Bacterial Toxin Lecture PDF

 Bacterial Toxins Glen Carter [email protected] Department of Microbiology & Immunology Peter Doherty Institute for Infection and Immunity Learning Objectives At the completion of this lecture students should be able to: Explain what bacterial toxi...

 Bacterial Toxins Glen Carter [email protected] Department of Microbiology & Immunology Peter Doherty Institute for Infection and Immunity Learning Objectives At the completion of this lecture students should be able to: Explain what bacterial toxins are and how they can be classified. Use examples to describe how A-B toxins function. Understand that not all toxins are proteinaceous. Use examples to describe the beneficial uses of toxins in medicine. Definition Bacterial toxins are toxic substances that are produced and released by bacteria to target other bacterial or host cells. Major Toxin Types Endotoxin Lipopolysaccharides associated with the cell wall of Gram-negative bacteria. Exotoxin: Mostly proteins and secreted by pathogenic bacteria. Effector Proteins Injected into host cell via specialised secretion systems (T3SS). Endotoxin Integral part of Gram-negative bacterial cell wall. Mainly released when bacteria are lysed. Small amounts released as part of normal metabolism. Consists of three parts: O antigen – immunogenic. Core. Lipid A – toxicity (endotoxin). LPS Structure www.sigmaaldrich.com/technical- documents/articles/biology/what-is-endotoxin.html Endotoxin cont… Lipid A responsible for toxicity. TLR4 is LPS receptor. Pyrogenic. Less potent than exotoxins. Fatal in large doses. Can not be made into a toxoid vaccine. Can lead to fever, diarrhoea and septic shock. LPS Structure www.sigmaaldrich.com/technical- documents/articles/biology/what-is-endotoxin.html Mechanism of Endotoxin Endotoxin Macrophage Complement Factor XII IL-1 TNF NO C3a C5a Coagulation Cascade Hypotension Neutrophil Disseminated Fever Fever & Hypotension & Edema Chemotaxis Intravascular hypotension INFLAMMATION Coagulation Effects of Endotoxin Fever. caused by production of IL-1 by macrophages. Leukopenia followed by leukocytosis. Hypotension. early marker of Gram negative bacteraemia. Septic Shock. Activation of complement alternative pathway leads to inflammatory response. Disseminated Intravascular Coagulation (DIC) can lead to organ failure. Death https://www.verywellhealth.com/sepsis-and- septic-shock-diagnosis-and-treatment-3156827 Septic Shock or DIC. Exotoxins Produced by Gram-positive and Gram-negative bacteria. Important bacterial virulence factors. Avirulent strains have often lost the ability to make toxins. Highly potent. Botulinum toxin lethal dose in humans 1-2 ng by iv route. Generally released/ secreted from the bacterial cell. Most have a specific mode of action. Exotoxins cont… Exotoxins resemble enzymes: Most often proteins although not always. Associated with high biological activity. Most act catalytically. Display specific activities. Can be classified according to activity. Exotoxin Classification According to their host cell tropism: Cytotoxin, haemolysin, leukotoxin. According to their site of action: Neurotoxin, cardiotoxin, enterotoxin. According to the disease/bacteria: Diptheria toxin, anthrax toxin, TSST. According to the enzymatic activity of the toxin: Mono glucosyltransferase, ADP-ribosyltransferase. Exotoxin Classification cont… According to how toxins interact with host cells: Bind to cell membrane and enter cell without damaging membrane: AB toxins. Bind to cell membrane and cause damage: pore forming toxins and phospholipase. Bind to cell membrane but do not enter the cell: super antigens (TSST). Pore Forming Toxins Peraro & van der Goot Nature Reviews Microbiology 14 77-92 (2016) Examples include: Streptolysin O and Perfringolysin. Phospholipase Examples include: phospholipase C (PLC) (α-, β- and γ-toxins of C. perfringens and some other haemolysins). phospholipase D (murine toxin of Y. pestis). A-B Toxins Although exotoxins are structurally and functionally very different, many consist of two components. One component (subunit A) is responsible for “Activity” One component (subunit B) is responsible for “Binding” Generally, in A-B toxins, the “A” subunit is not active until it is “released” into the cell by subunit “B”. A-B toxins can be single chained or multi-chained. Attachment & Entry of A-B Toxins Receptor mediated endocytosis – binding of “B” subunit induces endocytosis and endosome formation. The “A” subunit translocates from endosomes into cytosol and inhibits cellular target. Image credit: Jeongmin Song, in