Bio305 2012 Lecture 1 on E. coli

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The first of two lectures on E. coli as a pathogen given on the Bio305 module in the School of Biosciences, University of Birmingham

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Bio305 2012 Lecture 1 on E. coli

  1. 1. Bio305 Escherichia coli Lecture 1 Inside the gut; outside cells Professor Mark Pallen University of Birmingham
  2. 2. Wk Day Date Time Topic Staff Room Tue 10-Jan 1100 Introduction: Pathogen Biology Prof Pallen LC-LG321 Wed 11-Jan 1200 Introduction: Genetics of virulence Prof Pallen LC-LG32 Thur 12-Jan 1200 Introduction: Regulation of virulence Prof Pallen SPX-LT3 Tue 17-Jan 1100 spare LC-LG322 Wed 18-Jan 1200 Bacterial Genomics: Sequence Analysis Prof Pallen LC-LG32 Thur 19-Jan 1200 Tuberculosis 1 Dr Bhatt SPX-LT3 Tue 24-Jan 1100 Bacterial Genomics: Dynamics and Evolution Prof Pallen LC-LG323 Wed 25-Jan 1200 Tuberculosis 2 Dr Bhatt LC-LG32 Thur 26-Jan 1200 Cell envelope components 1: peptidoglycan Dr Lovering SPX-LT3 Tue 31-Jan 1100 Cell envelope components 2: teichoic acid Dr Lovering LC-LG324 Wed 01-Feb 1200 Cell envelope components 3: the mycobacterial cell wall 1 Dr Alderwick LC-LG32 Thur 02-Feb 1200 Cell envelope components 4: the mycobacterial cell wall 2 Dr Alderwick SPX-LT3 Tue 07-Feb 1000-1200 Bioinformatics Practical Session LC-LG045 Thur 09-Feb 1200 Cell envelope components 5: LPS and capsules Dr Alderwick SPX-LT36 Tue 14-Feb 1100 E. coli 1 Prof Pallen LC-LG32 Thur 16-Feb 1200 E. coli 2 Prof Pallen SPX-LT3 Drs Mon 20-Feb 1500-1800 Lab Practical Session 1 Alderwick/Bhatt Lab E2047 Tue 21-Feb 1100 Bacterial protein secretion 1: An Overview Prof Pallen LC-LG32 Thur 23-Feb 1200 Bacterial protein secretion 2: The Bacterial Flagellum Prof Pallen SPX-LT3 Drs Mon 27-Feb 1500-1800 Lab Practical Session 2 Alderwick/Bhatt Lab E2048 Tue 28-Feb 1100 Bacterial protein secretion 3: Type III Secretion Prof Pallen LC-LG32 Thur 01-Mar 1200 Bacterial protein secretion 4: Esx Secretion Prof Pallen SPX-LT39 Tue 06-Mar 1100 Bacterial protein secretion 5: Sortase and LPXTG proteins Prof Pallen LC-LG32 Thur 08-Mar 1200 Pseudomonas aeruginosa 1 Prof Penn SPX-LT3 Tue 13-Mar 1100 Pseudomonas aeruginosa 2 Prof Penn LC-LG3210 Thur 15-Mar 1200 Staphylococcus aureus 1 Dr Lovering SPX-LT3 Tue 20-Mar 1100 Staphylococcus aureus 2 Dr Lovering LC-LG3211 Thur 22-Mar 1200 Spare SPX-LT3
  3. 3. Importance of Escherichia coli Biology‘s premier model organism • more known about this bacterium, esp. K-12, than any other organism clear favourite in the study of bacterial genetics, biochemistry and physiology • rapid growth rate, undemanding lab growth requirements, tractable genetics, metabolic versatility • DNA replication, transcription, translation, gene regulation, restriction enzymes and horizontal gene transfer (e.g. phage lambda) major enabling technology—so safe and user-friendly that even non-microbiologists can work with it! re-born as mathematical modelers favourite biological system
  4. 4. Importance of Escherichia coli Neidhardt‘s dictum  ‗All cell biologists have at least two cells of interest: the one they are studying and E. coli.‘ BUT!  E. coli K-12 now rough, poor gut colonizer  Life in lab nothing like life in a state of nature  E. coli K-12 stored in lab for several decades after 1922  Subjected to harsh mutagenesis to remove lambda phage and F plasmid
  5. 5. The real E. coli Highly versatile commensal, pathogen and environmental organism Probiotic and pathogen! Infects many hosts and organ systems weanling diarrhoea in piglets; avian colibacillosis; diarrhoeal disease, urinary tract infections, blood stream infection and meningitis in humans enteropathogenic, enterotoxigenic, enteroinvasive, enterohaemorrhagic, enteroaggregative and diffusely adherent varieties– plus Shigella!
