L/d • results in shock, acidosis, and death. • 25g cholera toxin  40 L watery diarrhoea • Ion transport pumps play critical role in maintaining control of ion fluxes across intestinal epithelial cells Na+ Cl- HCO3- K+ • Disrupted by CTx, which increases cAMP in enterocytes • Activates a protein kinase • Opens CFTR channel (Cl- secretion) • Excess fluid in lumen

  10. Cholera toxin & E. coli Heat-labile toxin (LT) A1 ADP-ribosylates Gs B-pentamer binds to GM1 facilitates A1 entry (not RME)

  11. How does CTxA reach its target ? Apical Tight junctions V. cholerae in gut lumen Basolateral CFTR Cl channels present in apical membrane, but the CTx target (Gs proteins + Adc) only on cytosolic surface of basolateral membrane.

  12. How does CTxA reach its target ? • EM with Gold • -labelled CTx non-coated vesicles (caveolae) clathrin-coated caveolae CT-A2 has a KDEL sequence Could suggests trafficking thro’ ER ???

  13. KDEL receptor • The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval of escaped ER proteins bearing a C-terminal KDEL sequence. • This occurs throughout retrograde traffic mediated by COPI-coated transport carriers.

  14. Sorting signal: KDEL ER-retrieval

  15. KDEL – retrieval of soluble ER proteins KDEL receptor KDEL ‘tagged’

  16. How does CTxA reach its target ? Binds to GM1 non-coated vesicles (caveolae) KDEL sequence ER (via golgi) BMV (Basolateral membrane vesicles) Point at which A1 enters cytosol unclear

  17. CHOLERA TOXIN Sufficient to account for symptoms, but by no means only virulence factor First illustrated by human volunteer studies on ‘candidate’ live vaccines – ctxA mutants mild diarrhoea in 25% search for other potential toxins

  18. ZOT toxin • Disrupts zonula occludens (i.e. tight junctions) between • apical regions of enterocytes • Identified as factor in ctxA mutant supernatants that increased • conductivity of rabbit ileal tissue in Ussing chambers • EM studies visualised leakage of high mol wt material via • paracellular route – i.e. tight junctions disrupted • BUT double ctxA zot mutant STILL capable of inducing • (mild) diarrhorea

  19. changes in Cl and HCO3 transport in T84 cells ACE toxin Accessory cholera enterotoxin • Increased conductance of tissue in Ussing chambers • Caused fluid accumulation in ‘sealed infant mouse’ model HOWEVER V. choleraetriplectxA zot ace mutant still causes residual diarrhoea in human volunteers - other unidentified toxins ? - are Zot & Ace really toxins ? see later

  20. Haemolysin: • Produced by most non-01V. cholerae and original El Tor • isolates – but not ‘classical’ or recent El Tor isolates • pore-forming toxin, shown to be ‘enterotoxic’ in some • animal models • BUT, in human volunteers ctxA hylA mutant no less reactogenic that ctxA alone • Produced by El Tor and O139 strains, but not O1 classical

  21. Other toxins S-CEP: Secreted CHO cell elongation protein • identified only in 2000, causes fluid accumulation in • infant mouse model – role in disease ?? WO7 toxin: • identified in 1999 in El Tor strain from cholera-like • disease, BUT lacking CTx, Zot or ACE • fluid-accumulation in ligated ileal loops • 2-subunits, but quite distinct from CTx

  22. Other Vibrio cholerae virulence factors Neuraminidase: • removes sialic acid residues from complex glycolipids • & glycoproteins - might facilitate adhesion or possibly • even increase in CTx binding by converting complex • glycolipids to structures similar to GM1 - speculative • Defective mutants displayed only small reductions in • virulence in animal models - significance ? LPS: • Unclear if it has any role additional to OM structure • – cannot exclude contribution to adhesion

  23. Vibrio cholerae Adhesins TCP:Toxin-coregulated pili • Long filamentous fimbriae bundles, localised to • one area of cell-surface - significance ? • Tcp-defective mutants are avirulent in human volunteers

  24. Vibrio cholerae Adhesins MFRHA:Mannose-fucose resistant haemagglutinin • Originally called simply ‘surface haemagglutinin’ • OM protein - defective mutants avirulent in infant mice • model, but not tested in humans MSHA - mannose-sensitive haemagglutinin • MSHA pili found only one some O1 strains (El Tor) • Defective mutants show no reduction in colonisation • of infant mice model, so role in virulence unclear

  25. Treatment • water and electrolyte replacement • Oral tetracycline to reduce stool output in cholera; but in some endemic areas, tetracycline resistance of V. cholerae has emerged – genes are carried by transmissible plasmids

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