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

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