  6. 6. Escherichia coli Rod-shaped, non- H antigen depends on variation in flagellin spore-forming Gram- O antigen depends on variation in LPS negative bacterium Lactose-fermenter Belongs to the family Enterobacteriaceae Serotypedusing O antigens (LPS side chains) and H antigens (flagellin variable domains) http://mgl.scripps.edu/people/goodsell/illustration/public © David S. Goodsell 1999
  7. 7. E. coli as a pathogen
  8. 8. E. coli as a pathogen DiarrhoeagenicPathotypes Extra-intestinal E. coli  • enterotoxigenicE. coli (ETEC) (ExPEC) • enteroaggregativeE. coli • neonatal meningitis E. coli (EAEC) (NMEC) • enteropathogenicE. coli • uropathogenicE. coli (UPEC) (EPEC) • avian pathogenic E. coli • enterohaemorrhagicE. coli (APEC) (EHEC) • E. coli as a cause of hospital • enteroinvasive E. coli (EIEC) infection • Shigella
  9. 9. EnterotoxigenicE. coli (ETEC) Epidemiology and clinical features • causes watery diarrhoea • sometimes with vomiting and fever • range from mild to severe profuse cholera-like illness • common in underdeveloped world, rivalling rotavirus • infants living in the Nile delta area experience between 4.6 and 8.8 diarrhoeal episodes per year, with ETEC accounting for 66% of these episodes • can be fatal, especially in infants and young children • 100,000s of deaths, 100m of cases worldwide annually • in non-native adults, causes traveller‘sdiarrhoea
  10. 10. Heat-labile toxin (LT) Plasmid-encoded heterohexamericholotoxin, closely related to cholera toxin single A subunit, two domains linked by disulfide bridge  A1: active toxin molecule  A2: helical anchor to B pentamer intact A not enzymatically active until nicked to Al, A2  Al subunit released by reduction of disulfide bond pentameric B subunit  binds to GM1 gangliosides centered in caveolae on host cell surface  triggers endocytosis of holotoxin
  11. 11. Heat-labile toxin (LT) A1 domain translocated across intracellular membrane  allosterically interacts with ADP-ribosylating factors  ADP-ribosylatesGsα, an intracellular guanine nucleotide protein Gs regulates activity of host cell adenylatecyclase  active (GTP-bound) form of Gs increases activity of AC  inactive GDP-bound form renders adenylatecyclase inactive ADP-ribosylation of Gs short-circuits off-on control by locking Gs in "on" form, leads to constitutive activation of adenylatecyclase  increased levels of intracellular cAMP activate CFTR chloride channel  secretion of electrolytes and water leads to diarrhoea
  12. 12. LT secretion LT secreted through OM via two-step process  sec-dependent transport of monomers across IM to periplasmwhere they assemble into holotoxin  secretion across OM relies on type II secretion Polarized secretion of LT to ganglioside receptors  LT and secretion apparatus polarize to one end of the bacterium  anti-LT antibodies no effect on LT delivered by adherent organisms  LT-bearing outer membrane vesicles can enter host cells via lipid raft dependent endocytosis LT down-regulates innate host responses including defensins  enhances ETEC adherence to epithelial cells and colonization of the small intestine  powerful adjuvant
  13. 13. Heat-stable toxin (ST) small cysteine-rich peptide secreted by ETEC  can be boiled! binds to extracellular domain of guanylylcyclase C  molecular mimicry: resembles endogenous ligandguanylin activates intracellular catalytic domain of guanylylcyclase  intracellular accumulation of cGMP  activates cGMP-dependent protein kinase II  leads to phosphorylation of CFTR Cl- secretion and inhibition of NaCl absorption leads to osmotic diarrhea
  14. 14. Heat-stable toxins (ST) Human ETEC strains produce  STa(STI), ST-Ia (ST-P), ST-Ib (ST-H) STI molecules share core structure of 13 amino acids with 3 disulfide bonds required for biologic effect structure of the active ST-P toxin domain predicts  hexamericring  GC-C binding region residues Asn11– Ala13  promotes GC-C clustering and activation? STb or STII molecules typically associated with porcine  binds to different receptors  not clearly linked to human disease
  15. 15. Heat-stable toxins (ST) ST-H and ST-P are plasmid encoded  often in transposons synthesized as 72 amino acid precursor molecules  with19-aa signal peptide for Sec-dependent transport into periplasm export of STI peptides through OM requires trimericTolC protein exporter similar heat-stable toxin, EAST1, found in enteroaggregative strains
