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Lecture 4

Lecture 4 - Bacterial TOXINS

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University of Guelph
MICR 4010
Rob Foster

BACTERIAL TOXINS What are bacterial toxins? - A diverse collection of proteins responsible for many of the symptoms caused by pathogens during the course of infection. - Today we’ll look specifically at exotoxins - Exotoxins are actively exported by the bacteria or released upon cell lysis. Endotoxin - Part of the outer membrane of the cell wall of Gram negative bacteria  Lipopolysaccharide (LPS) – the toxicity is associated with the lipid A component - Toxic whether the bacterium is pathogenic or not - Generally only a minute amount of LPS is released when bacterium is growing (not enough to cause damage if you are taking antibiotics) - Lots produced when the bacteria are lysed e.g. by the action of phagocytosis - Mechanism of toxicity is complex - Low degree of specificity Where do toxins come from? - In many bacteria, the toxin genes are ‘foreign’ genes – i.e. part of the genome through integration of a lysogenic phage or acquisition of a plasmid - Maybe toxins have important roles in phage or plasmid biology - Impact on the human body may be accidental Exotoxins – classification Some bacterial toxins may fall into more than one category. Classification of toxins is a bit like herding cats! Nevertheless, newly discovered toxins are assigned a category  comparing new toxins to toxins whose mechanisms of action have been elucidated can be very useful (because you may elucidate their mechanisms and pathways as well). Pore-formers & hydrolyzers  Cause direct damage to the extracellular matrix or the plasma membrane  May form a pore – e.g. listeriolysin O  May be hydrolyzers – e.g. streptokinase (S. pyogenes); plasminogen -> plasmin Protein synthesis inhibitors  Can bind to ribosomal RNA or elongation factors (EFs)  Bind to chosen substrate and inhibit protein synthesis  Arrest of protein synthesis eventually leads to host cell death  E.g. Diphteria toxin; Shiga toxin; Pseudomonas exotoxin A Second messenger pathway activators  These are toxins that target and alter cellular functions without directly killing the affected cell  Bind key proteins involved in signal transduction pathways  Cause dramatic alterations to cell structure and function  E.g. B. anthracis edema factor; C. Botulinum toxin; Pertussis toxin; Cholera toxin. Immune response activators  These are the ‘superantigens’  Act directly on T-cells and antigen-presenting cells  Stimulate cytokine response, pyrogenicity (produces fever), and enhance endotoxic shock.  E.g. toxic shock syndrome toxin Protease toxins  Bind to specific protein targets and cleave them  Often are metalloproteases (require a metal ion for full activity, usually zinc)  Proteolysis inhibits cell function  Best examples are BoNT and TeNT Bacterial effectors  Secreted directly into the host cell by type 3, 4 (and maybe 6) secretion subvert host cell processes  Secreted effectors represent a further class of toxins Cytholethal distending toxin (CDT)  It is in a class of its own  Produced by e.g. C. jejuni, E. coli, S. enterica, Typhi, S. dysenteriae, H. pylori, Haemophilus ducreyi and Actinobacillus actinomycetemcomitans  CDT A, B and C subunits  B is the active subunit (holotoxin)  On delivery, CdtB is taken up by the cell nucleus via active transport, where it damages chromatin (DNAse I – like activity)  DNA damage leads to cell-cycle arrest, cell distension and eventually cell death. Toxins: friends or foes? Toxins can be bad news during infection, but science has taken advantage of their mechanisms of action: - Cell biologists love toxins as they allow the study of cellular processes - Can form part of ‘magic bullet’ therapies for e.g. cancer treatment - BoTox - Streptokinase treatment for heart attack and stroke victims (dissolve clots in the arteries of the heart wall) Vaccination using toxins - Usually partially purified toxin preparations obtained from culture supernatants - The toxin activity is ablated (change a couple of epitopes but it is still recognized by the antibodies) – e.g. using formaldehyde to form a toxoid (a bacterial toxin (usually an exotoxin) whose toxicity has been inactivated or suppressed either by chemical (formalin) or heat treatment, while other properties, typically immunogenicity, are maintained). - Next generation of toxoid vaccines include: o Purified, inactivated toxoids o Live attenuated strains that produce a genetically engineered toxoid o Live attenuated unrelated bacterial vector strains that are engineered to express the target toxoid. The pathogenic Clostridia - Clostridia are Gram positive, anaerobic, spore-forming bacteria found in soil or the digestive tract of animals. - Notable as producers of highly potent toxins -> BoNT is most potent toxin known to man. Sporulation - Clostridia and Bacillus produce endospores as a way to survive hostile or unfavorable environmental conditions - Sporulation is an interruption of the normal cell cycle -> it is not a necessity - A complex series of events takes place to enable sporulation to take place (very energetically unfavorable) - Bacterial sporulation is not a virulence determinant per se, but it contributes to disease prevalence (mainly for C. difficile) Stages of sporulation 1. Cell is happy, plenty of nutrients, actively growing 2. Then it gets a signal from the environment -> asymmetric cell division. Cell is stressed and sets sporulation cascade in motion 3. Prespore is engulfed by mother cell 4. A cortex forms around the prespore (this is where