Bio305 2012 Lecture 1 on E. coli
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This document outlines a comprehensive lecture series on Escherichia coli, covering its biology, genetics, pathogenesis, and role in various infections. It discusses E. coli as a vital research organism, its diverse pathotypes, and associated diseases, with a focus on mechanisms of virulence and clinical implications. Additionally, it highlights the importance of understanding these bacteria in the context of public health due to their impact on both developed and developing nations.
Bio305 2012 Lecture 1 on E. coli
- 1. Bio305 Escherichia coli Lecture 1 Inside the gut; outside cells Professor Mark Pallen University of Birmingham
- 2. Wk Day Date Time Topic Staff Room Tue 10-Jan 1100 Introduction: Pathogen Biology Prof Pallen LC-LG32 1 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-LG32 2 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-LG32 3 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-LG32 4 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-LG04 5 Thur 09-Feb 1200 Cell envelope components 5: LPS and capsules Dr Alderwick SPX-LT3 6 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 E204 7 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 E204 8 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-LT3 9 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-LG32 10 Thur 15-Mar 1200 Staphylococcus aureus 1 Dr Lovering SPX-LT3 Tue 20-Mar 1100 Staphylococcus aureus 2 Dr Lovering LC-LG32 11 Thur 22-Mar 1200 Spare SPX-LT3
- 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 modeler's favourite biological system
- 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
- 6. 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!
- 7. 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
- 8. E. coli as a pathogen
- 9. 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
- 10. 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
- 12. 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
- 13. 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
- 14. 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
- 15. 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
- 16. 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
- 17. 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
- 18. 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)
- 19. EnterotoxigenicE. coli adhesins CFA 1 and III CS3 fibrils
- 20. Non-fimbrial surface structures Invasins 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
- 21. 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
- 22. 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
- 24. In a phylogeny based on chromosomal house-keeping genes ETEC strains scattered among all lineages; not a monophyletic group!
- 25. 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
- 26. 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
- 27. 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
- 28. 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.
- 29. 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
- 30. 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
- 31. EHEC virulence factors Shiga-like Toxin A/E Lesion (aka SLT; Vero-Toxin; VT; Stx) Type III secretion plus pO157-encoded ToxB
- 32. A subunit, cleaves rRNA inhibits protein synthesis B subunits, bind Gb3 Holotoxin http://www.rpc.msoe.edu/cbm/smartteams/remote/
- 33. 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
- 34. 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
- 35. EHEC genetics of virulence Shiga Toxin and T3SS effectors encoded by bacteriophages
- 37. 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
- 39. 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)
- 40. 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
- 41. Time to avoid typological thinking? ETEC not a monophyletic group… STEC EAEC ESBL AE-lesion-forming + E. coli E. coli EPEC
- 42. 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
- 43. 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