Mechanism of Bacterial Damage & Bacterial Toxins

1. Mechanism of Bacterial Damage & Bacterial Toxins Special Lecture Topic

2. Microbial Damage • Damage during infection occurs • Direct action of pathogen • Host response to infection • Combination of both

3. Microbial Damage • Mechanical • Blockage by pathogen • Worm infections • Blockage by inflammation • Host cell death • Direct cell lysis • Chlamydia • Rickettsia • Early apoptosis • HIV and herpes • Toxins

4. Bacterial Toxins • Many different types of toxins • Exotoxins • Neurotoxins • Endotoxins • Exoenzymes • Toxins are dispensable because they are not required for growth • Genes for toxins are usually on plasmids

5. Also see page 399 of textbook Bacterial Exotoxins • Exotoxins • Initial location outside cells • Transported into host cells • Alter host cell physiology and metabolism • Typical A – B toxins AB toxin enters cells via: 1) Receptor mediated endocytosis 2) Fusion of vesicle with lysosome 3) Acid environment of lysosome reduces disulfide bonds and releases A into cell 4) A has various cellular activities

6. Corynebacterium diphtheriae • Corynebacterium diptheriae • Produces AB exotoxin • Gram positive rod w/ tapered ends • Significant cause of mortality until 1950s • Decline due to vaccination with toxoid • Spread by close contact via droplets from human carriers or humans with active infection • Common location upper respiratory tract

7. Symptoms of diphtheria • Symptoms • Local infection • Severe inflammatory reaction • Severe swelling in back of neck • Sore throat, nausea, vomiting • Formation of pseudomembrane • Systemic • Toxemia as toxin is absorbed from throat and carried by blood to target organs • Heart and nervous system

8. Mechanism of Action of diphtheriae toxin • A subunit… • Enzyme which ADP ribosylates EF-2 • EF-2 is needed for protein synthesis • ADP ribosylation inactivates EF-2 • One is sufficient to inactivate all EF-2 • Halts protein synthesis and kills the cell

9. Vibrio cholerae • Vibrio cholerae • Produces A + 5B exotoxin • Gram negative vibrio • Unusual disease • Cholerae does not invade tissue • Cholerae does not damage tissue • Lives in estuaries on copepods • Humans are incidentally infected when ingesting contaminated food or water

10. Symptoms of Vibrio cholerae • Symptoms • Secretory or watery diarrhea • No blood in diarrhea • Large watery bowel movements • Loss of electrolytes • Muscle cramps • Low blood pressure • Rapid heart rate & feeble pulse • Vomiting • White blood cell count usually normal • Treatment • Usually self limiting symptoms as long as IV fluids are administered with oral rehydration solutions

11. Mechanism of Action of cholerae toxin • Normally • Adenylate cyclase (AC) enzyme which makes cAMP • Epithelial cells secrete digestive fluid (HCO3-) in response to small increases in cAMP levels • cholerae exotoxin • Over activation of AC • ADP ribosylation of AC • Causes 100X increase in cAMP • Causes huge amounts of water and Cl- to leave via channels

12. Bacillus anthracis • Bacillus anthracis • Produces 2A + B exotoxin • Gram positive spore forming bacteria • Found in soil • Anthrax disease – direct exposure to spores • Inhalation – pulmonary • Ingestion – gastrointestinal • Invasion into surface wound – cutaneous • No cases involve person to person spread

13. Symptoms of Bacillus anthracis • Cutaneous • Spores enter abrasions or cut in skin • Germination of spore causes local ulceration of the skin • Painless black eschar with edema • Antibiotics prevent invasion into blood stream • Usually heals completely without scarring

14. Symptoms of Bacillus anthracis • Pulmonary • Life cycle • Macrophages engulf spores • Travel to nodes • Spores germinate en route • Cells are released spreading toxins and vegetative cells into the blood stream • Symptoms – caused by toxins • Fever and chills • Shortness of breath, & cough • Massive pleural effusions • Sepsis, shock & death

15. Mechanism of Action of anthracis Toxins • Two primary toxins & capsule gene • All three genes are located on plasmids • Edema Factor A – toxin • Adenylyl cyclase enzyme – increase in cAMP • Causes edema and pro-inflammatory response • Lethal Factor A – toxin • Metalloprotease • Cleaves MAP kinase required from cell division and signaling • Causes an overall suppression of immune system

16. Mechanism of Action of anthracis Toxins B. Anthracis EF LF B EDEMA Increased expression of pro-inflammatory mediators LF B EF B Endosome Acidic Environment EF cAMP B MAPK LF IMMUNE SUPPRESSION WBCs do not divide in the presence of pathogens; overall decrease in phagocytosis

17. Normal Signaling Pathway Receptor Adenylate Cyclase GTP cAMP GDP P PKA Catalytic Subunit PKA Regulatory Subunit Leads to a temporary increase in pro-inflamatory proteins and an overall activation of the immune system. Nucleus

