Bacterial Toxins and Toxin classification

Bacterial

Toxins Classification

Dr.Faghri

Meisam.Roozbahani

Introduction

Toxins were the first bacterial virulence factors to be identified and were also the first link between bacteria and cell biology.

Introduction

Cellular microbiology was, in fact, naturally born a long time ago with the study of toxins, and only recently, thanks to the sophisticated new technologies, has it expanded to include the study of many other aspects of the interactions between bacteria and host cells.

Two Main Categories of Toxins

Specific Host Site Exotoxins

Neurotoxins Enterotoxins Cytotoxins

Nephrotoxin

Hepatotoxin

Cardiotoxin

Classification by Entrance Mechanism

Acting on intracellular

targets

Injected into eukaryotic cells

Unknown mechanism of

action

Acting on the cell surface

Immune system (Superantigens)

Clas

sTa

rget

Surface molecules

Cell membrane

Large pore- forming toxins

Small pore- forming toxins

Insecticidal toxins

Membrane-perturbing toxins

Other pore- forming toxins

RTX toxins

Protein synthesis Mediators of apoptosis

Signal transduction

Cytoskeleton structure

Intracellular trafficking

Inositol phosphate metabolism

Cytoskeleton

Signal transduction

Toxins acting on the cell surface: Immune system (Superantigens)

Superantigens are bacterial and viral proteins that share the ability to activate a large fraction of T-lymphocytes.

Toxins acting on the cell surface: Immune system (Superantigens)Toxin Organism Activity Consequence

SEA-SEI, TSST-1, SPEA, SPEC, SPEL, SPEM, SSA, and SMEZ

Staphylococcusaureus and Streptococcuspyogenes

Binding to MHC class II molecules and to Vβ or Vγ of T cell receptor

T cell activation and cytokines secretion

MAM Mycoplasmaarthritidis


Chronic inflammation

YPMa Yersiniapseudotuberculosis


Chronic Inflammation

SPEB S. pyogenes Cysteine proteaseAlteration in Immunoglobulin binding properties

ETA, ETB, and ETD S. aureus Trypsin-like serineproteases

T-cell proliferation,intraepidermal layer separation

Agr Regulatory System

Toxins acting on the cell surface: Surface molecules

BFT enterotoxin: The pathogenicity of ETBF is ascribed to a heat-labile ∼20-kDa toxin (B. fragilis toxin [BFT], also called fragilysin).

This toxin binds to a specific intestinal epithelial cell receptor and stimulates cell proliferation.

Zonula

BFT enterotoxin

Toxins acting on the cell surface: Surface moleculesToxin Organism Activity Consequence

BFT enterotoxin Bacteroides fragilis Metalloprotease, cleavage of E-cadherin

Alteration of epithelialpermeability

AhyB Aeromonashydrophyla

E l a s t a s e ,metalloprotease

Hydrolization of casein and elastine

Aminopeptidase Pseudomonasaeruginosa

E l a s t a s e ,metalloprotease

Corneal infection, inflammation and ulceration

ColH Clostridiumhistolyticum

Collagenase,metalloprotease

Collagenolytic activity

Nhe Bacillus cereus Metalloprotease andcollagenase

Collagenolytic activity

Toxins acting on the cell surface:Pore-Forming Protein toxins forming pores in biological membranes

occur frequently in Gram-positive and Gram-negative bacteria.

Pore-forming toxins, also known as "lytic factors". Some of them are also called "hemolysins“.

Toxins acting on the cell surface:Large Pore-Forming Toxins

Generally secreted by diverse species of Gram-positive bacteria.

Binding selectively to cholesterol on the eukaryotic cell membrane.

Toxins acting on the cell surface: Large pore forming toxinsToxin Organism Activity Consequence

PFO C. perfringensThiol-activated cytolysin, cholesterol Binding

Gas gangrene

SLO S. pyogenesThiol-activated cytolysin, cholesterol Binding

Transfer of other toxins, cell death

LLO Listeria monocytogenesInduction of Lymphocyte apoptosis Membrane damage

Pneumolysin S. pneumoniae Induction of Lymphocyte Apoptosis

Complement activation, cytokine production, apoptosis

Toxins acting on the cell surface: Small pore forming toxins

Creating very small pores 1-1.5 nm diameter. Selective permeabilization to solutes with a

molecular mass less than 2 kDa.

