Bacterial toxins

bacterial toxins.....

1. Bacterial Toxins Presented by Nagaraj.S M.Sc. 3 rd year microbiology

2. TOXINS: Are major determinants of virulence. Usually virulent strains of the bacterium produce the toxin while non virulent stains do not. Toxigenesis : The ability to produce toxins, is an underlying mechanism by which many bacterial pathogens produce disease

3. Bacterial toxins are the soluble antigens, secreted by a number of pathogenic bacteria Toxins can modulate the cellular functions by selectively targeting a number of signaling pathways within the host cell in order to tilt the balance in bacteria favor. Since the discovery of the first diphtheria toxin by Emile Roux and Alexandre Yersin in 1888 from the bacteria- Corynebacterium diphtheriae, more than 500 bacterial toxins are known till date.

4. There are two types of bacterial toxins. ENDOTOXINS : LIPOPOLYSACCHARIDES - Are associated with the cell walls of gram – ve bacteria EXOTOXINS : PROTEINS - Released into the extracellular environment of pathogenic bacteria

5. Endotoxins Endotoxins are the integral part of the cell walls of Gram-negative bacteria, and are liberated when bacteria are disintegrated (lysed). Cell wall of Gram negative bacteria contain lipopolysaccharides (LPS, endotoxin) which consists of (1) Lipid A. This is the endotoxin and is the core, and (2) Polysaccharide form coat. This is the O antigen which can induce specific immunity .

6. Features of endotoxins Endotoxin: Integral part of the cell wall of Gram-negative bacteria. Released on bacterial death and in part during growth. Formed only by Gram-negative bacteria Lipopolysaccharides. Lipid A portion is responsible for toxicity No specific receptor. Moderately toxic. Fatal to animals in large doses. Relatively heat stable. Toxicity is not destroyed above 60°C for hours. Weakly antigenic. Antibodies are protective. Not converted to toxoid. Synthesis directed by chromosomal genes. Usually produce fever in the host by release of interleukin-1 and other mediators

7. All endotoxins can produce the same signs and symptoms: Chills, fever, weakness, general aches, blood clotting and tissue death, shock, and even death Can also induce miscarriage. The biological activity is associated with the lipopolysaccharide( LPS ) . the toxicity is associated with the lipid component ( lipid A ) and the immunogenicity is associated with the polysaccharide components. Endotoxins are heat stable. But certain powerful oxidising agents such as super oxides, peroxide and hypochlorite are neutralise them.

8. Mechanism of endotoxins Begins with CD14 binding of receptors on Macrophages that: 1. Induces cytokine production: IL-1, IL-6, IL-8, TNF, PAF 2. Activation of complement cascade (C3a, C5a or alternate pathway) 3. Activation of coagulation cascade (Hageman factor; Factor XII)

9. Mechanism of endotoxin

10. Endotoxin and pyrogenic response The cytokines induce the hypothalamus to release lipids called prostaglandins, which reset the thermostat in the hypothalamus at a higher temperature.

11. Organisms that produce endotoxins include: Salmonella typhi Proteus spp. Pseudomonas spp. Neisseria spp .

12. Complications of endotoxins 1. Fever.  The endotoxin acts on mononuclear phagocytes (monocytes /macrophages), with liberation of interleukin-1. Interleukin-1 acts on thermoregulatory center. Chill is due to widespread arteriolar and venular constriction. 2. Leucopenia   occurs early with onset of fever. It may be followed by leukocytosis . 3. Hypoglycemia.   LPS enhances glycolysis in many cell types and can lead to hypoglycemia. 4.  Hypotension   occurs early in Gram-negative bacteremia. 5.  Shock.   ' Endotoxic ' or 'septic' shock may develop in severe Gram-negative bacteremia.

13. 6.      Activation of complement .  Endotoxin activates complement system by alternative pathway. 7.  Disseminated Intravascular Coagulation (DIC).  DIC may occur in Gram negative bacteremia. It is initiated on activation of factor XII (Hageman factor) 8.  Death  may occur due to shock and/or DIC.

14. Exotoxins Bacterial protein toxins are the most potent poisons may show activity at very high dilutions. Protein toxins are resemble enzymes. like enzymes , bacterial exotoxins are: Proteins. Denatured by heat, acid, proteolytic enzymes. Have a high biological activity (most act catalytically). Exhibit specificity of action. Bacterial exotoxins have enterotoxic , cytotoxic, hemolytic and neurotoxic effect.

