Bacterial Toxins

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Bacterial Toxins
Medical Microbiology

• BIOL 533
• Lecture 9

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Bacterial Toxins General Aspects

• Definition
• Soluble substances that alter normal metabolism
  of host cells with deleterious effects on the
  host
• Host range
• Known for bacteria, but possible that they play a
  role in diseases caused by fungi, protozoa, and
  worms

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Bacterial Toxins General Aspects

• Toxin type
• Exotoxinprotein produced by bacteria either
  excreted or bound to bacterial surface and
  released when lysed
• Endotoxinlps of the outer membrane of Gram
  bacteria
• Acts as toxin only under special circumstances

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Bacterial Toxins General Aspects

• Specificity
• Some act on certain cell types
• Other affect wide range of cells and tissues
• Numbers produced by single bacterium
• Some produce none
• Pneumococci

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Is Toxin Important in Infection?

• Questions to ask
• Is virulence quantitatively correlated with toxin
  production?
• Does the purified toxin produce damage?
• Can a specific antibody (antitoxin) prevent or
  alleviate the manifestations of the disease?
• If toxin production is impaired by a mutation, is
  the disease process affected?

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If So, What are Toxin Properties?

• Questions to ask
• What is the mechanism of action?
• Why is it specific for certain cells or tissues?
• Does the pathogen make other toxins, and if so,
  do they interact with one another?
• Some make none pneumococci
• Some make only one agents that cause cholera,
  diphtheria, tetanus, and botulism
• Other make many staphylococci, streptococci

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Toxin Production

• Properties
• Dispensable, but essential under certain
  situations where survival and spread are at stake
• Genes frequently carried on plasmids and
  temperate bacteriophage

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Toxin Production

• Found on phage toxin genes for
• Diphtheria
• Botulism
• Scarlet fever
• Toxic streptococci (flesh-eating)
• Found on plasmids
• E. coli toxin causes diarrhea
• S. aureus toxin causes scalded skin syndrome
• E. coli 0157H7

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Toxin Production

• Properties
• Mobile elements ensure that genes can be spread
  to nontoxigenic derivatives or be lost from cell
• Experimentally called curingget nontoxigenic
  derivatives
• Phase of production
• Some produced continuously by growing bacteria
• Other synthesized when cells enter stationary
  phase (true also for many antibiotics)

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Toxin Production

• Explanation
• Certain toxins may help bacteria get scarce
  nutrients
• Example high levels of diphtheria toxin produced
  when cell depleted of iron
• Very little free iron in normal tissue
• Is this a way for organisms to obtain it from
  dead tissue?

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Toxin Production

• Sporulating bacteria sometimes release toxins
  during spore formation
• Bacterial cells eventually lyse and liberate
  cytoplasmic proteins, including toxins
• Examples organisms that cause botulism, gas
  gangrene, or tetanus
• In contaminated wound, some organisms are growing
  and some are sporulating
• End result is continual production

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Mechanism of Action

• General aspects
• Sphere of influence
• Some act locally, killing wbc nearby
• Others help organism to spread in host itssues by
  degrading connective tissue
• Still others are disseminated very far from site
  where synthesized
• Diphtheria toxin made in throat, but acts on
  heart and brain

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Mechanism of Action

• Level of toxicity
• Work at extremely low levels include strongest
  poisons known
• 1 g tetanus, botulinus, or Shiga toxin is enough
  to kill 10 million people
• 100-fold more is required for diptheria
• 1000-fold more for Pseudomonas A

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Mechanism of Action

• Mechanisms of damage
• Lysis of host cells
• Stop or interfere with cell growth
• Exaggerate normal physiological mechanisms
• By depressing or augmenting particular functions,
  toxins can kill without damaging any cells
• Tetanus toxin paralyzes body without affecting
  target neurons
• Cholera toxin speeds up normal excretory process,
  resulting in massive loss of water

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Mechanism of Action

• Toxins that assist bacterial spread in tissues
• Properties
• Do not target any type of cell
• Include degradative enzymes that allow spreading

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Mechanism of Action

• Examples
• Streptococci
• Some secrete
• Hyaluronidasebreaks down hyaluronic acid
  (connective tissue)
• DNasethins out pus made viscous by DNA from dead
  white blood cell
• Streptokinase (protease)cleaves precursor of
  plasminogen activator to active form
• Converts plasminogen to plasmin (serum protease
  that dissolves fibrin clots)

