the diversity of escherichia coliinfections prof. michael

The Diversity of Escherichia coli Infections Prof. Michael S. Donnenberg

1The screen versions of these slides have full details of copyright and acknowledgements

The Diversity of Escherichia coli Infections

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Prof. Michael S. Donnenberg, MDProfessor of Medicine, Professor of Microbiology & Immunology

University of MarylandSchool of MedicineBaltimore, MD USA

A microbial Dr. Jekyll…

• Ubiquitous

• Innocuous commensal

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• Contributes to colonization resistance

• Produces beneficial metabolites

…and Mr. Hyde

• Diarrheagenic

– Enterotoxigenic (ETEC)

– Enteropathogenic (EPEC)

– Enterohemorrhagic (EHEC) d th Shi t i

• Extraintestinal (ExPEC)

– Uropathogenic (UPEC)

– Strains associated with neonatal meningitis (MAEC)

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and other Shiga toxin producing (STEC)

– Enteroaggregative (EAEC)

– Enteroinvasive (EIEC) and Shigella

– Diffuse adhering (DAEC)



How can E. coli cause so many diseases?

• Genetic plasticity

– Enumerable recombination events

• Distinct virulence factors specific for particular pathotypes

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• Virulence factors encoded on mobile genetic elements

– Plasmids

– Bacteriophages

– Pathogenicity islands

How do E. coli strains differ from one another?

• Analysis of 17 E. coli genomes

• 5020 ± 446 genes per strain

• Core genome = 2344 ± 43 genes

• About 1700 “optional” genes

r of

Gen

esN

umbe

r of

Con

serv

ed G

enes

5

per strain

• Pangenome > 13,000 genes; Open

Rasko et al., J. Bacteriol.2008; 190: 6881-6893Number of genomes

Num

ber o

f Gen

es

in P

an-G

enom

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ExPEC

• Can cause a variety of extraintestinal infections

– UTI, meningitis, pneumonia, cholecystitis, intraabdominal abscess

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• These strains have similar virulence factors

• Clonal groups that cause neonatal meningitis (MAEC) can also cause UTI (UPEC)

– MAEC = ExPEC subset that have K1 capsule



UPEC pathogenesis

• Leading cause of UTI

• Specific strains

– Outbreaks/clones

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• Ascending route

– Begins with colonization of periurethral mucosa

• Host factors

– Genetic/behavioral

UPEC virulence factors

• Numerous adhesins

– Type-1 fimbriae

– P-fimbriae

– Dr adhesins

– Many more! Fe

HemolysinCNFSAT

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• Toxins

– Hemolysin

– Cytotoxic necrotizing factor

• Fe acquisition

• Capsule

• Flagella

Fe

Arrays259

STM5

6

fimphoU manB

Complementary techniques for ExPEC gene discovery

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phoU, manB,wecE,yaiT,pyrD

Genomics1778

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kpswzy 45



Sugar coating

• Many E. coli strains have a capsule made of polysaccharides

• Capsule may inhibit phagocytosis t t f

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or protect from serum

• Deletion of K2 capsule –specific genes reduces ability to cause UTI

Buckles et al., J. Infect. Dis. 2009; 199: 1689-1697

K2 capsule and serum resistance

11Buckles et al., J. Infect. Dis. 2009; 199: 1689-1697

ETEC disease

• Watery diarrhea

• Children in developing countries

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• Visitors to those countries



ETEC Pathogenesis

• Fecal-oral route

• Plasmid virulence factors

– Fimbriae/fibrillae

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CFA

CS

– Enterotoxins

Heat-labile

Heat-stable

Chromosomal factors in ETEC pathogenesis

• Type II secretion system

• Outer membrane vesicles

• Secreted EtpA adhesin and flagella

14Roy et al., Nature 2009; 457: 594-598

EPEC disease

• Once a feared cause of deadly outbreaks

• Affects infants in developing countries

• Re-emerging in developed countries

C t di h

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• Causes watery diarrhea

– Vomiting can be severe

– Variable fever

– Can be protracted

• Spread person-person



EPEC pathogenesis

• Localized adherence

– Typical strains

– Plasmid-encoded pilus

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• Attaching and effacing

– Type III secretion system on pathogenicity island

The bundle-forming pilus

• Found in “typical strains”