  16. 16. Colonisation Factors >25 different CFs: antigenically, structurally diverse proteinacioussurface structures  mostly plasmid-encoded  implicated in adhesion to small bowel  diversity hampers understanding of pathogenesis and vaccine design fimbrial, fibrillar and helical structures  lengths ranging from 1-to more than 20 μm  thought to bind to glycoprotein conjugates on host cells CFA/I fimbriae best characterised  ~1 μm long  1000 copies of the major fimbrial subunit CfaB  CfaEadhesin molecule, located at distal tip  periplasmicchaperone (CfaA)/outer membrane usher (CfaC)
  17. 17. EnterotoxigenicE. coli adhesins CFA 1 and III CS3 fibrils
  18. 18. Non-fimbrial surface structuresInvasins  role played by invasion of epithelial cells to molecular pathogenesis of ETEC remains uncertain Tia  25 kD OMP encoded on a large pathogenicity island inserted in the selCtRNA gene of H10407  interacts with host cell surface proteoglycans  promotes adherence and epithelial cell invasion when cloned into lab strains of E. coli TibA  autotransporter  synthesized as a 100 kD precursor protein, preTibA,  glycosylated by TibC, putative glycosyltransferase
  19. 19. Non-fimbrial surface structures Flagella  >30 different flagellar (H-antigen) types among ETEC strains  intact flagellar structures  essential for TEC adherence and heat-labile toxin delivery in vitro  contribute significantly to intestinal colonization EatA  plasmid-encoded  belongs to a family of SPATE proteins (Serine Protease Autotransporters of Enterobacteriaciae)  contributes to virulence in ileal loop studies  role and targets uncertain
  20. 20. Non-fimbrial surface structures Two-partner secretion (TPS) locus encoded on large virulence plasmid of ETEC H10407  EtpA, a 170 kDa secreted glycoprotein  EtpBa transport pore  EtpC, a putative glycosyltransferase required for secretion and glycosylation of EtpA EtpAfunctions as a molecular bridge  linking host cell receptors and highly conserved regions of flagellin proteins  required for optimal adhesion of H10407 in vitro, and for intestinal colonization in a murine model  Immunogen and target for vaccine development
  21. 21. In a phylogeny based on chromosomalhouse-keeping genes ETEC strains scatteredamong all lineages; not a monophyleticgroup!
  22. 22. EnteroaggregativeE. coli Linked to persistent diarrhoea in children  unclear whether well-defined group Like ETEC strains bind to enterocytes: do not invade Differ from ETEC strains  do not adhere uniformly to mucosal surface; form biofilms  auto-aggregative: clump in small aggregates (stacked-brick appearance)  relies on aggregative adherence fimbriae (AAFs, related to Dr family), dispersin Produce  ST-like toxin EAST, but also found in many commensals  Autotransporters Pet and Pic Kaur, Chakraborti, Asea (2010): doi:10.1155/2010/2541
  23. 23. Enteropathogenic E. coli First pathotype of E. coli to be described  Bray in UK in 1945  serologically distinct strains from children with diarrhoea but not from healthy children  Remains important cause of potentially fatal infant diarrhoea in developing countries Patchy adherence in bundles  In classical strains, microcoloniesformed via bundle- forming pili
  24. 24. Enteropathogenic E. coli A characteristic intestinal histopathology  ‗attaching and effacing‘ (A/E) lesion  effacement of microvilli  bacteria intimately attach to intestinal epithelial cells  cause striking cytoskeletal changes, polymerized actin beneath the adherent bacteria  pedestal-like structures on which the bacteria perch rise up from the epithelial cell
  25. 25. Enteropathogenic E. coli A/E lesion depends on 35-kb pathogenicity island, the locus of enterocyte effacement (LEE)  encodes a 94-kDa outer-membrane protein called intimin, which mediates the intimate attachment of EPEC to epithelial cells  encodes a type III secretion system (see later talk) Diarrhoea in EPEC infections probablyresults from multiple mechanisms, including activeion secretion, increased intestinal permeability,intestinal inflammation and loss of absorptive surfacearea resulting from microvillus effacement.
