Bacterial toxins Flashcards
What is the sphere of influence?
The area in a host which is affected by a toxin.
- Some act locally killing cells nearby.
- Some help the pathogen to spread in host tissue, i.e. degarding connective tissue.
- Some disseminate far from the site of synthesis, e.g. diptheria toxin is made in the throat, but acts on the heart and brain.
Toxins are commonly found on mobile genetic elements. Name two elements and examples of each.
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Phage
- Diptheria toxin
- Botulism toxin
- Scarlet fever toxin
- Toxic streptococci (“flesh-eating” haemolysins)
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Plasmids
- E. coli toxin (diarrhoea)
- S. aureus toxin (“scalded-skin syndrome”)
- E. coli 0157:H7 (severe GI infection)
What is the difference in release of exotoxins and endotoxins?
Exotoxins are actively released from a bacterium, whereas endotoxins are part of the cell wall and are released when the microbe dies.
Outline exotoxins.
Proteins are released extracellularly as the microbe grows, and may travel from the site of infection to other parts of the body, e.g. diptheria, tetanus. They mainly fall under three categories:
- Cytolytic toxins
- A-B toxins
- Superantigen toxins
Outline endotoxins.
Endotoxins (lipopolysaccaride) are mostly released from gram-negative bacteria when the cells die, causing fever, inflammation, and in severe cases septic shock.
They have three components:
- Lipid A
- Core polysaccharide
- O side chains (oligosaccharides)
Outline cytolytic toxins. Give an example.
Cytolytic toxins act on cell membranes and have a cytolytic effect. Examples include haemolysins, leukocydins, phospholipases, lecithinases, and other pore-forming toxins.
Outline superantigen toxins. Give an example.
Superantigen toxins have a structure that is similar to cytokines, and are able to stimulate large numbers of immune cells (lymphocytes) and induce a cytokine storm. This causes fever, systemic toxicitiy, and immune suppression. An example is Staphylococcus aureus TSST (toxic shock syndrome toxin).
Name some examples of diseases caused by A-B toxins and the microbe that causes them.
- Diptheria (Corynebacterium diptheriae): toxin inhibits protein synthesis in heart muscle and other cells.
- Tetanus (Clostridium tetani): toxin affects neuromuscular junctions leading to a constant release of acetlycholine, which leads to irreversible contraction of muscles and spastic paralysis.
- Botulism (Clostridium botulinum): toxin affects neuromuscular junctions, preventing the release of acetylcholine leading to a lack of stimulus to muscles, causing flaccid paralysis.
- Cholera (Vibrio cholerae): toxin activates adenyl cyclase in intestinal cells, leading to the disruption of soidium ion influx and loss of water to the lumen, and diarrhoea.
Outline the structure and method of action of A-B toxins.
Bacterial toxins often act distanced from the site of infection and are physically organized into distinct domains that recognize receptors on the surface of sensitive cells and possess enzymatic capacity to modulate the action of an intracellular host target, often a protein, termed A–B structure-function organization.
- The A domain, also described as an effector, is usually an enzyme or a factor that functions through protein-protein interactions within the cell.
- The B domain comprises the receptor-binding function, providing tropism to specific cell types through receptor binding capacity. The B domain also includes a domain that translocates the A domain across a lipid bilayer, either at the plasma membrane or within the endosomal compartment. Translocation of the A domain across the lipid bilayer is hypothesized in most cases to occur through a pore/channel formed by the B domain. The B domain can be a single subunit (B) or an oligomeric (B5) form.
The A and B domains may be linked by a disulfide bond or associated by non-covalent interactions. Diptheria toxin is AB, cholera toxin is A+5B, and anthrax toxin is 2A+B.
What are some common intracellular signalling pathway targets of toxins?
These proteins interfere with important pathways and the important enzymes that are involved, inhibiting, inactivating, or hyperactivating them.
- Adenylate cyclase (AC): a common toxin target, a membrane enzyme that makes cAMP.
