1. SBM 2044: Lecture 10 AIMS: To provide
Brief introduction to E. coli: a versatile pathogen
Overview of Enterotoxigenic E. coli (ETEC)

3. 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

4. EAEC First recognised in 1987 as distinct from other ETEC
Characteristic adhesion pattern in vitro reflects bacteria-
bacteria (in addition to bacteria-host) adhesion
Known virulence factors:
AAF - Aggregative adherence fimbriae
EAST-1 – EAEC heat stable toxin (similar to ETEC Sta)
Pet – Plasmid-encoded enterotoxin mucus release
Pic – role in intestinal colonisation? - various activities
(mucinase, serum resistance, haemagglutinin)

5. EAEC

6. Strains displaying a distinct adhesion pattern
Enteroaggregrative E. coli (EAEC)

7. Diffusely adhering E. coli (DAEC)

8. DAEC Doubts about their importance as pathogens
Like EAEC and ETEC, appear to be heterogeneous
group of strains, sharing certain common factors
Four different adhesins identified – F1845 fimbriae &
3 non-fimbrial adhesins
No significant information on potential toxins

9. Enteropathogenic E. coli (EPEC)
First associated with diarrhoea in 1940s
Major cause of watery diarrhoea in infants (< 6 months)
Developing countries - endemic
Developed countries – sporadic outbreaks in nurseries,
paediatric wards, day centres, etc
No detectable enterotoxin
1970s – first seen by EM to produce unique ‘attaching
& effacing’ effects on enterocytes - producing unique
lesions, now called A/E lesions

10. EPEC interactions with epithelial cells
1. Initial (non-intimate)
attachment
2. Intimate adhesion
+ effacement
3. Formation of
pedestals
Studies on mechanisms very limited until late 1980s

11. EPEC interactions with epithelial cells
1. Initial (non-intimate)
attachment
2. Intimate adhesion
+ effacement
3. Formation of
pedestals
NDP-phalloidin
Intimin (EaeA)
[OM protein]
Actin
rearrangements
Involved:
BFP

12. Filaments ‘connecting’
Knutton et al (1998) EMBO Journal 17:
Anti-EspA
Activation of EPEC
Type III secretion EM
Filaments ‘connecting’
EPEC & host cells
Immuno-gold antibodies
Filaments composed
of EspA

13. EPEC Type III secretion filament EspB/D pore
Host cell membrane
Deduced from studies combining:
Mutants
protein-protein binding
Labelling with specific Ab
Electron microscope
EPEC OM
EscC
EPEC IM
EscJ
IM-associated ‘machinery’
Esc R,S,T,U,V,N,D

14. Salmonella Type III ‘needles’
Kubori et al (1998) Science 280:
Salmonella Type III ‘needles’
Similar ‘needles’ in EPEC, but Salmonella lack ‘filament’

15. Tir: Translocated intimin receptor
EPEC: Kenny et al (1997) Cell 91:
Remarkable discovery:
Receptor for intimin (adhesin) is NOT a host cell protein
It is an EPEC protein
Translocated into host cell, phosphorylated, & inserted
into the host cell membrane
Tir: Translocated intimin receptor

16. EPEC: Model of A/E Lesion Formation
Initial adhesion (via Bfp )
Quorum sensing ?
Activation of Type III secretion
filaments
EspB/D pore
Tir + ? signals translocated
into host cell Tir- P
Ca++
Intimate adhesion by
EaeA to Tir- P
Details of signalling still unclear
Actin polymerization
pedestal formation

17. Enterohaemorrhagic E. coli (EHEC)
1982: two cases of severe food-poisoning (hamburgers)
in USA associated with rare serotype of E. coli - O157:H7
1983: E. coli O157:H7 produces a Shiga-like toxin
1986: Like EPEC, O157:H7 produce A/E lesions
‘Enterohaemorrhagic E. coli’ (EHEC) first used in 1987 to
describe what seemed to be a new type of pathogenic E. coli
Main difference with EPEC is production of Shiga-toxin

18. Origin of E. coli O157:H7 ?
First isolated in US in now worldwide
Quantitative population genetic studies (MLEE)
O157:H7 isolates throughout world are single clone that
emerged relatively recently - closely related to a much
less virulent EPEC clone
Both SLT-I & SLT-II genes encoded by bacteriophages,
- could facilitate their transmission into new strains
O157:H7 probably emerged when an EPEC strain (already
capable of producing A/E lesions & diarrhoea) acquired
slt phages, resulting in a dramatic increase in virulence

19. A1 Shiga-toxins Typical A-B subunit toxins A1 subunit - N-glycosidase
27kDa
Hydrolyses eucaryotic cell 28S rRNA S
4kDa A2
Pentameric B subunit (5 x 7kDa) B
Receptor: glycolipid called Gb-3
Gb3-rich cells (particularly sensitive to STx) include
vascular endothelial cells
haemorrhage
absorptive enterocytes
diarrhoea
kidney endothelial cells
renal failure

20. STx: Entry via RME retrograde transport via Golgi & ER
From: Groisman

21. E. coli Shiga-toxins (STx)
Role in disease
haemorrhage
diarrhoea
Local effects of toxin in colon
Absorption – systemic effects
Renal failure
(10% - 20% cases)
Haemolytic uraemic syndrome (HUS)
Very serious – often fatal.
S. dysenteriae
E. coli producing high levels of STx
Strongly associated:

22. E. coli O157:H7 Clinical reports that therapy with certain antibiotics
increased severity of O157 infections
WHY ?
Some antibiotics (e.g. quinolones) inhibit DNA replication.
DNA damage
SOS response
Induces lysogenic phage
higher levels of STx in gut

23. Urinary tract infections
(community acquired)

24. source: UPEC in colon - harmless commensals
Uropathogenic E. coli (UPEC)
Bloodstream
septicemia
Kidneys
pyelonephritis
Urine
Flushing
Ureter
ureteritis
Bladder
cystitis
Removes
non-adherent
bacteria
Urethra
urethritis
Ascending infection
source: UPEC in colon - harmless commensals

25. Uropathogenic E. coli - virulence mechanisms
Adhesion - essential to avoid removal by urine
Survival - studies limited to some aspects only
iron-acquisition (siderophores)
capsules (evasion of host defences)
- more important in kidney than lower UT
- critical if organism enters bloodstream
Toxicity inflammation
LPS (endotoxin)
a-haemolysin (membrane damaging toxin)
cytotoxic necrotizing factor 1(CNF-1)

26. E. coli a-haemolysin (HlyA)
Membrane-damaging toxin:
Produces small hydrophilic pores in mammalian cells
high concentrations ion leakage osmotic lysis
lower concentrations more subtle cytotoxic effects
Produced by about:
50% strains from upper UTI – most tissue damage
Note: Do not confuse with “a-haemolysins” produced by other species
30% strains from lower UTI
10% GI strains
Suggests strong association with UTI

27. E. coli a-haemolysin (HlyA)
Role in kidney damage supported by studies in mouse
models:
Parent strain expressing P fimbriae + HlyA
colonized bladder + kidneys 66% mice died
Isogenic mutant expressing P fimbriae, but not HlyA
colonized as above, but no apparent damage or deaths
Note: Do not confuse with “a-haemolysins” produced by other species

28. Cytotoxic necrotizing factor 1 (CNF-1)
Large (110,000Da) protein, produced by ca 30% UPEC
Studies in vitro:
epithelial cells: rearrangements of actin filaments &
membrane ruffling
bladder cells: reduced migration & proliferation –
- impede repair of damage bladder ??
No direct evidence for role in UTI