Escherichia coli

Molecular
biology of
Escherichia coli
Infections
http://www.medicine.tcd.ie/clinical_microbiology/
courses/student_area/MolMed.php
Introduction to enterobacterial gene regulation
and protein export systems

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Lecture overview
Escherichia coli as a pathogen
Genome structure of E. coli
Regulation of gene expression in enterobacteria
Co-ordinating protein export in bacteria

Escherichia coli as
a pathogen

Asymptomatic
Intestinal
colonization
Clinical Manifestation
Diarrhea
Extraintestinal
infection
Commensal ++++ - +
Intestinal-
Pathogenic
Extraintestinal
ExPEC
Pathogenic behavior E. coli
- ++++ _
+ - ++++

Top Ten Infectious Diseases -- Globe: Frequency
Condition Frequency (x 1000)
Diarrhea 4,000,000
Tuberculosis 1,900,000
Worms 1,400,000
Malaria 500,000
Hepatitis (HBV & HCV) 450,000
Pneumonia 395,000
Sexually transmitted diseases 330,000
Measles 42,000
Pertussis 40,000
HIV (new infections) 3,100
Meningococcal meningitis 350
5
Escherichia coli
Rotavirus

Top six killers: infections responsible for greatest
numbers of deaths (WHO 2004)
Acute Respiratory Infections AIDS Diarrheal Diseases
TB Malaria Measles
Could all be eradicated by vaccination
Clean water for South America $200 billion
6

• Intestinal IPEC
ETEC toxigenic
EPEC pathogenic
EAEC aggregative
EIEC invasive
DAEC diffuse adherent
Escherichia coli PATHOTYPES
EHEC/VTEC/STEC haemorrhagic
• Extra-intestinal EXPEC
• Uropathogenic E. coli
– UPEC
• Neonatal meningitic E. coli
– NMEC
•Commensal

Enteric E. coli Pathotypes
Shigella

EPEC
Forms an Attaching-Effacing (A/E) lesion

EPEC
• Bind to enterocyte with bundle forming pili BFP
• Intimin to attach closely
• Protein hypodermic syringe
• Type three secretion system TTSS
• Injects the Intimin receptor Tir into the epithelial cell
• Actin rearrangement and engulfment

Bundle-
Forming Pili
Bundle-Forming Pili (Bfp) (Fimbriae)

EPEC attachment and effacement
12
EspA
EspB

Pedestals with EPEC Showing EspA Tubules on
their Surfaces
EspA
Pedestal

The EspA
Translocation
Tube and
EspB/EspD
Pore in the
Host Cell

Tir
Actin
EPEC
EPEC-Blue
Tir-Red
Actin-Green

Actin Polymerisation mediated by Actin
Nucleating Factors (WASP & Arp2/3)
Interacting with Tir

EPEC
• Effacement leads to malabsorption

EPEC Diarrhoea

Enteric E. coli Pathotypes
Shigella
Produce
Hly
Cnf

EHEC/VTEC
• Low infective dose
• Disease -mild uncomplicated diarrhea
• Hemorrhagic colitis with severe abdominal pain
• Hemolytic uremic syndrome (HUS)
• > 50 Serotypes
• O157:H7
• O26, O111, O103 and O145

EHEC
VTEC

Virulence factors
• Adhesion via the EPEC method
• Toxins
• Shiga-like toxins SLT1, SLT2 and SLT2v
• a.k.a. Verocytotoxins VT1 VT2 and VT2v
• A and B subunits
• VT1 and 2 bind to globotriosylceramide Gb3

VTEC toxins
• VT binds to GB3 on
kidney cells - HUS
• GB3 also expressed on
blood cells by 70% of the
population
• Protective
• Toxicity associated with
inhibition of protein
synthesis
B binding
Pentamer
A (Active)Domain
–N-Glycosidase

Enteric E. coli Pathotypes
Shigella

ETEC
• > 300,000 deaths per annum
• High infectious dose
• Mainly in children
• Traveler’s diarrhea
• 80,000 US travelers
• Virulence factors
• Toxins
• Fimbriae/Pili

ETEC Virulence factors
• Fimbriae - Pili
• Plasmid encoded
• Colonization factor antigens CFAs
• CFA/I
• CFA/II (CS1, CS2, CS3) coli-surface
• CFA/III
• CFA/IV (CS4, CS5, CS6)

RNS H-NS
+ve -ve

ETEC Virulence factors
• Toxins - Plasmid encoded
• Heat-Labile Toxin LT
• Heat-Stable toxin ST

Heat-Labile Toxin LT
Binds to
GM1
Cl -
Crypt
cell
ADPr
Gs Gs
NaCl
Absorptive
cell
Protein
Phosphorylation
cAMP
Adenylate
cyclase