15. Features of Exotoxins Exotoxins: Excreted by living cells Produced by Gram-positive and Gram-negative bacteria Polypeptides Usually bind to specific receptors on cells Highly toxic. Fatal to animals in very small doses Relatively heat labile. Toxicity destroyed over 60°C Highly antigenic. Stimulate formation of antitoxin. Antitoxin neutralizes the toxin Converted to toxoid by formalin. Toxoid is nontoxic but antigenic used to immunize, e.g. tetanus toxoid Usually controlled by extra-chromosomal genes, e.g. plasmids, phage gene do not produce fever in the host

16. A+B subunit arrangement Many protein toxins, notably those act intracellularly (with regard to host cells), consist of two components: one component ( subunit A ) is responsible for the  enzymatic activity  of the toxin; the other component ( subunit B ) is concerned with  binding  to a specific receptor on the host cell membrane and transferring the enzyme across the membrane.  The enzymatic component is not active until it is released from the native ( A+B)  toxin.

17. Attachment and Entry of Toxins There are at least  two mechanisms of toxin entry into target cells. Direct entry , the B subunit of the native (A+B) toxin binds to a specific receptor on the target cell and induces the formation of a pore in the membrane through which the A subunit is transferred into the cell cytoplasm.   Alternative mechanism : receptor-mediated endocytosis ( RME ). 

18. Attachment and entry of toxin

19. Staphylococcal enterotoxin It is responsible for the manifestations of staphylococcal food poisoning- nausea, vomiting and diarrhoea 2-6 hrs after consuming food contaminated by toxin. Toxin is relatively heat stable, resisting 100°c for 10-40 mins depending on the concentration of the toxin and the nature of the medium. About 2/3 rd of staph.aureus strains, growing in carbohydrate and protein foods, secret the toxin. Milk and milk products is also contaminated. The source of infection is usually a food handler, become a carriers.

20. 8 antigenic types of enterotoxins are currently known, named A,B,C1-3, D,E and H. they are formed by toxigenic strains either by singly or in combination. The toxin acts directly on autonomic nervous system to cause illness. The toxin is very potent . The toxin also exhibit pyrogenic, mitogenic, hypotensive, thrombocytopenic and cytotoxic effect. Sensitive serological tests such as latex agglutination and ELISA are available for detection of toxin.

21. Toxic shock syndrome It is potentially fatal multisystem disease presenting with fever, hypotension, myalgia, vomiting, diarrhea , mucosal hyperaemia and an erythromatous rash. TSST type 1 is often more responsible, through enterotoxin B or C may also cause the syndrome. TSST 1 antibody seen in convalescents

22. Staphylococcal enterotoxins and TSST-1 are super antigens which are potent activators of T lymphocytes This leads to an excessive and dysregulated immune response, with release of cytokines interlukin1-2,TNF and interferon gamma This explain the multisystem involvement and florid manifestations in staphylococcal food poisoning and TSS.

23. Clostridium. perfringens Toxin Perfringolysin O Cholesterol-dependent cytolysins (CDCs) constitute a family of pore forming toxins secreted by Gram positive bacteria. These toxins form transmembrane pores by inserting a large β-barrel into cholesterol-containing membrane bilayers. Binding of water-soluble CDCs to the membrane triggers, formation of oligomers containing 35-50 monomers. The coordinated insertion of more than seventy β-hairpins into the membrane requires multiple structural conformational changes and leads to pore formation. Perfringolysin O(PFO), secreted by Clostridium perfringens, has become the prototype for the CDCs.

24. Streptococcus exotoxin Streptococci produce 2 types of hemolysins , they are streptolysin O&S. Streptolysin O is oxygen and heat labile. It is inactivate in the oxidised form but may be reactivated by treatment with mild reducing agents. It is lethal on intravenous injection into animals and has specific cardiotoxic activity and also has leucotoxic activity. In its biological action, streptolysin O resembles the oxygen labile hemolysins of C.perfringens , C.tetani and S.pnemoniae .