Toxins acting on the cell surface: Small pore forming toxinsToxin Organism Activity Consequence

Alveolysin B. alveis Induction of lymphocyte Apoptosis

Complement activation, cytokine production, apoptosis

ALO B. anthracis Induction of lymphocyte apoptosis

Complement activation, cytokine production, Apoptosis

α-Toxin S. aureus Binding of erythrocytes Release of cytokines, cell lysis, apoptosis

PVL leukocidin(LukS-LukF) S. aureus Cell membrane

permeabilizationNecrotic enteritis, rapid shock-like syndrome

γ-Hemolysins(HlgA- HlgB andHlgC- HlgB)

S. aureus Cell membrane permeabilization

Necrotic enteritis, rapid shock-like syndrome

β-Toxin C. perfringens Cell membrane permeabilization Necrotic enteritis, neurologic effects

Toxins acting on the cell surface: RTX toxins

The RTX toxin family is a group of cytotoxins produced by Gram-negative bacteria.

There are over 1000 known members with a variety of functions.

Toxins acting on the cell surface: RTX toxins The RTX family is defined by two common features:

characteristic repeats in the toxin protein sequences, and extracellular secretion by the type I secretion system (T1SS).

The name RTX (repeats in toxin) refers to the glycine and aspartate-rich repeats located at the C-terminus of the toxin proteins.

Genomic Structure

The toxin is encoded by four genes, one of which, hlyA, encodes the 110-kDa hemolysin. The other genes are required for its posttranslational modification (hlyC) and secretion (hlyB and hlyD).

Toxins acting on the cell surface: RTX toxins

Toxin Organism Activity Consequence

Hemolysin II

B. cereus Cell membrane permeabilization Hemolytic activity

CytK B. cereus Cell membrane Permeabilization Necrotic enteritis

HlyA E. coli Calcium-dependent formation of transmembrane Pores

Cell permeabilization and lysis

Toxins acting on the cell surface: Membrane perturbing toxins

Soap like structure. The toxin binds nonspecifically parallel to the

surface of any membrane without forming transmembrane channels.

Cells first become permeable to small solutes and eventually swell and lyse, releasing cell intracellular content.

Toxins acting on the cell surface: Membrane perturbing toxinsToxin Organism Activity Consequence

ApxI, ApxII, and ApxIII A.pleuropneumoniae Calcium-dependent formation

of transmembrane PoresLysis of erythrocytes and other nucleated Cells

LtxA A.actinomycetemcomitans

Calcium-dependent formation of transmembrane Pores Apoptosis

LtxA P.Haemolytica Calcium-dependent formation of transmembrane Pores

Activity specific versus ruminant leukocytes

Toxins acting on the cell surface: Other pore forming toxins Like other functionally related toxins, aerolysin

changes its topology in a multi-step process from a completely water-soluble form to a membrane-soluble heptameric transmembrane channel that destroys sensitive cells by breaking their permeability barriers.

Toxins acting on the cell surface: Other pore forming toxinsToxin Organism Activity Consequence

δ-Hemolysin S. aureus Perturbation of the lipid

bilayer Cell permeabilization and lysis

Aerolysin A. hydrophila Perturbation of the lipid bilayer Cell permeabilization and lysis

AT C. septicum Perturbation of the lipid bilayer Cellpermeabilization and lysis

Toxins acting on the cell surface: Insecticidal toxins

The class of insecticidal proteins, also known as

δ-endotoxins, includes a number of toxins produced by species of Bacillus thuringiensis. These exert their toxic activity by making pores in

the epithelial cell membrane of the insect midgut.

Toxins acting on the cell surface: Insecticidal toxins

δ-Endotoxins form two multigenic families, cry and cyt; members of the cry family are toxic to insects of

Lepidoptera, Diptera and Coleoptera orders (Hofmann et al., 1988),

whereas members of the cyt family are lethal specifically to the larvae of Dipteran insects (Koni and Ellar, 1994).

Lepidoptera is a large order of insects that includes moths and butterflies.True flies are insects of the order Diptera.Coleoptera is an order of insects commonly called beetles.