Heat-Stable toxin ST
• STa
• Peptide resembling Guanylin
• Bind to Guanylate cyclase
• Stimulates conversion of GTP to cGMP
• Chloride efflux

Enteric E. coli Pathotypes
Shigella
Produce
Hly
Cnf

EIEC and Shigella

Severity
S. dysenteriae
fever, malaise,
tenesmus, bloody diarrhea with mucus
prostration, febrile convulsions
S. flexneri/ S. boydii Enteroinvasive E. coli
above mainly developing nations
S. sonnei
HUS due to
SHIGA toxin
passage of some loose stools, vague cramps

Pathogenesis
1 Invade thru’ microfold M cells
2 Engulfed by macrophage but induce apoptosis
this is facilitated by a EFFECTORS secreted by a TTSS
3 Exit
4 Reach basolateral surface of epithelial cell

Pathogenesis
5 Bacteria move from cell to cell by polymerizing
actin
6 Dissemination

Cell to cell spread
• Requires IcsA outer membrane protein

Cell to cell spread
No IcsA

Pathogenesis
A: Release of IL-1 by apoptotic macrophage (LPS)
B: IL-8 released by infected enterocytes
C: Promotes recruitment of monocytes
D/E: Further increases bacterial entry

Shiga toxin

Genome structure
of E. coli

Typical characteristics of E. coli genomes
Not all E. coli are the same!

Genomic
comparisons
reveals
extensive
rearrangement

Evolution of E. coli
non-pathogenic uro-pathogenic
585
39% in all 3
12% in 2
49% unique
514
193
2996
1364
204
enterohaemorrhagic
45
1623

Evolution in bacteria
Horizontal transfer
• En bloc exchange of large pieces of DNA
• Remodels the genomic backbone
• Cross-links distinct branches of clones
• Breaks down the clonal frame
46

Evolution of pathotypes

Extra-chromosomal elements
• Plasmids
✴ Independent & self replicating
✴ Circular and double-stranded
✴ Inherited
✴ Copy number 1- 400
✴ Size 60-120 kbp, 1.5 to 15 kbp.
✴ Can encode antibiotic resistance
✴ Can encode virulence factors
✴ Transmissible
48

pO157

Gene transfer
50

Evolution of
pathotypes

Extra-chromosomal elements
Bacteriophage
52

Gene transfer
53

Evolution of
pathotypes

Genomic islands - pathogenicity islands
Usually inserted in vicinity of tRNA
Flanked by direct repeats
Encodes several traits that increase fitness or adaptability
Multiple IS elements
Integrase

Genomic islands - pathogenicity islands

Evolution of pathotypes

transposition

Relationship between
E. coli pathotypes
DAEC
Typical
EPEC
AEEC
Atypical
EPEC
EHEC
UPEC
0157
STEC
EAEC
NMEC
EIEC
Shigella
ETEC

Regulation of gene
expression in
enterobacteria

Transcription and Translation are
coupled and non-compartmentalized in
DNA
mRNA
RNA polymerase
Ribosome
bacteria
mRNA degraded soon after translation
62

CRP
IHF
RNA polymerase
sigma 70 subunit (RpoD) recognizes TTGACA-17bp-TATAAT
deviations lead to alterations in gene expression
less complex than eukaryotic transcription
63

Transcription in bacteria-alternate sigma factors
Stress/Stationary phase sigma factor Rpos
Sigma 38
-13 CTACACT -7
Nitogen Limitation RpoN
Sigma 54
CTGGCAC-5bp-TTGCA (6-11bp)
Heat shock RpoH
Sigma 32
Extracellular stress ECF
Sigma E

Transcription levels can be altered
Recruitment
Anti repressor
65
Occlusion
Block open complex
Roadblock

Multiple genes can be regulated simultaneously
This methodology is used to control virulence
66
PhoP Salmonella
regulates ~ 50 genes
mutants are avirulent

Global Regulators

Regulation of transcription in bacteria- nucleoid proteins

Regulation of transcription in bacteria -nucleoid proteins
Protein Structure
Copy Consensus
binding
H-NS α2 15.4 kDa 20000 Curved
HU αβ 9.5 kDa 20000 Cruciform/H
IHF αβ 11.2/10.5 INTEGRATION
HOST FACTOR 100,000 C/TAAN3TTGATA/T
FIS α2 11.2 kDa
FACTOR FOR
INVERSION
STIMULATION
Early
stationary
10,000
GN2AN2A/
TN2TN3C
Genes
regulated
Represses
~1000 genes
Activates >
150
300

Specific Regulators
Two component regulators - winged helix
AraC-like regulators - helix-turn-helix