25. S.pyogenus exotoxin This toxin acts as a super antigen.super antigens are another group of extracellular protein toxins produced by S.aureus or A beta –haemolytic streptococci Binds to structural peptides on host T-lymphocyte receptors and macrophage class 2 histocompatibility antigens This binding induces the release of cytokines (TSST-1, staphylococcal enterotoxins A&B and group A strep.toxin .) Which acts as a super antigens and induce high levels of T-cell proliferation, IL-2 and TNF-alpha release cause shock syndrome

26. Diphtheria toxin Produced by Corynebacterium. Diphtheriae The toxin has 58KDa of molecular weight. And divided into 2 subunits depending upon their mol.wt . The large subunit B (mol.wt.37KDa) which helps the attachment of diphtheria toxin to the host membrane, contains C terminal receptor binding domain The small subunit A (mol.wt.21KDa) actively participate in catalytic action. toxic effect mediated by this unit.

27. Elongation factor is required for the protein synthesis on both prokaryotes and eukaryotes. EF-2 + New A.A. sequence Transamination one place to another place. adenorybosyl phosphate So, for the stopping of protein synthesis, the diphtheria toxin inactivates the elongation factor-2. As a result of stopping of protein synthesis and cell dyes with in minutes.

28. Receptor binding domain B binds to host membrane Membrane bound toxin A+B enters the cell via RME Endosome vesicle acidifies ,disulphide bonds are reduced. A Subunit hydrolysed inside the vesicle and diffuse through the vesicle on the cytoplasm of the host cell A Subunit transfer the adenosyl group from NAD EF2 EF2 loss their function and inhibition of protein synthesis Cell dyes with in a minute A B

29. Clostridium.perfringens toxin C.perfringens is one of the most prolific of toxin producing bacteria, forming at least 12 distinct toxins. The four major toxins Alpha , Beta , Epsilon and Eta are predominantly responsible for pathogenicity. The alpha toxin is produced by all types of C.perfringens and most abundantly by type A strain. This is the most important toxin biologically and is responsible for the profound toxaemia of gas gangrene. It is lethal, dermo necrotic, haemolytic.

30. alpha toxin contains phospholipidase enzyme ( lecithinase C activity) lecithin Ca++, Mg++ phosphoryl choline, di glyceride lecithinase and phosphoribosyl activity myo-necrosis

31. Tetanus toxin Tetanus is caused by the systemic effect of tetanus neuro toxin, the toxin produced by clostridium.tetani Tetanus usually occurs as a result of wound infection by c.tetani or rarely, from injection of materials contaminated with c.tetani vegetative cells and spores Tetanus neurotoxin is released upon cell lysis after bacterial growth under anaerobic conditions (deep wounds).

32. L H H Tetanus neurotoxin(150KDa) L H 50 Kda 100 Kda proteolytic enzymes generalised spastic paralysis receptor binding site sensitivity to excitatory Internalise and move from peripheral to CNS by impulse Retrograde axonal transport Toxin reaches neurons in spinal cord, brain stem, elevated muscle tone and and cerebellum hyper active reflexes binds to its receptor, a ganglioside containing stearic acid , Sphingosine, glucose, galactose, N-acetyl glucosamine and sialic acid it blocks the normal post synaptic inhibition of spinal motor neurons synaptic blockage prevention of releasing inhibitory neurotransmitters of CNS alpha amino butyric acid and glycine L

33. Botulinum toxin The botulism is caused by the ingestion of performed toxin. The types of the bacillus and the nature of the food responsible vary in different regions. Human disease is usually caused by types A, B, E and rarely F. Type C,D associated with out breaks of cattle and wild fowl. Type G has been associated with sudden death in few patients. Type E is associated with fish and other sea foods. The source of food is usually food- meat and meat products, canned vegetables in Europe and japan.

34. Symptoms usually begin 12-36 hrs after ingestion of food. No vomiting or diarrhoea are present. Coma or death delirium may occurs. Death is due to respiratory failure and occurs 1-7 days after onset. Case fatality varies at 25-70%

35. Improperly canned fruits, botulinum toxin production colonize the intestinal tract vegetables and fish products in wounds , in parenteral drug users of infants( infant botulism) progenitor protein( mol.wt.150KDa) light chain 50 KDa , heavy chain 100 Kda absorption from GIT susceptible neurons via blood stream fatigue, dizziness, nausea , blurring of vision slurring of speech, dilatation of pupils, bound to pre-synaptic terminals urinary retention,flaccid paralysis of skeletal muscles and respiratory paralysis enters NMJ & peripheral autonomic synapse blocks the release of Ach . from cholinergic nerve endings

36. Toxins of E.coli E.coli (heat labile toxin) Heat labile toxin of E.coli was discovered in 1956 by De and colleagues in isolates from adult diarrheal cases in Calcutta, by the rabbit ileac loop method. E.coli LT resembles the cholera toxin in its structure, antigenic properties and mode of action.