- cAMP: an intracellular second messenger that bind various cellular components, including the regulatory subunit of protein kinase A (PKA). Toxins can affect the phosphorylating activity of PKA.
- G-proteins: a group of proteins that activate AC. Inactive when bound to GDP, active when bound to GTP. Tightly regulates cAMP, GDP/GTP exchange is under hormonal regulation.
- PKA: an enzyme that phosphorylates other proteins, which can inactivate or activate them.
- Transcription factors: DNA binding proteins that increase or increase gene expression, in some cases phosphorylation is needed.
Outline the diptheria toxin.
Corynebacterium diptheriae is a gram-positive rod (‘comma’) that produces an AB toxin. The B portion is used in vaccines against the disease, stimulating antibodies against the toxin.
It causes upper respiratory tract infection in children, forming a pseudo-membrane in the throat. Systemic infection can arise if left untreated, affecting the heart and nervous system.
The A portion of the toxin interferes with protein synthesis:
- EF-2 is required for protein synthesis, and the A portion (enzyme) attaches ADP to EF-2, inactivating the elongation factor.
- This means EF-2 cannot be involved in polypeptide chain synthesis, so elongation does not occur and protein synthesis is deactivated causing problems for the cell.
Outline the cholera toxin.
Vibrio cholerae is a gram-negative vibrio bacterium that produces an A+5B exotoxin (CTx). No tissue invasion occurs during infection, and infection occurs through ingesting contaminated food or water. Infection causes watery diarrhoea, low blood pressure and vommiting.
The A-subunit of bacterial AB5 toxins is a single polypeptide composed of two domains (A1 and A2) that are also linked together via a disulfide bond. The A1 domain comprises the catalytic domain responsible for the toxicity to the host cell. The A2 domain consists of an α-helix that penetrates into the central pore of the pentameric B-subunit, thereby non-covalently anchoring the A- and B-subunits together to create the holotoxin.
In order to reach their molecular targets in the cytosol, the Ctx toxin recognizes its cognate cell surface receptor via the pentameric B-subunit; this recognition event triggers endocytosis, followed by retrograde transport via the Golgi to the ER. In the ER, protein disulfide isomerase (PDI) unfolds the A-subunit (A1), which is then retro-translocated into the cytosol via the Sec61 channel 20, or via the derlin-1–Hrd1 complex. It then undergoes refolding and finally causes cellular toxicity.
Normally, adenylate cyclase (AC) makes cAMP and epithelial cells secrete digestive fluid (HCO3-) in response to small increases in cAMP levels. The CTx toxin overactivates AC via ADP ribosylation of AC, leading to a 100x increase in cAMP. This causes huge amounts of water and Cl- to leave the cell via channels.
Outline the anthrax toxin and disease.
Bacillus anthracis, a gram-positive spore-forming bacterium, is the etiologic agent of anthrax. It procudes a 2A+B exotoxin that can cause different forms of disease depending upon the route of entry.
- In most cases of anthrax acquired by inhalation, the spores are thought to be transported from their site of deposition in the lungs to regional lymph nodes (via macrophages) where they germinate and outgrow into the vegetative bacilli . The bacilli multiply within the lymph nodes and are then released, rapidly spread systemically, and produce large amounts of the anthrax toxins.
- The large majority of reported anthrax cases are cutaneous infections. Although cutaneous infections can be fatal, they are more typically self-limited, with mortality rates in untreated cases of about 20%.
- Gastrointestinal infections in humans have been reported, but are considered rare.
Anthrax toxin consists of three nontoxic proteins that associate to form toxic complexes at the surface of mammalian cells. One of these proteins, protective antigen (PA), transports the other two, edema factor (EF) and lethal factor (LF), to the cytosol.