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Mechanism of Action

• Examples
• Similar roles suggested for elastases and
  collagenases of other organisms
• In this case, are unregulated forms of enzymes
  that also exist in uninfected host (activity is
  normally under control)

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Mechanism of Action

• Toxins that lyse cells
• General aspects
• Large class kill host cells by destroying their
  membranes act as lipases
• Example of lipase type Clostridium perfringens
  (gas gangrene) lecithinase
• Lyses cells indiscriminately because
  phosphatidylcholine (lecithin) is ubiquitous in
  mammalian membranes
• Also hemolysins are of this type lyse both red
  blood cells and white blood cells

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Mechanism of Action

• Act by inserting themselves in membrane forming
  pores
• Mechanism make membrane more permeable, water
  pours into cytoplasm, cell begins to swell, and
  eventually bursts
• At very low concentrations (not enough to cause
  lysis), cell functions may be severely damaged.
  Slight perturbations of permeability cause
• Leakage of potassium ions needed for protein
  synthesis and cell viability
• Low levels inhibit phagocyte functioning

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Mechanism of Action

• Examples
• Staphylococci ?-toxin (homogeneous pore former)
• Receptors existcells show 100-fold range in
  sensitivity
• Consequences of action aggregation of platelets
  and narrowing of blood vessels leads to necrosis

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Mechanism of Action

• Examples
• Streptococcal streptolysin 0 (heterogeneous pore
  former)
• Binds to cholesterol in cell membrane
• Free toxin can be inactivated by cholesterol, but
  once bound by membrane, it is impervious
• Consequences of the action lyses red blood
  cells, but not neutrophils or macrophage
• White blood cells are killed by low levels of
  toxin because it acts preferentially on membranes
  of lysosomes, releasing hydrolytic enzymes

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Mechanism of Action

• Toxins that block protein synthesis
• Structure and mode of action
• Toxins that work outside the cell are variable in
  structure and mode of action
• Toxins that work inside have a number of
  similarities

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Mechanism of Action

• Similarities
• Most have two portions (A-B toxins)
• Subunits
• Toxic activity (A)
• Binding to cell membrane (B)
• Can be one polypeptide chain or many
• Binding to membrane may be followed by
  receptor-mediated endocytosis and internaliztion
  of the toxin (some investigators propose direct
  passage through pore)

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Mechanism of Action

• A moity is often latent, even after engulfment
• May be activated by proteoytic cleavage and
  reduction of disulfide bridges
• Toxins of diphtheria, cholera, tetanus, and
  Shigella are synthesized as inactive precusors

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Mechanism of Action

• May have common mode of action
• Catalyze transfer of adenosine-diphosphate group
  from NAD to target proteins
• Examples of ADP-ribosyltransferasestoxins of
• Diphtheria
• Cholera
• Exotoxin A (Pseudomonas aeruginosa)

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Diphtheria Toxin

• How does toxin enter cell?
• A and B are single polypeptide chain
• Hydrophobic B portion binds to receptor on
  membrane
• By this time, molecule is cleaved at sensitive
  site between A and B portions, but is still
  covalently associated by disulfide linkage
• Entire receptor-toxin complex enters cell by
  receptor-mediated endocytosis

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Diphtheria Toxin

• Once toxin enters, reduction S-S bond separates A
  and B portion
• Acidic conditions within endosomal vesicles
  promote insertion of B chain into endosomal
  membrane
• Somehow, this facilitates passage of A into
  cytosol
• Resistant to denaturation and is long-lived
  within cells
• Accounts in part for potency (single molecule can
  kill cell)

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Diphtheria Toxin

• Mechanism of killing
• ADP-ribosylation of EF2 (protein that catalyzes
  hydrolysis of GTP that drives movement of
  ribosomes on eucaryotic mRNA)
• Reaction is EF-2 NAD? ADPR-EF2 H

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Diphtheria Toxin

• EF2 is only known substrate for diphtheria toxin
• EF2 contains rare modification of one of
  histidine residues and this is site recognized by
  toxin
• Mutant cells that cannot modify site are
  resistant
• Addition of ADP-ribose inactivates EF2
• Kills cells by irreversible block of protein
  synthesis
• P. aeruginosa exotoxin A works same as diphtheria
  toxin

