TOXINS PRODUCED BY E. COLI AND

MECHANISMS BY WHICH E.COLIPRODUCE DIARRHEAPRESENTED BY : DR. KUMAR VIKRAM

MD (MICROBIOLOGY) IGIMS, PATNA

HISTORY:Escherich’s legacy to Science

• German pediatrician Theodore Escherich described the organism in 1885 as Bacterium coli commune.

1885

• Castellani and Chalmers defined the genus Escherichia and established the type species E. coli (Castellani and Chalmers 1919).

1919

• Bray established the causative role of a specific type of E.coli during a hospital outbreak of childhood diarrhea in London.

1945

GENUS AND SPECIES DEFINITION

GENUS DEFINITION

• gram-negative,• rod-shaped bacteria, • oxidase-negative,• usually motile by peritrichous flagella • do not produce spores.• facultatively anaerobic; • gas is usually produced from

fermentable carbohydrates. • They are methyl red-positive and

Voges–Proskauer-negative. • Many strains produce polysaccharide

capsules or microcapsules

SPECIES DEFINITION(Escherichia Coli)

• Most strains of this species promptly ferment lactose or give a positive o-nitrophenyl-b-D-galactopyranoside (ONPG) reaction.

• They produce indole,• fail to hydrolyze urea.• H2S production is not detectable on

triple sugar iron (TSI) agar • phenylalanine is not deaminated,• Gelatin is not liquefied.• Most strains decarboxylate lysine.• they do not grow on Simmons’ citrate

agar

E. Coli : The Good

E.coli: The Enemy within

Despite the fact that Escherichia coli as a commensal bacteria can be found in intestinal microflora of a variety of animals including man, not all the strains are harmless, and some can cause debilitating and sometimes fatal diseases in humans.

WHO (April 2013):• Diarrhoeal disease is the second leading cause of death in children

under five years old.• Each year diarrhoea kills around 760 000 children under five.• Rotavirus and Escherichia coli are the two most common etiological

agents of diarrhoea in developing countries.

• INTESTINAL PATHOGENS(ENTEROVIRULENT E.COLI)• Enteropathogenic E.coli• Enterohaemorrhagic E.coli• Enterotoxigenic E.coli• Enteroaggregative E.coli• Enteroinvasive E.coli• Diffusely adherent E.coli

• EXTRAINTESTINAL PATHOGENS• Uropathogenic E.coli• Meningitis• septicemia

PATHOGENIC E.COLI

Common themes in E. Coli virulence

• Like most mucosal pathogens, E. coli can be said to follow a requisite strategy of infection:

(i) colonization of a mucosal site, (ii) evasion of host defenses, (iii) multiplication, and (iv) host damage.• The most highly conserved feature of diarrheagenic E. coli strains is

their ability to colonize the intestinal mucosal surface despite peristalsis and competition for nutrients by the indigenous flora of the gut.

• Diarrheagenic E. coli strains possess specific fimbrial antigens that enhance their intestinal colonizing ability and allow adherence to the small bowel mucosa.

• Once colonization is established, E. Coli may cause diarrhea by three general paradigms: (i) enterotoxin production (ETEC and EAEC),

(ii) invasion (EIEC), and/or (iii) intimate adherence with membrane

signalling (EPEC and EHEC).

• First recognized in the late 1960s as a cause of cholera-like diarrhoea in india.

• Resemble vibrio cholerae in that they induce profuse,watery diarrhea by elaboration of toxins that act on the mucosal cells.

• Adhere to the small intestinal mucosa, but do not invade.

ENTEROTOXIGENIC E. COLI

• no apparent histological changes and little inflammation.

• a common cause of dehydrating diarrhea in children in developing countries, particularly when they are weaned.

• considered to be the leading cause of travelers’ diarrhea accounting upto 75% of these cases

VIRULENCE FACTORS2 classes:

Enterotoxins

Heat labile toxin(LT)

LT I

LT II

Heat Stable Toxin(ST)

ST I

ST II

adhesive factors termed colonization factor antigens (CFA) or coli surface (CS) associated antigens

• Oligomeric toxins that are closely related in structure and function to the cholera enterotoxin (CT) expressed by vibrio cholerae.

• Heat-labile toxin (lt) is inactivated by incubation at 100°c for 30 min.

• 2 major serogroups :– LT I– LT II

Heat Labile Toxin

• LT-I is expressed by E. coli strains that are pathogenic for both humans and animals.

• Composed of :– One A subunit• The A1 domain constitutes the

active toxin and is linked to the A2 domain via a disulfide bond

• The A2 fragment is the helical portion of the molecule and anchors the A subunit to the B pentamer

– Five B subunits• Bind to ganglioside GM 1

receptor.