most of the genes takes place, because the cortex is very complex) 5. Spore is given a coat, and mother cell disintegrates Spores in the soil: the truth about rusty nails - Spores pass through out digestive tracts often, but do not germinate, and therefore do no harm - Danger: deep puncture wounds (C. tetani); poorly canned foods (C. botulinum). Under these anaerobic conditions bacteria will germinate and produce toxins. The disease: tetanus • Although fairly rare nowadays, tetanus is a greatly feared disease – by the time you got th symptoms, it is too late • Major (only) virulence determinant of C. tetani is TeNT • Causes spastic paralysis; painful spasms of the facial, neck, arm and abdominal muscles • Spasms can be severe enough to break bones • Disease is unusually fatal, depends on speed of treatment, dose, etc. Therapy for tetanus - Anti-toxin: to mop up any remaining circulating toxin - Antibiotics: in case any bacteria are left at the site of infection (it takes a while for the bacteria to grow and for the toxins to find their binding sites at the body) - Supportive care: aimed at ensuring adequate hydration and nutrition (lockjaw) - Prevention is the best therapy! TeNT in medicine - Glial cell-line derived neurotrophic factor (GCNF) is recognized to promote survival of CNS motor neurons -> potential therapeutic effects have been realized e.g. ALS - The problem is how to get the GCNF to the motor neurons in the brain and spinal cord - Answer: engineer GCNF bound to the non-toxic fragment C of TeNT (which is known to have a very specific actions in particular neurons). Clostridium botulinums • 7 toxins types (A, B, C, D, E, F & G) -> same effects, serologically different. • Most cases of human botulism are caused by: o C. botulinum & toxins A – most severe, B & E o C. baratii & toxin F o C. butyricum & toxin E C. botulinum toxins - It actually produces 3 distinct toxins: o BoNT: causes the major symptoms of botulism o C2 and C3 toxins: responsible for ADP-ribosylation activities; far less potent than BoNT. Types of botulism - Foodborne botulism – most common type, a public health emergency (because if one person is recognized to have botulism, chances are high that another bunch of people have the same disease, and it is very hard to isolate the source) - Wound botulism – it is a little bit like tetanus - Infant botulism - Adult infectious botulism - Botulinum toxin can also be aerosolized and absorbed through the lungs – potential biological weapon Clinical features of botulism • Usually caused by ingesting home-canned products – spores survive canning process and germinate in anaerobic environment • Symptoms start 6h – 2 weeks after ingestion o Initial symptoms: nausea, vomiting, abdominal cramps, diarrhea o Later: neurologic symptoms include constipation, dry mouth, blurred vision, confusion o Paralysis starts at the head and ends at the feet – characteristic for BoNT intoxication Treatment of botulism - Antitoxin: to mop up any remaining circulating toxin - Supportive therapy - In severe cases assisted ventilation required -> need to wait for new presynaptic end plates and neuromuscular junctions to be generated BoNT as a chemical weapon Lethal doses are extremely low. 100g of BoNT enough to kill the entire human race. BoNT in medicine - Used to help spasticity – injection in specific muscles to help release them - Can be used to help reduce excess sweating - Can be used to treat chronic migraines (change the way the muscles control the vessels and the way they influence the pressure in the brain) - BoNT is also being investigated as a carrier of medicines from the GI tract to the general circulation TeNT and BoNT: close relatives • Many similarities: common origin, closely related structure, similar mechanisms of action • Both toxins are zinc-requiring endopeptidases that cleave synaptobrevins (one of the SNARE proteins) -> found in synaptic vesicles of neurons, responsible for neurotransmitter release. Molecular structure 1. Blue = light chain = a zinc endopeptidase specific for SNARE proteins 2. Green = heavy chain = responsible for membrane translocation 3. Orange & red = heavy chains = responsible for neurospecific binding and retrograde transport I. Normal neurotransmitter release+ SNARE proteins form a complex that allows the vesicle to fuse with the membrane and release its content. Result = muscle fiber contracts II. Exposure to botulinum toxin Highly specific neurotoxins cleave the SNARE proteins, preventing the formation of SNARE complex so that the vesicle cannot fuse with the synaptic membrane and release neurotransmitters. If acetylcholine can’t be released, the contraction of the muscle doesn’t occur. Result = muscle fiber paralyzed. Flaccid (BoNT) vs. spastic (TeNT) paralysis - BoNT and TeNT have different cell specificities o BoNT targets peripheral neurons – if TeNT is administered peripherally it too causes flaccid paralysis.  It affects neurotransmitter release o TeNT finds its way to the CNS  It has additional effects on neuroactive compounds (an area of ongoing research) C. perfringens This species describes a group of strains that together produce 12 different toxins (usually a strain doesn’t produce all of them). - 4 lethal toxins - Hemolysins - Proteases - Neuroaminidase - Enterotoxin Gram variable, aerotolerant anaerobe, non-motile, widely distributed in soil and GI tracts of animals. C. perfringens: gas gangrene - An extreme and rare form of wound infection - Causes an expanding zone of dead tissue: gas is the waste product of the bacteria from digestion of fl
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