Advances in Microbial Physiology, 2022 Examples of A-B Toxins Toxin Organization Activity Diphtheria toxin AB ADP-ribosylation Exotoxin A AB ADP-ribosylation Botulinum toxin AB Zn2+ protease Tetanus toxin AB Zn2+ protease Cholera toxin AB5 ADP-ribosylation Shiga toxin AB5 Cleaves 23S rNA Anthrax toxin LF A-B Zn2+ protease Anthrax toxin EF A-B Adenylate cyclase CDT binary toxin A-B ADP-ribosylation Diphtheria Toxin (DT) Produce by Corynebacterium diphtheria. Causative agent of diphtheria. Encoded by a prophage. First identified toxin: https://www.verywellhealth.com/overview-of-diphtheria-4175810 Discovered in 1888 by Émile Roux and Alexandre Yersin. Lethal dose in humans is 100 ng/kg. Causes inflammation of the heart muscle and nerves. Diphtheria Toxin www.researchgate.net/figure/Structural-properties-of-diphtheria-toxin-DT-DT-is-organized-as-an- AB-toxin-PDB_fig1_23252758 DT is a single-chain AB toxin. ADP-ribosyl transferase. Ribosylates EF2. www.wwnorton.com/college/biology/microbiology2/ch/25/etopics.aspx ADP-Ribosyltransferases ADP-ribosyltransferase is a common activity of exotoxins. Transfer of an ADP-ribose moiety from NAD+ to target proteins of intoxicated eukaryotic cells. Target + NAD+ Target-ADP-ribose + Nicotinamide + H+ ADP-Ribosylation inhibits function of target protein. Examples of ADP-Ribosyltransferases ADP-ribosyltransferase Toxin Effect Diphtheria toxin Inhibits protein synthesis Pseudomonas exotoxin A Inhibits protein synthesis Cholera toxin Activates adenylate cyclase TEC heat-labile toxin Activates adenylate cyclase Pertussis toxin Activates adenylate cyclase C. botulinum C2 toxin Inhibits actin polymerisation S. aureus EDIN toxin Disassembles Golgi CDT binary toxin Inhibits actin polymerisation Anthrax Toxin Produced by Bacillus anthracis. Causative agent of Anthrax. Encoded by a plasmid. B. Anthracis spores - image credit: CDC/Janice Haney Carr Four types depending on route of infection Skin – small blister. Inhalation – fever, chest pain, death. Intestinal – nausea, vomiting, diarrhea, death. Injection – fever, abscess. Image Credit: Kateryna Kon/Shuttestock.com Anthrax Toxin Anthrax toxin is a multi-chain A-B exotoxin. Anthrax toxin is made up of three proteins: Protective Antigen (PA). Edema Factor (EF) – adenylate cyclase. Lethal Factor (LF) – Zn2+ dependent endopeptidase. PA is “B” component, EF & LF both “A” components. www.semanticscholar.org/paper/Anthrax-toxin%3A-pathologic- effects-on-the-system.-Golden- Watson/151284362fd339456c9fe8decd614aa3009d9cdf/figure/0 Anthrax Toxin https://oncohemakey.com/anthrax-and-other-bacillus-species/ EF converts cellular ATP to cAMP – increase in fluid LF inactivates MAPKK leading to apoptosis and death. Not all Exotoxins are Proteins For many years exotoxins were considered to be just proteins. Recently several non-protein toxins identified. Natural products/ secondary metabolites Mycolactone Tilivallin Produced by PKS and NRPS. Mycolactone Produced by Mycobacterium ulcerans. Causative agent of Buruli ulcer. Mycolactone is encoded on aplasmid pMUM001 Mycolactone is immunosuppressive and cytotoxic. Mycolactone cont… 12-membered macrolide with an ester linked polyketide tail. There are five different Mycolactones. Plasmid encoded. Synthesised by the mlsA1, mlsA2 & mlsB polyketide synthase genes. https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-9-462 Tilivalline Tilivalline is a Pyrrolobenzodiazepine. Enterotoxin. Produced by cytotoxic strains of K. oxytoca. Non-toxigenic strains are avirulent. Causes antibiotic associated haemorrhagic colitis. https://www.cib.csic.es/news/research/revealed-molecular- mechanism-colitis-caused-intestinal-bacteria-klebsiella-oxytoca How Do We Know Exotoxins are Important in Disease? Toxic activity against cell lines. Eg Vero or HT29 cells. Virulence in animal models. mouse, chicken etc. Activity of purified toxins. Can be misleading!!! Toxin neutralisation. toxin specific antibodies. Isogenic mutants. only definitive proof (TcdA vs TcdB) Toxin-Mediated Disease Toxins can be are entirely responsible for symptoms of disease: TcdA & TcdB - Clostridium difficile infection (CDI). BoNT – Botulism. TeNT – Tetanus. DT – Diphtheria. ETA & ETB -Staphylococcal scalded skin syndrome. Can also play a supplementary role: PFO – Myonecrosis. Beneficial Uses of Toxins To make toxoid vaccine: Diphtheria & Tetanus. To directly treat certain medical conditions: hyperhidrosis, reduce muscle spasms. To treat cancer: Immunotoxins. To clear blocked arteries after heart attack: streptokinase. Used extensively in cell biology. QUESTIONS???