  26. 26. EnterohaemorrhagicE. coli (EHEC) Bind tightly to cells  same type of attachment-effacement as EPEC Difference from EPEC: produce Shiga toxin (Stx) Disease: closer to Shigella infection than ETEC or EPEC diarrhoea  haemorrhagic colitis; hemolytic-uremic syndrome can follow Very low infectious dose (<100 cells) In UK and US, predominant serotype is O157:H7, but O111 and O26 have caused problems elsewhere
  27. 27. EnterohaemorrhagicE. coli first recognised in 1982 common in developed countries ―Sakai‖ strain caused Sakai/Osaka outbreak in 1996  >9000 cases, 12 deaths. Several other outbreaks of EHEC (e.g. in California spinach, Sept 2006) Outbreaks usually linked to manure from infected animals Commensal in cattle
  28. 28. EHEC virulence factors Shiga-like Toxin A/E Lesion(aka SLT; Vero-Toxin; VT; Stx) Type III secretion plus pO157-encoded ToxB
  29. 29. A subunit, cleaves rRNAinhibits protein synthesis B subunits, bind Gb3 Holotoxin http://www.rpc.msoe.edu/cbm/smartteams/remote/
  30. 30. Shiga Toxin (Stx) Release of Stx occurs through lambdoid phage- mediated lysisin response to DNA damage  Antibiotic therapy discouraged, as may trigger toxin release Targets endothelium of small blood vessels Clinical effects  bloody diarrhoea  damage to kidneys and brain  anaemia  loss of platelets
  31. 31. Stx trafficking Stx diffuses through body fluids and actively transported by phagocytes Stxbinds GB3 (globotriaosylceramides) on plasma membrane  Internalised within clathrin-coated vesicles In sensitive cells  trafficked via endosomes to Golgi then ER  in ER, Stx nicked by furin  retro-translocation of StxA1 fragment into cytosol  cleaves rRNA, inhibits protein synthesis In insensitive cells, degraded in lysosomes
  32. 32. EHEC genetics of virulence Shiga Toxin and T3SS effectors encoded by bacteriophages
  33. 33. STEC beyond EHEC… the German E. coli O104:H4 outbreak May-July 2011 >4000 cases >40 deaths Link to sprouting seeds High risk of haemolytic- uraemic syndrome Females particularly at risk
  34. 34. Take-away messages Infection still presents threat even in the most advanced societies Pathogens don‘t bother with passports!  Not a new strain: something similar seen in Germany ten years ago and in Korea  closest genome-sequenced strain was isolated from Central African Republic in late 1990s, belongs to an enteroaggregative lineage German STEC probably comes from a lineage circulating in human populations rather than from an animal source (unlike E. coli O157)
  35. 35. Take-away messages Bacteria evolve quickly  Virulence factors in E. coli can jump from one lineage to another on mobile genetic elements  Pathotypes can overlap and evolve  Antibiotic resistance seen where no obvious prior use of antibiotics
  36. 36. Time to avoid typological thinking? ETEC not a monophyletic group… STEC EAEC ESBL AE-lesion-forming + E. coli E. coli EPEC
  37. 37. E. coli as a pathogen DiarrhoeagenicPathotypes Extra-intestinal E. coli  • enterotoxigenicE. coli (ETEC) (ExPEC) • enteroaggregativeE. coli • neonatal meningitis E. coli (EAEC) (NMEC) • enteropathogenicE. coli • uropathogenicE. coli (UPEC) (EPEC) • avian pathogenic E. coli • enterohaemorrhagicE. coli (APEC) (EHEC) • E. coli as a cause of hospital • enteroinvasive E. coli (EIEC) infection • Shigella
  38. 38. Further reading, video and audio Slides and slidecast available online Reading just the lecture notes will get you a 2.2 or 2.1 Showing external reading is needed to get a first Suggestions for further reading on WebCT

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