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Mechanism of Action

• Phamacological toxins (elevation of cAMP-cholera)
• Excess of cAMP interferes with phagocyte
  functioning (chemotaxis and phagocytosis)
• Methods of increasing
• Secretion of cAMP
• Secretion of adenyl cyclase to make more cAMP
• Secretion of toxin alters activity of host adenyl
  cyclase (cholera)

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Cholera Toxin

• Target tissue is small intestine epithelium
• Structure and mechanism of toxin
• Toxin has separate A and B subunits
• B has affinity for intestinal epithelial mucosa
• A ADP-ribosylates GTPase (part of complex that
  makes cAMP)
• Synthesis of cAMP becomes unregulated made in
  large amounts
• Provokes loss of fluids and copious diarrhea

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Cholera Toxin

• Structure of subunits
• Five B subunits and one A subunit
• A subunit is synthesized as single chain
• Then, after secretion, cleaved into two fragments
  (A1 and A2 held together by disulfide bonds)

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Cholera Toxin

• Mechanism
• Whole toxin binds to 5 ganglioside receptors on
  surface of intestinal epithelial cells
• A1-A2 portion enters cell and is cleaved into A1
  and A2 pieces (by reduction of disulfide bonds)
• A1 fragment in enzymatically active

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Cholera Toxin

• Regulation
• Normal
• Adenylate cyclase complex is membrane bound and
  is composed of three proteins (Gs, R, cyclase)
• Gs protein is GTPase protein with two
  conformational states
• Binds GTPstimulates adenyl cyclase to make cAMP
• GTPase that cleaves GTP to GDP

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Cholera Toxin

• Balance is determined by binding of R protein
• Binding of GTP by Gs stimulated by binding R
  protein
• R is receptor for several different hormones
  (adenergics)
• Whole picturewhen R protein binds with hormone,
  interacts with Gs protein to increase its binding
  of GTP
• Gs remains in active state to stimulate adenyl
  cyclase

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Cholera Toxin

• Abnormal (cholera) normal action of R protein
  mimicked by cholera toxin
• Promotes active state of Gs protein by different
  mechanism
• ADP-ribosylates Gs at one of its arginine
  residues (Gs protein locked into active
  conformation)

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Mechanism of Action

• Other toxins that activate adenylate cyclase
• Number of enterotoxins that produce diarrhea
• LT (labile)E. coli
• Bordetella pertussis adenylate cyclase
• Raise level cAMP in leucocytes

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Mechanism of Action

• Toxins that block nerve function
• Most lethal toxins known are tetanus and
  botulinum toxins
• Tetanus toxin produces irreversible muscle
  contraction
• Botulinum toxin blocks muscle contraction

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Mechanism of Action

• General mechanism of both
• Consist of single polypeptide chains with A and B
  regions
• Binding to ganglioside receptors specific for
  nerve tissue
• Activated by proteolysis and disulfide reduction,
  and they function intracellularly

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Tetanus Toxin

• Acts at distance from central nervous system
• Once bound to cell membranes, toxin is
  internalized probably by receptor-mediated
  endocytosis

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Tetanus Toxin

• Transported through axonal processes to the
  spinal cord
• Toxin interferes with synaptic transmission by
  preferentially inhibiting release of
  neurotransmitter, such as glycine from inhibitory
  interneurons
• Excitory and inhibitory effects of motor neurons
  become increasingly unbalanced, causing rigid
  muscle contractions
• Cause of inhibitory synapse action unknown

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Botulinum Toxin

• General aspects
• Intoxication, not infection organism not needed
  after toxin produced
• Toxin not destroyed by proteases of digestive
  tract probably complexed with other proteins
• Mechanism
• Affects peripheral nerve endings

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Botulinum Toxin

• Once across the gut, it is carried in the blood
  to neuromuscular junctions
• Bind to gangliosides at motor nerve endpoints and
  is taken up by cell
• Subsequent events unknown
• Result in presynaptic block of release of
  acetylcholine
• Interruptions in nerve stimulation causes
  irreversible relaxation of musclesleads to
  respiratory arrest

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Lecture 9

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