• A member of the type IV pili family

• Encoded by 14 gene cluster

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• Encoded by 14 gene cluster on large EPEC plasmid

• Required for LA and auto-aggregation

• Required for virulence

The bfp gene cluster

-IPTG

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+IPTG

Stone et al., Molec. Microbiol. 1996; 20: 325-337Anantha et al., J. Bacteriol. 2000; 182: 2498-2506

• 12 genes required to make BFP

• 1 required for pilus retraction

• 1 pseudogene



A model of the BFP biogenesis machine

L

U G

B

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L

I J KC

DF

E

Diversifying selection of bundlin

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• 10 different bfpA alleles specifying bundlin variants

– 3 highly related alpha types; 7 distantly related beta types

• Surface-exposed region near C-terminus where non-synonymous substitutions predominate

Blank et al., Infect. Immun. 2000; 68: 7028-7038Ramboarina et al., J. Biol. Chem. 2005; 280: 40252-40260

Specificity of human antibody response against bundlin

• Veteran and naïve volunteers challenged with same strain

• Pre and post challenge titers tested against homologous and heterologous bundlin

• Naïve group: significant increase in antibody titer against homologous

Veteran groupNaïve group

Tite

r

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in antibody titer against homologous but not heterologous bundlin

• Veteran group: significantly higher pre-(re)challenge antibody titers against homologous v. heterologous bundlin

• Final titers against heterologous bundlin higher in veteran v. naïve group

Fernandes et al., Infect. Immun. 2007; 75: 4687-4696

Geo

met

ric M

ean

T



Attaching and effacing

• Hallmark of EPEC

• Destruction of microvilli

Formation of actin rich

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• Formation of actin-rich pedestals

• Observed in vivo and in vitro

• Encoded by LEE

The locus of enterocyte effacement

• 41 gene pathogenicity island

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• 41 gene pathogenicity island

• Necessary and sufficient for attaching and effacing

• Type III secretion system

• Secreted proteins:

– Adhesin/receptor pair

Intimin• 94-kDa outer membrane

adhesin encoded by eae

• Proven virulence factor in volunteers

– Wild type 11/11

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yp

– eae mutant 4/11

• Immunogenic

• Structure reveals 2 (3) Ig domains and a C-lectin binding domain

Luo et al., Nature 2000; 405: 1073-7

Donnenberg et al., J. Clin. Invest. 1993; 92: 1412-1417

Wild type eae



The translocon

• Composed of EspA, EspB, EspD

• EspA filament

• EspBD form pore

EspB pro en ir lence factor

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• EspB proven virulence factor

– Wild type 10/10

– espB mutant 1/10

Knutton et al., EMBO J. 1998; 17: 2166-2177

Tacket et al., Infect. Immun. 2000; 68: 3689-3695

EspB is required to damage the brush border in vivo

a-EPEC

espBrecipient

a-EPEC + actinActin

26Tacket et al., Infect. Immun. 2000; 68: 3689-3695

Wild typerecipient

The translocated intimin receptor

• Tir secreted/translocated via T3SS

• Tir inserted in host cell membrane

• Intimin binds Tir in host cell membrane

• Tir from some EPEC strains phosphorylated by host tyrosine kinase

• Ligand bound phosphorylated EPEC Tir binds host linker protein Nck

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• Ligand-bound phosphorylated EPEC Tir binds host linker protein Nck

• Tir/Nck → activates N-WASP → activates Arp2,3 → actin polymerization

Luo et al., Nature 2000; 405: 1073-7



EPEC summary

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EHEC

• Reservoir: cattle

• Contaminated food

– Ground beef

– Produce

Juice

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– Juice

• Water

– Drinking

– Swimming

• Petting zoos

• Person to person

– ID50 < 100 cfu

EHEC clinical features

• Broad spectrum of illness

– Asymptomatic to fatal

• Cramps, pain

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• Watery diarrhea

• Bloody diarrhea

• Fever absent or low-grade

• Fecal leukocytes often present



Hemolytic-uremic syndrome

• Thrombotic microangiopathy

• May affect any organ

• Risk of death or ESRD 12%, permanent renal damage 25%

• Complicates ~4% of cases

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Complicates 4% of cases

• Most common cause of renal failure in children

• Risk factors

– Age

– WBC count

– Vomiting

– Anti-motility agents

– Antibiotics

The relationship between EPEC and STEC

TypicalSTECAEEC

AtypicalEPEC

O157:H7

EHEC

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TypicalEPEC

AEEC – Attaching and effacing E. coli

STEC – Shiga toxin-producing E. coli

EHEC – Enterohemorrhagic E. coli

EPEC – Enteropathogenic E. coli

O157:H7

The role of eae in attachment in vivo

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Wild type eae mutant eae mutant+ eae gene

