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Escherichia coli

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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






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

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