Lecture 3. Anaerobiosis Flashcards
What does Clostridium mean?
Bunch of organisms that can be pathogenic or non-pathogenic
Why is oxygen important?
Oxygen is a universal component of cells and is always provided in large amounts by H₂O. However, prokaryotes display a wide range of responses to molecular oxygen O₂
What are obligate aerobes?
Require O₂ for growth; they use O₂ as a final electron acceptor in aerobic respiration
What are obligate anaerobes (occasionally called aerophobes)?
Do not need or use O₂ as a nutrient. In fact, O₂ is a toxic substance, which either kills or inhibits their growth. Obligately anaerobic prokaryotes may live by fermentation, anaerobic respiration, bacterial photosynthesis, or the process of methanogenesis
What are facultative anaerobes (or facultative aerobes)?
Organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions (no O₂) they grow by fermentation or anaerobic respiration, but in the presence of O₂ they switch to aerobic respiration
What are aerotolerant anaerobes?
Bacteria with an exclusively anaerobic (fermentative) type of metabolism but they are insensitive to the presence of O₂. They live by fermentation alone whether or not O₂ is present in their environment.
What are microaerophiles?
Grow in aerobic conditions, but only when O₂ levels are low (below 0.2 atm)
What determines an organisms response to oxygen in its environment?
Depends upon the occurrence and distribution of various enzymes which react with O₂ and various oxygen radicals that are invariably generated by cells in the presence of O₂
What do all cells contain?
All cells contain enzymes capable of reacting with O₂ e.g. oxidation of flavoproteins by O₂ invariably results in the formation of H₂O₂ (peroxide) as one major product and small quantities of an even more toxic free radical, superoxide or O₂⁻
When do chlorophyll and other pigments in cells react with O₂ and what does this generate?
In the presence of light
Generates a singlet oxygen, another radical form of oxygen which is a potent oxidising agent in biological systems
What prevents lethal accumulation of superoxide within aerobes and anaerobes?
The enzyme superoxide dismutase
What enzyme do nearly all organisms contain and what is this enzyme’s function?
Catalase. decomposes peroxide
How do certain aerotolerant bacteria (such as the lactic acid bacteria that do not possess catalase) decompose peroxide?
By means of peroxidase enzymes which derive electrons from NADH₂ to reduce peroxide to H₂O
Because obligate anaerobes lack superoxide dismutase, catalase and/or peroxidase, what do obligate anaerobes undergo?
Lethal oxidations by various oxygen radicals when they are exposed to O₂
What are carotenoid pigments?
Found in all photosynthetic (and some non-photosynthetic) organisms
Protect organisms from lethal oxidations of singlet oxygen through a physical reaction that lowers the singlet oxygen radical to its non-toxic “ground” (triplet) state
Carotenoids are said to “quench” singlet oxygen radicals
What is the chemical equation of superoxide dismutase activity?
[O₂⁻] + [O₂⁻] + 2H⁺ → O₂ + H₂O₂
What is the chemical equation of catalase activity?
O₂ + H₂O₂ → 2H₂O + O₂
What is the chemical equation of peroxidase activity?
O₂ + H₂O₂ → 2H₂O
NADH + H⁺ → NAD⁺
What do most Clostridia lack?
Respiratory chain cytochromes, catalase, peroxidases and superoxide dismutase
How do Clostridia obtain ATP?
Only by substrate-level phosphorylation (in which high energy phosphate bonds from organic intermediates are transferred to ADP)
What do a number of clostridia ferment?
Ferment sugars, producing butyric acid (and also acetone and butanol)
Others ferment amino acids
What does substrate level fermentation generate in Clostridium?
The energy they need to survive
What are the properties of Clostridium botulinum?
Gram-positive, spore producing
Commonly found in soil smaples and aquatic sediments
What is C. botulinum the causative agent of?
Botulinum food poising
How potent is botulinum toxin?
Extremely potent (1mg of pure toxin can kill 1 million guinea pigs)
Clostridia can form spores = many processed foods are processed based on the need to protect consumers from botulinum outbreaks in food
How do botulinum outbreaks in food occur?
Poorly canned foods create an anaerobic environment. Unkilled spores germinate and produce toxin
How many exotoxin types are produced by the different strains of Clostridium?