Toxins acting on the cell surface: Insecticidal toxinsToxin Organism Activity Consequence

PA B. anthracis Perturbation of the lipid bilayer Cell permeabilization and lysis

HlyE E. coli Perturbation of the lipid bilayer Osmotic lysis of cells lining the Midgut

CryIA, CryIIA,CryIIIA, etc

Bacillus thuringiensis

Destruction of the transmembranePotential

Osmotic lysis of cells lining the Midgut

CytA, CytB B. thuringiensisDestruction of the transmembranePotential

Osmotic lysis of cells lining the Midgut

BT toxin B. thuringiensisDestruction of the transmembranePotential

Cytocidal activity on human cells

Toxins Acting on Intracellular Targets

The group of toxins with an intracellular target (A/B toxins) contains many toxins with different structures that have only one general feature in common: they are composed of two domains generally identified as "A" and "B.“


targets



action



Clas

sTa

rget

Surface molecules

Cell membrane



Insecticidal toxins



RTX toxins


Signal transduction




Cytoskeleton

Signal transduction

Toxins Acting on Intracellular Targets

The A domain is the active portion of the toxin; it usually has enzymatic activity and can recognize and modify a target molecule within the cytosol of eukaryotic cells.

The B domain is usually the carrier for the A subunit; it bind the receptor on the cell surface and facilitates the translocation of A across the cytoplasmic membrane.

Toxins acting on intracellular targets: Protein synthesis

These toxins are able to cause rapid cell death at extremely low concentrations.

This reaction leads to the formation of a completely inactive EF2-ADP-ribose complex.


A very important step in the elucidation of the mechanism of enzymatic activity has been the determination of the crystal structure for the complex of diphtheria toxin with NAD.

Upon the addition of NAD to nucleotide-free DT crystals, a significant structural change.

This change lead to recognition and binding of the acceptor substrate EF-2.

This would explain why DT recognizes EF-2 only after NAD has bound.


Toxins acting on intracellular targets: Protein synthesisToxin Organism Activity Consequence

DT Corynebacterium diphtheriae ADP-ribosylation of EF-2 Cell death

PAETA P. aeruginosa ADP-ribosylation of EF-2 Cell death

SHT S. dysenteriae N-glycosidase activity on 28S RNA Cell death, apoptosis

Toxins acting on intracellular targets: Signal transduction

Two types of transduction mechanism: Tyrosine phosphorylation

Modification of a receptor-coupled GTP-binding protein cyclic AMP

inositol triphosphate

diacylglycerol

Pertussis toxin

PT Subunits

A

B

Cholera toxin (CT) and E. coli heat-labile enterotoxins (LT-I and LT-II)

Cholera toxin (CT) and E. coli heat-labile enterotoxins (LT-I and LT-II) share an identical mechanism of action and homologous primary and 3D structures.

While V. cholerae exports the CT toxin into the culture medium, LT remains associated to the outer membrane bound to lipopolysaccharide.

The corresponding genes of CT and LT are organized in a bicistronic operon and are located on a filamentous bacteriophage and on a plasmid, respectively.

Clostridium difficile Toxins

Enterotoxin A (TcdA) and cytotoxin B (TcdB) of Clostridium difficile are the two virulence factors responsible for the induction of antibiotic-associated diarrhea.

The toxin genes tcdA and tcdB together with three accessory genes (tcdC-E) constitute the pathogenicity locus (PaLoc) of C. difficile.

Toxins acting on intracellular targets: Signal transduction 1Toxin Organism Activity Consequence

PT Bordetella pertussis

ADP-ribosylation of Gi cAMP increase

CT Vibrio cholerae ADP-ribosylation of Gi cAMP increase

LT E. coli ADP-ribosylation of Gi cAMP increase

α-Toxin (PLC) C. perfringens Zinc-phospholipase C, hydrolase Gas gangrene

Toxins A and B (TcdA and TcdB) C. difficile Monoglucosylation of Rho, Rac,

Cdc42Breakdown of cellular actin stress fibers

Adenylate cyclase (CyaA) B. pertussis Binding to calmodulin

ATP→cAMP conversion cAMP increase

Anthrax Edema and Lethal Factors

The EF and LF genes are located on a large plasmids.