37. Complex polypeptide subunit A B B B B B active binding GM1 ganglioside receptor on intestinal epithelial cells activated to yield 2 fragments A1 A2 activates adenylate cyclase in the enterocyte Cyclic adenosine mono phosphate(cAMP) inhibits the absorption of sodium transport system in the villus cells activates the chloride transport system in the crypt cells Water moves passively in the bowel lumen to maintain the osmolarity watery diarrhoea

38. E.coli heat stable toxin It comprises low mol.wt . polypeptides, which are poorly antigenic Two types of ST are known. ST A and ST B. ST A acts by activation of cGMP in the intestine. It acts as very rapidly and induces fluid accumulation in the intestines of infant mice within four hrs of intra-gastric administration. This infant mice test is the standard method for demonstration of ST A It is methanol soluble.

39. ST B ST B causes fluid accumulation in young piglets but not in infant mice The mode of action is not known but it is not through cAMP or cGMP It is not methanol soluble

40. Verocytotoxin of E.coli It was first detected by its cytotoxic effect on vero cells, a cell line derived from African green monkey kidney cells. It is also known as shiga like toxin(SLT) because it is similar to Shigella dysenteriae type1 toxin in its physical, antigenic and biological properties. It acts by inhibition of protein synthesis and shows enterotoxicity in rabbit ileal loops and mouse paralytic lethality VT is also composed of A and B sub units

41. Toxins of shigella Shiga toxin Shigella.dysenteriae type1 forms an exotoxin, it appears to much less important in pathogenesis than the ability of the bacillus to penetrate and multiply in colonic mucosa. It is also known as shiga toxin or verotoxin which is similar to EHEC verotoxin . It acts by inhibition of protein synthesis. It has A and B subunits They can be tested by toxicity in vero -cell lines

42. Shiga toxin A ,B5 B sub unit attaches the colonic epithelial cell A subunit enter in to the cell by endocytosis It inactivates the ribosomal function depurinated RNA inhibition of protein synthesis

43. Cholera toxin Cholera is an acute diarrheal disease caused by V.cholerae which produces cholera toxin on the intestinal epithelium. in severe cases leads to hypovolemic shock and death in less than 24 hrs. Cholera toxin is very similar to labile toxin of E.coli in several properties. CT is more potent than LT in biological activity.

44. Toxin molecule mol.wt.84000 A B B B B B yields GM1 ganglioside receptor on the intestinal epithelium A1 A2 helps to tethering A&B subunits activation of adenylate cyclase intracellular accumulation of cAMP cAMP, inhibits absorptive Na transport system in villus cells activate the chloride transport system in crypt cells watery diarrhea accumulation of NaCl in intestinal lumen accumulation of isotonic fluid water moves passively into the bowel lumen to maintain osmolarity

45. Characteristics of bacterial exotoxins and endotoxins Property Endotoxin Exotoxin Chemical nature Lipopolysaccharide m.W = 10 kda Protein m.W =50-1000 kda Relationship to cell Part of outer membrane Extracellular, diffusible Denaturated by boiling No Yes Antigenic Yes Yes Form toxoid No Yes Potency Relatively low (>100 ug ) Relatively high (1 ug ) Specificity Low degree High degree Enzymatic activity No Often Progenicity Yes occasionally

46. Toxin detection methods Diagnostic methods of toxins Bioassay methods Immunological assays Molecular techniques Cell cultures

47. Animal model for surgical procedures Ligated loop technique : B. cereus, cl. Perfringens, E.coli, V. parahaemolyticus . RITARD model : ETEC V. cholerae, C.jejuni , Aeromonas

48. Immunological assays Reverse passive hemagglutination antibody coupled to sheep RBC agglutination if sheep toxin is present sensitivity is 1.5 ng/ml Radioimmunoassay : solid phase RIA- staph ET (A – E). Sensitivity- 1-5 ng ELISA Double antibody “sandwich” ELISA: staphylococcus enterotoxin A- E.

