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Bacterial Toxins

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Medical Microbiology Bacterial Toxins BIOL 533 Lecture 9 Bacterial Toxins: General Aspects Definition Soluble substances that alter normal metabolism of host cells ... – PowerPoint PPT presentation

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Title: Bacterial Toxins


1
Bacterial Toxins
Medical Microbiology
  • BIOL 533
  • Lecture 9

2
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

3
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

4
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

5
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?

6
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

7
Toxin Production
  • Properties
  • Dispensable, but essential under certain
    situations where survival and spread are at stake
  • Genes frequently carried on plasmids and
    temperate bacteriophage

8
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

9
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)

10
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?

11
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

12
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

13
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

14
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

15
Mechanism of Action
  • Toxins that assist bacterial spread in tissues
  • Properties
  • Do not target any type of cell
  • Include degradative enzymes that allow spreading

16
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)

17
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)

18
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

19
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

20
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

21
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

22
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

23
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)

24
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

25
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)

26
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

27
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)

28
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

29
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

30
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)

31
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

32
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)

33
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

34
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

35
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

36
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)

37
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

38
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

39
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

40
Tetanus Toxin
  • Acts at distance from central nervous system
  • Once bound to cell membranes, toxin is
    internalized probably by receptor-mediated
    endocytosis

41
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

42
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

43
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

44
Lecture 9
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