Cleavage of the N-terminal signal peptides yields mature EF and LF proteins.

LF, is able to cause apoptosis in human endothelial cells.

E. coli Cytotoxin Necrotizing Factors and Bordetella Dermonecrotic Toxin

CNF1 & CNF2: produced by a number of uropathogenic and neonatal meningitis-causing pathogenic E. coli strains.

cnf1 is chromosomally encoded, cnf2 is carried on a large transmissible F-like plasmid called "Vir“.

DNT is a transglutaminase, which causes alteration of cell morphology, reorganization of stress fibers, and focal adhesions on a variety of animal models.

Cytolethal Distending Toxins

HdCDT is a complex of three proteins (CdtA, CdtB and CdtC) encoded by three genes that are part of an operon.

Members of this family have been identified in E. coli, Shigella, Salmonella, Campylobacter, Actinobacillus and Helicobacter hepaticus.

Toxins acting on intracellular targets: Signal transduction 2Toxin Organism Activity Consequence

Anthrax edema factor (EF) B. anthracis Binding to calmodulin

ATP→cAMP conversion cAMP increase

Anthrax lethal factor (LF) B. anthracis Cleavage of MAPKK1 and

MAPKK2 Cell death, apoptosis

Cytotoxin necrotizing factors 1 and 2 (CNF1, 2)

E. coli Deamidation of Rho, Rac and Cdc42

Ruffling, stress fiber formation.

DNT Bordetella species

Transglutaminase, deamidation or polyamination of Rho GTPase

Ruffling, stress fiber formation

CDT Several speciesDNA damage, formation of actin stress fibers via activation of RhoA

Cell-cycle arrest, cytotoxicity, apoptosis

Toxins acting on intracellular targets: Cytoskeleton structure

The cytoskeleton is a cellular structure that consists of a fiber network composed of microfilaments, microtubules, and the intermediate filaments.

It controls a number of essential functions in the eukaryotic cell: exo- and endocytosis

vesicle transport

cell-cell contact

and mitosis

Toxins acting on intracellular targets: Cytoskeleton structure

Most of them do it by modifying the regulatory, small G proteins, such as Ras, Rho, and Cdc42, which control cell shape.

Lymphostatin

Lymphostatin is a very recently identified protein in enteropathogenic strains of E. coli

Lymphostatin selectively block the production of interleukin-2, IL-4, IL-5 and γ interferon by human cells and inhibit proliferation of these cells, thus interfering with the cellular immune response.

Toxins acting on intracellular targets: Cytoskeleton structureToxin Organism Activity Consequence

Toxin C2 and related proteins C. botulinum ADP-ribosylation of monomeric G

actin Failure in actin polymerization

Lymphostatin E. coli Block of interleukin production Chronic diarrhea

Iota toxin and related proteins C. perfringens Block of interleukin production Chronic diarrhea

Toxins acting on intracellular targets: Intracellular trafficking

Vesicle structures are essential in: receptor-mediated endocytosis and exocytosis

One example of exocytic pathway is that involving the release of neurotransmitters

Mechanism of action of clostridial neurotoxins (CNT)

Synaptosomal-associated protein 25 (SNAP-25)

Helicobacter pylori Vacuolating Cytotoxin Vac A

This toxin is responsible for massive growth of vacuoles within epithelial cells.

VacA can insert into membranes forming hexameric, anion-selective pores.

Toxins acting on intracellular targets: Intracellular traffickingToxin Organism Activity Consequence

TeNT C. tetanii Cleavage of VAMP/ synaptobrevin Spastic paralysis

BoNT-B, D, G and F neurotoxins C. botulinum Cleavage of VAMP/ synaptobrevin Flaccid paralysis

BoNT-A, E neurotoxins C. botulinum Cleavage of SNAP-25 Flaccid paralysis

BoNT-C neurotoxin C. botulinum Cleavage of syntaxin, SNAP-25 Flaccid paralysis

Vacuolating cytotoxin VacA H. pylori Alteration in the endocytic pathway Vacuole formation,

apoptosis

NAD glycohydrolase S. pyogenes Keratinocyte apoptosis Enhancement of GAS proliferation

Toxins injected into eukaryotic cells

These bacteria intoxicate individual eukaryotic cells by using a contact-dependent secretion system to inject or deliver toxic proteins into the cytoplasm of eukaryotic cells.