Regulation of transcription in bacteria-
EnvZ
N
C
His-P
OmpR
Asp-P
N
Signal
C
two component
systems
Outer Membrane
Periplasm
Inner Membrane
Binding to OmpF/C promoters and
other regulon members

Regulation of transcription in bacteria-
PhoQ
N
C
His-P
PhoP
Asp-P
N
[Divalent cation]
macrophage environment
C
PhoP- salmonella
slide 52
Outer Membrane
Periplasm
Binding to PhoP-dependent
promoters and other regulon
members PAGS and PRGS
Inner Membrane

Regulation of transcription in bacteria -two component
systems
Nitrogen
low
N
C His-P
NtrB
Asp-P
N
NtrC
C
Outer Membrane
Periplasm
Inner Membrane
RpoN
dependent
promoters

Regulation of transcription in bacteria - AraC family

Typical features of AraC regulators
> 800 members - 34 in Escherichia coli
Signal Reception
Contact With RNAP
Dimerization
>100 aas
Sugar binding or physical stimulus
Helix-Turn-Helix Motifs in
carboxy-termini
At N-terminus associated with
repression
Homology to AraC
DNA binding
100 aas

RNS H-NS
+ve -ve

Co-ordinating protein
export in bacteria

Gram-negative cell envelope

Getting proteins out of gram-negative bacteria
Chaperone
Usher
OM insertion
Type II
MTB
terminal branches
Type IV
Type V
Type1 GSP TAT TypeIII
Outer Membrane
Periplasm
Inner Membrane

GSP the General Secretory Pathway
Signal sequence
Signal peptidase
++ Hydrophobic *
N H C
1-5 7-15 3-7 aas

Sec translocase
SecB chaperone binds nascent peptide over 150 kDa region
Binds to SecA thus delivering the protein to the translocase
Repeated nucleotide induced conformational changes in SecA
Pumping of peptide thru’ the machinery - 20 AAs
SecB mutants still proficient for translocation - why?

FtsY-SRP system Signal Recognition Particle
Homolog of Eukaryotic system
Ffh homology to SRP54 and receptor FtsY, 4.5 S RNA
Binds to first 150 aa of the nascent peptide
Arrests protein synthesis and targeted to the membrane
SRP released and protein delivered to sec
CO-TRANSLATIONAL

TAT system an alternative for folded proteins
Signal sequence
Signal peptidase
++ RRXFXK Hydrophobic *
N H C
5-24 12-20 3-7 aas
22 proteins in E. coli
Hydrophobic region is SEC avoidance
Translocated fully folded - even oligomeric
Tend to be periplasmic enzymes
Integral membrane proteins TatABC, cytoplasmic
TatD -pore 7 nm

Protein trans-location out of the cell

TOSS type 1 secretion pathway
Direct Export thru envelope
HlyA haemolysin A pore forming toxin
UROPATHOGENIC
Three components
Pore forming OMP
MFP membrane fusion protein HlyD
IM ABC protein HlyB
Specific SS binds to specific ABC
triggers interaction with MFP/TolC
Exported and folded at OM

TOSS type 1 secretion pathway

Type II secretion - the main terminal branch
Usually Sec-dependent occasionally Tat
PulA of Klebsiella oxytoca
D protein = SECRETIN
12-14 subunit beta barrel
E protein is regulatory kinase
G-K proteins for a pseudo pilus
N-terminal methylation by protein O
Multi subunit complex

Type II secretion -Labile toxin secretion

T3SS type three secretion system
First described in Yersinia
Contact dependent
Sec Independent
Secretion of Yops
Similar to flagella

TTSS type three secretion system
Organism
Secreted
proteins
Yersinia spp. Yop
Yersinia
enterocolitica
Salmonella
typhimurium
Salmonella
typhimurium
Shigella
flexneri
Virulence Location
Cytotoxicity
Phagocytosis inhibition
Ysp Invasion of Peyer’s patches C
Sip
Invasion
Apoptosis induction
P
C SPI-1
Sse Macrophage survival C SPI-2
Ipa
EPEC Esp Tir
Invasion
Apoptosis
Adhesion attaching and
effacing
P
C LEE PI

TFSS type four secretion system
Agrobacterium tumefaciens
Protein and DNA transport
Similarity to conjugal DNA transfer systems
Toxin translocation
CagA - pertussis toxin

Enterobacterial protein secretion systems

The chaperone usher pathway Pili and Fimbriae
Periplasm
Pap

The chaperone usher pathway Pili and Fimbriae

TypeV the Autotransporters

TypeV the Autotransporters
Very long signal sequences ~ 45 aa
Particularly in >100 kDa protein
Recognized by SRP - not by TAT
beta domain spontaneously forms in periplasm
inserts into OM

TypeV the Autotransporters