7 (A, B, C₁, C₂, D, E, F, G)
What botulinum exotoxin types are encoded by lysogenic bacteriophage?
C and D
What does botulism result from?
From the ingestion of bacterially produced neurotoxins.
What protein exotoxin types are the most toxic to humans and how are they often released?
Types A, B, E and F
Often released in an inactive form; proteolytic cleavage activates them
What is the most potent exotoxin known to man?
Botulinum type A (10 ng can kill a normal adult)
How do the botulinum toxins work?
These toxins block the release of the neurotransmitter acetylcholine resulting in double vision, slurred speech, decreased saliva, difficult swallowing and general weakness.
What occurs in about 20% of those who have botulism food poisoning?
Flaccid paralysis with accompanying respiratory failure which can be fatal
When can botulism food poisoning be observed following ingestion of heat liable preformed toxin?
18-36 hours
How is botulinum toxin expressed?
Expressed as an inactive 150 kDa polypeptide comprising a 100kDa heavy chain (HC) and a 50 kDa light chain (LC) linked through a disulphide bridge
What does the heavy chain of botulinum toxin bind to?
Binds to the presynaptic receptor; toxin enters the cell & the disulphide bond is cleaved
What happens when the disulphide bond in the botulinum toxin is cleaved?
Cleavage liberates the light chin into the cytoplasm and endosomal compartment
What does the light chain of botulinum toxin act as?
Acts as a zinc endopeptidase, cleaving the synaptosomal-associated protein (SNAP), vesicle-associated membrane protein (VAMP) and syntaxin. This prevents fusion of acetylcholine vesicles at the cell membrane
What is Clostridium tetani the causative agent of?
Tetanus (lockjaw)
What does tetanus result from?
Trauma or a puncture wound leading to tissue contamination
What is tetanus caused by?
By the release of a single antigenic type of exotoxin by C. tetani
The tetanus exotoxin circulates in the blood, but what does it adhere to?
Neuronal receptors
Specifically, the toxin fixes to gangliosides thereby blocking the release of the neurotransmitters glycine and γ-amino butyric acid (GABA). Glycine normally prevents contraction of antagonistic muscles; therefore, muscle spasms and convulsions (lockjaw) may occur
What is tetanus toxin synthesised as?
As a 150kDa polypeptide chain (100kDa heavy chain (HC) required for cell entry and a 50kDa light chain (LC) which causes disease
What do the C-terminal domain and N-terminal domains of the heavy chain of the tetanus toxin to?
C-terminal domain binds to gangliosides
N-terminal domain allows the LC to cross into the cell cytoplasm
Once in the cytoplasm, what does the light chain of the tetanus toxin interrupt?
The release of neurotransmitters LC is a zinc metalloprotease which cleaves synaptobrevin 2, a SNARE protein involved in the fusion of neurotransmitter vesicles with the neuronal membrane
What does the cleaving of synaptobrevin 2 caused by the light chain of the tetanus toxin cause?
Vesicles containing GABA and glycine are not allowed to dock and hence no neurotransmitter can be released
How does a host die from a C. tetani infection?
Host dies from respiratory paralysis though the underlying infection with C. tetani is located elsewhere & plays only an indirect role in the outcome of disease.
What does the killing of a human host allow C. tetani to do?
To multiply as these anaerobic microbes can only grow in tissues that are not circulated with oxygenated blood
Which group of people are particularly susceptible to tetanus?
Heroin addicts
What are the properties of Clostridium difficile?
Gram positive, obligately anaerobic, spore former
What can cause C. difficile infections?
Antibiotic use (e.g. β-lactam antibiotics) reduces concentration of normal microbiota. C. difficile overgrows
What toxins are produced by C. difficile and what do these toxins cause?
Produces toxins A and B
Toxins cause diarrhoea & lesions on colon surface which coalesce forming extensive tissue damage – known as pseudomembranous colitis
Toxins A and B – large exotoxins that modify host cell membrane G proteins
What is the mode of action of C. difficile?
Alter actin cytoskeleton of mammalian cells
What mediates the mode of action of C. difficile?
Mediated by toxins glucosylating G-proteins
Glucosyl group (from UDP-glucose) added to specific threonine residue on the G protein