This is done by using specialized secretion systems that in Gram-negative bacteria are called "type III" or "type IV,“.

Toxins injected into eukaryotic cells: Mediators of apoptosis: IpaB in Shigella

Shigella invasion plasmid antigen (Ipa) proteins: IpaA, IpaB, IpaC, IpaD.

Only IpaB is required to initiate cell death.


targets



action



Clas

sTa

rget

Surface molecules

Cell membrane



Insecticidal toxins



RTX toxins


Signal transduction




Cytoskeleton

Signal transduction

Toxins injected into eukaryotic cells: Mediators of apoptosis: SipB in Salmonella

An analog of Shigella invasin IpaB. In contrast to Shigella, Salmonella does not escape

from the phagosome, but it survives and multiplies within the macrophages.

Salmonella virulence genes are encoded by a chromosomal operon named sip containing five genes (sipEBCDA).

Toxins injected into eukaryotic cells: Mediators of apoptosisToxin Organism Activity Consequence

IpaB Shigella Binding to ICE Apoptosis

SipB Salmonella Cysteine proteases Apoptosis

YopP/YopJ

Yersinia species

Cysteine protease, blocks MAPK and NFkappaB pathways Apoptosis

Toxins injected into eukaryotic cells: Inositol phosphate metabolism

SopB: in Salmonella is homologous to the Shigella flexneri lpgD virulence factor.

Both proteins contain two regions of sequence similarities with human inositol polyphosphatases types I and II.

Toxins injected into eukaryotic cells: Inositol phosphate metabolismToxin Organism Activity Consequence

SopB Salmonella species

Inositol phosphate phosphatase, cytoskeleton rearrangements

Increased chloride secretion (diarrhea)

IpgD S. flexneri Inositol phosphate phosphatase, cytoskeleton rearrangements

Increased chloride secretion (diarrhea)

Toxins injected into eukaryotic cells: Signal transductionToxin Organism Activity Consequence

ExoS P. aeruginosa ADP-ribosylation of Ras, Rho GTPase Collapse of cytoskeleton

C3 exotoxin C. botulinum ADP-ribosylation of Rho Breakdown of cellular actin stress

fibers

EDIN-A, B and C S. aureus ADP-ribosylation of Rho Modification of actin cytoskeleton

SopES. typhimurium

Rac and Cdc42 activationMembrane ruffling, cytoskeletal reorganization, proinflammatory cytokines production

SipAS. typhimurium

Rac and Cdc42 activationMembrane ruffling, cytoskeletal reorganization, proinflammatory cytokines production

IpaA Shigella species Vinculin binding Depolymerization of actin

filaments

YopE Yersinia species

GAP activity towards RhoA, Rac1 or Cdc42

Cytotoxicity, actin depolymerization

YopT Yersinia species

Cysteine protease, cleaves RhoA, Rac, and Cdc42 releasing them from the membrane

Disruption of actin cytoskeleton

VirA Shigella flexneri Inhibition of tubulin polymerization Microtubule destabilization

and membrane ruffling

Toxins injected into eukaryotic cells: Signal transductionToxin Organism Activity Consequence

YpkA Yersinia species Protein serine/threonine kinase Inhibition of phagocytosis

YopH Yersinia species Tyrosine phosphatase Inhibition of phagocytosis

Tir E. coli EPEC Receptor for intimin Actin nucleation and pedestalformation

CagA H. pylori Tyrosine phosphorylated Cortactin dephosphorylation

YopM Yersinia species

Interaction with PRK2 and RSK1 kinases Cytotoxicity

SptP S. typhimurium

Inhibition of the MAP kinase pathway

Enhancement of Salmonella capacity to induce TNF-alpha secretion

ExoU P. aeruginosa Lysophospholipase A activity Lung injury

Toxins with unknown mechanism of actionToxin Organism Activity Consequence

Zot V. cholerae ? Modification of intestinal tight junction permeability

Hemolysin BL (HBL) B. cereus Hemolytic, dermonecrotic and

vascular permeability activitiesFood poisoning, fluid accumulation and diarrhea

BSH L. monocytogenes ? Increased bacterial survival and

intestinal colonization

Abbreviations

SEA-SEI, staphylococcal enterotoxins SHT, Shiga toxin;