(1) PA binds to a receptor, ATR or CMG2; (2) cleavage by a furin protease removes PA20; (3) PA63 self-associates to form the heptameric prepore; (4) up to three molecules of EF and/or LF bind to the prepore; (5) the complex is endocytosed and trafficked to an acidic intracellular compartment; (6) under the influence of low pH the prepore converts to a pore, and EF and LF are translocated to the cytosol. There, EF catalyzes the formation of cAMP, and LF proteolytically inactivates MAPKKs (needed for cell division and signalling).
EF leads to oedema (temporarily increased expression of pro-inflammatory mediators) and LF leads to suppression of the immune system (WBC division inhibited).
Botulism results from intoxication by _________ __________ (BoNT) and is characterized by descending _______ paralysis as a result of inhibition of _______________ release at the neuromuscular junction. There are seven botulinum neurotoxin serotypes (A–G) produced by bacteria of the genus ___________, and one of the factors in recovery from botulism depends on the BoNT serotype involved. Together with _______ __________ (TeNT) produced by ____________ ______, the BoNTs make up the clostridial neurotoxin (CNT) family. TeNT exhibits a high degree of sequence and structural homology to the BoNTs, in particular to BoNT/B, and is the causative agent of tetanus, which is characterized by _______ paralysis.
Although differing in clinical manifestation, the fundamental mode of action – inhibition of neurotransmission – is common to all CNTs. Inhibition of neurotransmitter release by the CNTs is caused by the specific cleavage of a group of proteins integral to the exocytotic process, the ______ proteins (soluble NSF-attachment protein receptors). Cleavage of one or more of the ______ proteins leads to a block in the release of vesicular contents to the extracellular environment. Therefore, in the case of BoNT action on the motorneuron, release of ____________ is prevented.
Botulism results from intoxication by botulinum neurotoxin (BoNT) and is characterized by descending flaccid paralysis as a result of inhibition of acetylcholine release at the neuromuscular junction. There are seven botulinum neurotoxin serotypes (A–G) produced by bacteria of the genus Clostridium, and one of the factors in recovery from botulism depends on the BoNT serotype involved. Together with tetanus neurotoxin (TeNT) produced by Clostridium tetani, the BoNTs make up the clostridial neurotoxin (CNT) family. TeNT exhibits a high degree of sequence and structural homology to the BoNTs, in particular to BoNT/B, and is the causative agent of tetanus, which is characterized by spastic paralysis.
Although differing in clinical manifestation, the fundamental mode of action – inhibition of neurotransmission – is common to all CNTs. Inhibition of neurotransmitter release by the CNTs is caused by the specific cleavage of a group of proteins integral to the exocytotic process, the SNARE proteins (soluble NSF-attachment protein receptors). Cleavage of one or more of the SNARE proteins leads to a block in the release of vesicular contents to the extracellular environment. Therefore, in the case of BoNT action on the motorneuron, release of acetylcholine is prevented.
Outline the mechanism of action of botulinum toxin.
BoNT (an AB exotoxin) intoxication occurs through a multi-step process involving each of the toxin functional domains, and can be described as the outcome of three discrete stages.
- BoNTs bind to cholinergic nerve terminals by their HC domains and are subsequently internalized, possibly by receptor-mediated endocytosis. HC-C mediates the neurospecific binding of the toxin to the presynaptic membrane via two independent receptors: a polysialoganglioside (PSG) and the luminal domain of a synaptic vesicles (SV) membrane protein.
- After endocytosis is completed, the SV lumen and the lumen of synaptic endosomes with which SV may have fused is acidified by the proton pumping action of the v-ATPase present on the SV membrane. This pH gradient drives the uptake of neurotransmitters from the cytosol into SV and it is used by BoNTs to translocate their LC (catalytic domain) domain from the SV interior, or synaptic endosome interior, into the cytosol. This process is made possible by the HN domain, also known as translocation domain. Active LC is translocated into the cytosol, where it interacts with, and subsequently cleaves, SNAREs.