49. Gel-diffusion assay microslide gel double diffusion test b.cereus diarrheal enterotoxin cl.perfringens enterotoxin staph B enterotoxin

50. Electro immunodiffusion SET-A B.cereus enterotoxin Cl.botulinum enterotoxin A-E

51. Elek’s gel precipitation test For the diagnose of diphtheria toxin

52. Radial immunodiffusion assay Detection of ST- A-B Sensitivity – 0.3 mg/ml

53. Duplex SYBR green I real time PCR assay For one step differention between emetic B.cereus and staph. Aureus Real time PCR based assay BoNT A,B,E and F gene fragments

54. Reverse passive latex agglutinaton Detection of soluble antigen Antibody + latex particle Presence of toxin : diffuse layer at base Negative : button formation Simple, rapid B. cereus emetic toxin .

55. Cell cultures Type of cell culture organisms Human foetal intestine v. parahaemolyticus CHO monolayer E.Coli LT, v. Cholerae ET , salmonella ET Verocells E.Coli LT, Cl.perfringens ET , salmonella cytotoxin Y1 adrenal cell E.Coli ET, V. cholerae toxin

56. Treatment for toxins Botulinum toxin Food born and wound born botulism is treated with antitoxin, that blocks the neurotoxin circulated in the blood. Trivalent antitoxin (eff. Against A,B & E). Heptavalent antitoxin (eff. Against A,B,C,D,E,F &G). When contaminated food in the gut, inducing vomiting or by using enemas (used to remove unabsorbed toxins). Antibiotics (high dose IV penicillin) used in wound botulism. Antibiotics not used in food born botulism.

57. Diphtheria toxin Antidiphtheritic serum- to neutralise the toxin released by the bacteria. antibiotics - erythromycin or penicillin to eradicate the bacteria and stop it from spreading. 2 strengths of diphtheria toxoid are used in routine diphtheria vaccines. D – higher dose vaccine for children under 10. Three doses at 2,3&4 months of age, d – lower dose version. primary VACCINE for children over 10 and as a booster about 3 yrs after the primary vaccine, normally between 3.5 and 5 yrs of age.

58. Cholera toxin Doxycycline – first line treatment for adults. Azithromycin – first line treatment for children and pregnant women. Tetracycline – effective treatment against cholera. Superior to furazolidone, chloramphenicol, erythromycin is effective for cholera toxin. Cholera vaccine. As per CDC & WHO it may not protect from the half of the people, who receive it and it lasts only in a few months.

59. Staphylococcal enterotoxin B In emergency, cough suppressants and acetaminophens for fever. Anti- emitics for nausea. Vasopressors and diuretics in severe cases. Antibiotics have not demonstrated efficacy in SEB intoxification .

60. Tetanus toxoid Active immunization is possible by spaced infections of formal toxoid, which is available either as plain toxoid, or absorbed on aluminium hydroxide or phosphate. The adsorbed toxoid is a better antigen. Dosage: three doses of tetanus toxoid given intramuscularly (interval 4-6weeks). Triple vaccine : Tetanus toxoid + diphtheria toxoid + pertussis (adjuvant). Dosage: DPT1:6wks, DPT2: 10wks, DPT3: 14 wks. Booster 24 months.

61. Dermatological use of botulinum toxin Since the 1970 when Alan Scott introduced botulinum neurotoxin A as a therapeutic agent, the number of different uses of this drug has increased exponentially. FDA has approved Botox not only for facial aesthetics but also for neurogenic hyperactive bladder function . Incobotulinumtoxin A is a commercially available botulinum toxin preparation widely used to cure glabellar frown lines

62. Non-dermatological uses of Botulinum toxin in ophthalmology- include Blepharospasm Apraxia of lid opening Intermittent exotropia Congenital nystagmus Lacrimal hypersecretion Pain relief in acute angle closure glaucoma. Whereas in neurology and gastrointestinal it is used for Hemifacial spasm Oromandibular dystonia Spasmodic torticollis, Gustatory sweating Achalasia

63. Role of toxins as immunotoxins in treatment of cancer Immunotoxins are the hybrid molecules that combined the binding specificities of antibodies and the cytotoxicity effects of toxins for the target cells . On the basis of complete structure of toxin, immunotoxins can be divided into three generations. First generation immunotoxins are conjugate of antibodies/ligand to intact bacterial toxin (Enzyme domain and binding domain). In second generation immunotoxins, bacterial toxin is lacking binding domain. Binding domain of toxin part has been replaced by Fv portion of antibody in the third generation of immunotoxins to reduce the overall size of immunotoxin and better penetrability into solid cancer tumors

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