Donnenberg et al., J. Clin. Invest. 1993; 92: 1418-1424



Shiga toxins

• Encoded by lambda-like bacteriophages

• 5 identical B, 1 A subunit

• B subunits bind to GB3

– Present on many cell types

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Present on many cell types

• Transported retrograde to endoplasmic reticulum

• A subunit is an N-glycosidase

• Depurinate 28S ribosomal subunit

• Protein synthesis ceases

Shiga toxin production

• Under control of late promoters of bacteriophage

• Induced by SOS response

– Oxidative stress

– Antibiotics

• Induction leads to phage production cell lysis

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Induction leads to phage production, cell lysis

Zhang et al., JID 2000; 181: 661-70

Do antibiotics increase HUS risk?

• Early studies suggested an association

– Bias?

• Prospective study affirmed

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– Not random allocation

• Meta-analysis cast doubt

– Strong influence of a single large negative study

• As yet no evidence of benefit

– “Primum non nocere”



EHEC diagnosis• Diagnostic challenge

– Mimics non-infectious illnesses

– Results in unnecessary interventions

• Critical for patient and public health

• Sorbitol-MacConkey Agar plates

– O157:H7 strains ferment sorbitol

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– O157:H7 strains ferment sorbitol slowly if at all

– Think/request

• ELISA for toxin

– Especially non-O157:H7 STEC

• Confirmation in specialty labs

– PCR

– Serotyping

EHEC therapy

• Fluid replacement

• Monitoring/supportive care

• Antibiotics

Anti motility agents

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• Anti-motility agents

• Future

– Toxin receptor analogues

– Human monoclonal antibodies

– Safer antibiotic subclasses

EHEC prevention• Good hygiene

– Cook ground beef to 68.3°C

– Don’t cross-contaminate food

– Wash hands

– Responsible infant bathing practices

• Food safety

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• Food safety

– Control bovine colonization

– Better meat processing practices/irradiation

– More research into other routes of transmission

• Vaccines

– Toxoid

– O157 polysaccharide conjugate

– Attenuated strains



EAEC

• Diarrhea

– Developing and developed countries

– Sporadic and epidemic

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p p

– Acute and persistent

• Growth retardation

• HIV

• Increasing importance in travelers

EAEC pathogenesis

• Aggregative adherence

– Mediated by plasmid-encoded fimbriae

• Dispersin

• Damage to intestinal cells

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Damage to intestinal cells

• Toxins

– Pet

– EAST

• Inflammation

– IL-8

EIEC/Shigella• Plasmid-encoded invasion

– Type III secretion system

– Intracellular motility via IcsA/VirG

Usurps same machinery as does EPEC

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• Genome tells a novel tale

– Definitely E. coli

– Smaller than K-12

4,599,354 bp; 4084 genes

Lacks 357 K-12 genes, only 195 specific genes

– Many transposons



DAEC

• Case-control studies equivocal

– More likely of significance in older children in developed countries

• Volunteer studies negative

Pathogenesis

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• Pathogenesis

– Adhesins

Dr family

Autotransporter family

Summary: common themes in E. coli pathogenesis

• Specific pathotypes cause specific disease

• Virulence factors encoded on plasmids, b t i h th i it i l d

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bacteriophages or pathogenicity islands

• Adhesins, fimbrial or afimbrial

• Secretion systems/toxins

MAECDAEC

Relationships among pathogenic E. coli

ExPEC

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EIECShigella

TypicalEPEC

EHECSTECAEEC EAEC

ETEC S. dysenteriae

DAEC

AtypicalEPEC

UPEC

O157:H7



Acknowledgements• Lab members

– Ravi Anantha, Farah Bahrani, Rick Blank, Eric Buckles, Paula Fernandes, Kelly Stone

• Support

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the diversity of escherichia coliinfections prof. michael

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