18. Mechanism of Action of Edema Toxin Cell Membrane – toxin has already entered through RME EF Over expression of cAMP by edema factor A PKA Catalytic Subunit cAMP P Results in overall systemic edema Leads to an abnormal increase in pro-inflamatory proteins, increased capillary permeability & decrease in phagocytosis Nucleus

19. Intracellular Signaling Pathways • Terms • Adenylate Cyclase (AC) • Membrane enzyme which makes cAMP • cAMP • Inracellular second messenger • Binds to the regulatory subunit of PKA and releases an active catalytic subunit • G – proteins • A group of proteins which activates AC • When GDP is bound not active • When GTP is bound is active – short lived • GTP is self hydrolyzed by the G protein back to GDP • This ensures that the levels of cAMP are tightly regulated • Exchange of GDP to GTP requires hormonal activation

20. Intracellular Signaling Pathways • Terms • PKA – protein kinase A • Enzyme which phosphorylates (P) other proteins • Phosphorylation activates some proteins • Phosphorylation inactivates other proteins • Transcription Factor • DNA binding protein which binds to genes to increase or decrease the amount of gene expression • In some cases phosphorylation (P) is necessary for the transcription factor to bind DNA

21. Mechanism of Action of Lethal Toxin Cell Membrane – toxin has already entered through RME LF Protease Needed for cell division of WBCs during an immune response MAP Kinase Needed for expression of TNF – α Tumor necrosis factor which increases chemotaxis and phagocytosis Due to LF protease activity MAP kinase is not active and normal cellular responses are lost Degraded MAP kinase Protein

22. Clostridium botulium • Clostridium botulium • Produces AB exotoxin • Produces irreversible muscle relaxation • Flaccid paralysis • Symptoms result entirely from toxin • Anaerobic gram + rod • Usually ingested in contaminated food • Does not involve fever or sepsis • Patients die of paralysis and respiratory failure

23. Normal Neuronal Signaling

24. Mechanism of Action of botulinum toxin

25. Clostridium tetanus • Clostridium tetanus • Produces AB exotoxin • Produces irreversible muscle contraction • Spastic paralysis • Symptoms result entirely from toxin • Anaerobic gram + spore forming rod • Lives in soil usually on rusty metal • Enters from puncture wound or cut • Organism does not spread form entry point • Begins with stiff back and neck muscles • Death results from respiratory failure

26. Mechanism of Action of tetanus toxin • Mechanistically • Blocks release of inhibitory neurotransmitters • GABA – γ aminobutyric acid • Protease prevents release of synaptic vesicles • Acts on a different set of neurons • Results in exactly the opposite symptoms as botulinum toxin

27. Bacterial Endotoxins • Endotoxins • Misleading name • Toxin is not internalized • Toxin is located on outside of microorganisms • LPS of gram – bacteria • Lipoteichoic acid or gram + bacteria • Only toxic at high levels • Results in the activation of innate immune system • Endotoxin has different pharmacological effects when delivered at low or high levels

28. Mechanism of Action of Endotoxins • Endotoxins bind to • Receptors on • Macrophages • Neutrophils • Lymphocytes • Proteins of complement • Complement is a group of proteins which circulate at constant levels in the blood • When activated complement is a powerful tool against invading pathogens • Increased inflamation, opsonization, & MAC

29. Bacterial Endotoxins • Endotoxins • Host cell receptors (TLR) bind to components of pathogen • Pathogen associated molecular patterns PAMPS • LPS – gram - cell walls • Flagella • Lipoteichoic acid – gram + cell walls • Signal transduction pathways begin to illicit a cellular response • Production of cytokines

30. Inflammation Opsonization MAC

32. Bacterial Exoenzymes • Enzymes secreted by bacterial cells into the extra cellular matrix of host • Membrane Damaging Toxins • Enzyme destruction of host cell membranes • Lyse red blood cells • Membrane pore forming complex • Enzymes which act in the extra cellular matrix • Spreading factors • Breaks down connective tissue • Attacks blood clots • Thins pus filled with DNA • Enzymes which subvert drug therapy in patients • Penicillinase

33. αtoxin Pore forming toxin Common in Staphylococcus aureus Hemolysins Destroy red blood cells Streptolysins – group of hemolysins excreted by Streptococcus Streptokinase Attacks fibrin clots From Streptococcus pyogenes Hyaluronidase Breaks down hyaluronic acids in connective tissue Similar function for Collagenase Elastases DNase DNA is viscous Thins pus (DNA & debris) released from WBC Some Common Exoenzymes

34. Clostridium perfringens • Clostridium perfringens • Ananerobic gram + spore forming rod • Widely distributed in nature • Myonecrosis • Entry of spores by traumatic injury • Not highly invasive so it requires exoenzymes for a supportive growth environment • Exoenzymes • Lecithinase lipase c – major toxin • Lyses mammalian cells indiscriminately • Substrate is phophatidylcholine • Collagenase & hyaluronidase • DNAase