LT - I

MECHANISM OF ACTION

endocytosis and translocation of toxin through the cell in a process involving trans-Golgi vesicular transport.

permanent activation of adenylate cyclase

increased levels of intracellular cyclic AMP (cAMP).

Activation of cAMP dependent protein kinase (A kinase).

supranormal phosphorylation of chloride channels located in the apical epithelial cell membranes.

stimulation of Cl2 secretion from secretory crypt cells and inhibition of NaCl absorption by villus tip cells.

The increased luminal ion content draws water passively through the paracellular pathway, resulting in osmotic diarrhea.

Mechanism of Action

OTHER MECHANISMS

• Prostaglandins (PGE1 and PGE2): Stimulate electrolyte transport and intestinal motility.

• Enteric nervous system: Serotonin and vasoactive intestinal polypeptide, both of which can stimulate intestinal epithelial cell secretion via the ENS, are released into the human small bowel after treatment with CT.

• Intestinal inflammatory response:CT has been reported to stimulate production of the proinflammatory cytokine interleukin-6 (IL-6), thereby activating the enteric immune system and potentially generating arachidonic acid metabolites that stimulate secretion.

• 55 to 57% identity to LT-I and CT in the A subunit.• no homology to LT-I or CT in the B subunits.• LT-II increases intracellular cAMP levels by similar

mechanisms to those involved with LT-I toxicity.• But LT-II uses GD1 as its receptor rather than GM1• There is no evidence that LT-II is associated with

human or animal disease.

LT - II

• Small, monomeric toxins that contain multiple cysteine residues, whose disulfide bonds account for the heat stability of these toxins.

• 2 classes:– ST I (ST a) – soluble in methanol• ST Ip (porcine)• ST Ih (human)

– ST II (ST b) – insoluble in methanol

Heat stable Toxins

ST - I

ST - II

•ST -II is associated primarily with ETEC strains isolated from pigs.•induces histologic damage in the intestinal epithelium, consisting of loss of villus epithelial cells and partial villus atrophy.• stimulates the secretion of bicarbonate from intestinal cells . •stimulate increases in intracellular calcium levels from extracellular sources . • also stimulates the release of PGE2 and serotonin.

COLONIZATION FACTORS

• A heterogenous group of proteinacious surface structures

• Fimbrial, non-fimbrial or fibrillar.• The more recent nomenclature refer to these

structures as coli surface (CS) antigen.• At least 21 colonization factor antigens and coli

surface antigens specific for humans have been defined.

• CFA/I and CFA/ II are the most commonly found adhesins in human ETEC.

CFAsOf

HumanETEC

strains

Vero cytotoxin-producing E.coli(VTEC)

Vero cytotoxin-producing E.coli(VTEC)

• Described first by Konowalchuk in 1977.• In 1983, one particular serotype of E. Coli

(O157:H7) was identified as the causative agent involved in two outbreaks of a distinctive bloody diarrheal syndrome (Riley et al. 1983).

• Since then, these organisms have received much attention as a cause of epidemic or sporadic bloody and non-bloody diarrhea, HUS, and thrombotic thrombocytopenic purpura.

• Also called as shiga toxin producing E. Coli(STEC). - Because toxins are closely related to shiga toxin and that have a cytotoxic effect on vero cells,

• The term enterohaemorrhagic E. Coli (EHEC) is applied to those STEC serotypes that have the same clinical, epidemiological and pathogenetic features associated with the prototype strain E. Coli O157:H7.

Virulence factors

•VT 1 and VT2•Main virulence factor •associated with HUS

Vero cytotoxin

•Mediates attachmentIntimin•EnterohemolysinEHEC plasmid

Vero cytotoxin• Encoded on Stx

bacteriophage• Originally discovered in

Shigella dysenteriae (Stx1-like)

• Multiple variants-VT1, VT2 (VT2c, d, e, f, g)

• AB-5 toxin (5 B components and one A component)

Vero cytotoxin

Enterohemolysin

• Found in nearly all O157:H7 strains and is widely distributed among non-O157 VT-producing E. coli strains.

• Two other genetically distinct phage encoded hemolysins, called Ehly1 and Ehly2, have been reported to be produced by many VT-producing E. coli strains.

• There are no data to suggest in vivo expression or any role in pathogenesis for these hemolysins.

• EPEC is an important category of diarrheagenic E. Coli which has been linked to infant diarrhea in the developing world.

• Most EPEC infections occur in the first 3 years of life.

• EPEC infections show a marked seasonality and are associated with warm season peaks.

