Bacterial Toxins

1. Medical Microbiology Bacterial Toxins 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 • Exotoxin—protein produced by bacteria either excreted or bound to bacterial surface and released when lysed • Endotoxin—lps 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 0157:H7

9. Toxin Production • Properties • Mobile elements ensure that genes can be spread to nontoxigenic derivatives or be lost from cell • Experimentally called “curing”—get 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 • Hyaluronidase—breaks down hyaluronic acid (connective tissue) • DNase—thins 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 exist—cells 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-ribosyltransferases—toxins 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 GTP—stimulates 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 picture—when 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 muscles—leads to respiratory arrest

44. Lecture 9 • Questions? • Comments? • Assignments...