TSST, toxic shock syndrome toxin PT, pertussis toxin;

SPE, streptococcal exotoxin CT, cholera toxin;

SSA, streptococcal superantigen LT, heat-labile enterotoxin;

SMEZ, streptococcal mitogenic exotoxin z DNT, dermonecrotic toxin;

MAM, Mycoplasma arthritidis mitogen CDT, cytolethal distending toxin;

YPMa, Y. pseudotuberculosis-derived mitogen TeNT, tetanus neurotoxin;

ETA and ETB, exfoliative toxins RTX, repeats in the structural toxin;

ColH, collagenase Hly, hemolysin;

Nhe, nonhemolytic entertoxin Cry, crystal;

PFO, perfringolysin O; BoNT, botulinum neurotoxin;

SLO, streptolysin O; Ipa, invasion plasmid antigen;

Abbreviations

LLO, listeriolysin O; Sip, Salmonella invasion protein;

ALO, anthrolisin O; EDIN, epidermal cell differentiation inhibitor;

AT, α-toxin; Sop, Salmonella outer protein;

PA, protective antigen; Ipg, invasion plasmid gene;

DT, diphtheria toxin; Yop, Yersinia outer protein;

PAETA, Pseudomonas aeruginosa exotoxin A; GAP, GTPase-activating protein;

GAS, group A Streptococcus; Vir, virulence protein;

YpkA, Yersinia protein kinase A; Tir, translocated intimin receptor;

EPEC, enteropathogenic E. coli; CagA, cytotoxin-associated gene A;

SptP, Salmonella protein tyrosine phosphatase; VAMP, vesicle-associated membrane protein;

ICE, interleukin-1β-converting enzyme; SNAP, synaptosome-associated protein;

MAPKK, mitogen-activated protein kinase ; Zot, zonula. occludens toxin; and BSH, bile salt hydrolase.

Enzymatic activities

Glucosyl-transferases

Deamidases

ADP-ribosyltransferases

N-Glycosidases

Metalloproteases

DT Elongation factor EF-2 Cell death

PAETA Elongation factor EF-2 Cell death

PT Gi, Go and transducin

CTGs, Gt and Golf

cAMP increase

E. coli LT

Clostridium botulinum C2 Actin Failure in actin

P. aeruginosa ExoS Ras Collapse of cytoskeleton

Clostridium botulinum C3 Rho Breakdown of cellular actin stress fibers

ADP-

ribos

yltr

ansf

eras

esToxin Substrate Effect

Clostridium difficile toxins A and B Rho/Ras GTPases Breakdown of cytoskeletal structure

Toxin Substrate Effect

Glucosyl-transferases

DeamidasesE. coli CNF1 Rho, Rac and CdC42

Bordetella DNT Rho, Stress fiber formation

Shiga toxin Ribosomal RNA

Disruption of normal homoeostatic functions

N-Glycosidases

Metalloproteases

Bacillus anthracis LF Macrophages

Clostridium tetanii TeNT VAMP/synaptobrevin Spastic paralysis

Flaccid paralysisC. botulinum BoNTs VAMP/synaptobrevin, SNAP-25

Stop of protein synthesis

Abbreviations

SNAP-25, synaptosome-associated protein of 25 kDa.

CNF1, cytotoxin necrotizing factor 1;

DNT, dermonecrotic factor; DT, diphtheria toxin;

PAETA, Pseudomonas aeruginosa exotoxin A; PT, pertussis toxin;

CT, cholera toxin; LT, heat-labile enterotoxin;

ExoS, exoenzyme S; LF, lethal factor;

TeNT, tetanus neurotoxin; BoNT, botulinum neurotoxin;

VAMP, vesicle associated membrane protein;

Thank You

Bacterial Toxins and Toxin classification

Health & Medicine

protein toxins

introduction toxins

study of toxins

cell lysis

cell biology

eukaryotic cell membrane

main categories of toxins

rtx toxin family