Enteropathogenic E. Coli

Pathogenesis• Attaching-and-effacing histopathology– hallmark of infections due to EPEC– characterized by effacement of microvilli and intimate adherence between the bacterium and the epithelial cell membrane.– Marked cytoskeletal changes, including accumulation of polymerized actin, are seen

directly beneath the adherent bacteria; the bacteria sometimes sit upon a pedestal-like structure

Pathogenesis

• In 1992, Donnenberg and Kaper proposed a three-stage model.

• In 1998 Knutton et al. proposed a four-stage model.

Localized adherence.

• nonintimate adherence between the bacterium and the epithelial cell • mediated by the bundle-forming pilus (BFP) and intimin along with EspA filaments (short, surface-associated filaments).• The adherence pattern is called ‘localized’ because organisms attach to one or two small areas of the cell surface in microcolonies

Signal transduction

• The injection of the translocated intimin receptor (Tir) via a type III secretion system directly into the host cell,• Result in tyrosine protein kinase activation and formation of the intimin receptor (tyrosine phosphorylated Tir) and to

actin rearrangements.

Intimate adherence.

• intimin binds to phosphorylated Tir and polymerized actin and other cytoskeletal elements accumulate beneath the intimately attached bacteria

Pedestal formation

• further actin polymerization and accumulation of cytoskeletal elements at the site of bacterial attachment results in the production of the mature A/E lesion with the characteristic pedestal structure.

• Loss of tight-junction integrity and mitochondrial function, resulting in electrolyte loss and cell death.

• Have the capacity to invade interstitial epithelial cells in vivo.• EIEC strains are biochemically, genetically, and

pathogenetically related closely to Shigella spp.• Like Shigella spp.,EIEC strains are generally lysine

decarboxylase negative, nonmotile, and lactose negative.• Except – Lower acid resistant– Inability to produce shiga toxin

• Presentation:– Majority - watery diarrhea.– Occasionally - Dysentery syndrome, manifested as blood, mucus,

and leukocytes in the stool; tenesmus; and fever

ENTEROINVASIVE E. COLI

Pathogenesis

• Acquisition of the invasive plasmid (pINV) encodes the ability to invade host tissues.

• Comprises– (i) epithelial cell penetration, – (ii) lysis of the endocytic vacuole, – (iii) intracellular multiplication, – (iv) directional movement through the cytoplasm, – (v) extension into adjacent epithelial cells

Cellular pathogenesis

Sereny test

• Instillation of a suspension of freshly isolated EIEC or Shigella into the eyes of guinea pigs

• Mucopurulent conjunctivitis

Enteroaggregative E. coli

• Named so because they appear aggregated in a stacked brick formation on Hep – 2 cells.

• second most common cause of travelers’ diarrhea after ETEC in both developed and developing countries.

• commonly being recognized as a cause of endemic and epidemic diarrhea worldwide.

• associated with persistent diarrhea.

Pathogenesis

colonization of intestinal mucosa,

mucoid biofilm

formation

elaboration of various enterotoxins,

cytotoxins and mucosal

inflammation.

aggregative adherence fimbriae (AAF)• Plamid encoded

Enteroaggregative heat stable toxin (EAST)•Fluid secretion•Related to ST of ETEC

Diffusely Adherent E.coli• heterogenous group that generates a diffuse adherence pattern on HeLa and HEp-2 cells.• associated with the watery diarrhea that can become persistent in young children in both developing and developed countries as well as recurring urinary tract infections.

•The association of the bacteria with themembrane and the formation of long finger-like projections emanating from the cell. •These projections wrap around the bacterium in a phenotype termed “embedding.”Invasion is rarely seen.

2 classes

Afa/Dr adhesins (Afa/Dr DAEC)

urinary tract infections (UTIs) (pyelonephritis, cystitis, and

asymptomatic bacteriuria) and with various enteric infections.

adhesin involved in diffuse adherence

(AIDA-I)

a potential cause of infantile diarrhea.

Conclusion• A wealth of data concerning the virulence mechanisms

of diarrheagenic E. Coli has been accumulated over the years even though these complicated phenomena are not yet fully understood.

• This versatile organism affects a wide range of eukaryotic cell processes via an array of diverse genetic elements enabling each pathotype to colonize, multiply, and disseminate and understanding each pathogenic step at molecular level may help in devising effective measures for intervention in infection.

References

• Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev 1998; 11: 142-201.

• Topley and Wilson’s Microbiology and Microbial infections, 10th Edition

• Jafari et al , Escherichia coli: a brief review of diarrheagenic pathotypes and their role in diarrheal diseases in Iran, IRAN. J. MICROBIOL. 4 (3) : 102-117

• WHO Fact Sheet (April 2013) on diarrhoeal disease

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