- SNARE proteins are involved in the fusion of synaptic vesicles with the plasma membrane and thus the action of BoNT-LC is to prevent exocytosis. At a more specific level, cleavage of SNARE proteins by BoNT inhibits the release of acetylcholine (ACh) at the neuromuscular junction, leading to inhibition of neurotransmission. Cleavage of individual SNARE proteins does not prevent SNARE complex formation, but results in a non-functional complex where the coupling between Ca2+ influx and fusion is disrupted
The neurotransmitter vesicle fusion with the membrane is inhibited leading to no release of neurotransmitter, causing flaccid paralysis.
Outline the tetanus toxin.
Tetanus neurotoxin (TeNT) is a protein toxin (AB) produced by Clostridium tetani that causes tetanus, a neuroparalytic disease. The only natural source of TeNT is C. tetani, an anaerobic gram-positive bacterium, which is commonly found in soil samples throughout the world in the form of spores which are resistant to heat and desiccation.
TeNT comprises two polypeptide chains: a H (heavy chain) and L (light chain) joined by a single interchain disulfide bond. The L chain is the N-terminal part of the toxins and it is folded around the active site zinc atom. The heavy chain is folded into three domains : i) an N-terminal termed HN (similar to those of the botulinum neurotoxins), ii) a 2HC–N domain, whose function is not known, and iii) a C-terminal domain, termed HC–C which harbours two polysialoganglioside binding sites and mediates the binding of the toxin to the presynaptic membrane of peripheral motor axons.
- The C-terminal domain of TeNT is capable of binding two polysialogangliosides via their oligosaccharide portion. These glycolipids are highly enriched on peripheral nerve terminals and are considered to be sufficient to determine the toxin binding to the presynaptic membrane
- The endocytosis of TeNT inside a vesicle that moves in a retrograde direction inside the axon up to the dendrites departing from the cellular body located within the spinal cord.
- Retroaxonal transport to the postsynaptic dendrites of the motor neurons that synapse with the inhibitory interneurons.
- Discharge of TeNT in the intersynaptic space between the motor neuron and the inhibitory interneuron.
- Binding of TeNT to the presynaptic membrane of the latter cell.
- Binding is followed by endocytosis inside a synaptic vesicle whose lumen becomes acidic.
- Low pH induces the entry of the HN and L domains into the membrane and the translocation of L into cytosol where it is freed by reduction of the interchain disulfide bond.
The spastic paralysis that results from intoxication with TeNT arises from the loss of spinal inhibitory control of motorneuron activity. Prevents inhibitory transmittor (GABA) being released from being secreted, resulting in continuous stimulation by the excitatory transmittor.
Endotoxin, more accurately referred to as __________________ (LPS), is recognized as the most potent microbial mediator implicated in the pathogenesis of sepsis and septic shock. LPS release in the circulation provokes a vigorous systemic ____________ response. It is the host response to LPS, rather than the intrinsic properties of endotoxin itself, that accounts for the potentially lethal consequences attributable to LPS. As a species, humans are particularly susceptible to the immunostimulant capacity of LPS; even minute doses may be lethal.
LPS is a constituent of the _____ membrane of Gram-________ bacteria cells. Endotoxin consists of a polysaccharide part attached to a lipid (_____ _). The polysaccharide part facilitates the solubility of the molecule in water. It is made up of two parts, an oligosaccharide with a composition varying with bacterial species (accounts for the specificity of the different lipopolysaccharides) and a rather invariable core section, which is located between the oligosaccharide and the lipid A. The inner core region linking the lipid, together with the lipid A, provides the immunomodulating and toxic effects induced by LPS.
Endotoxin, more accurately referred to as lipopolysaccharide (LPS), is recognized as the most potent microbial mediator implicated in the pathogenesis of sepsis and septic shock. LPS release in the circulation provokes a vigorous systemic inflammatory response. It is the host response to LPS, rather than the intrinsic properties of endotoxin itself, that accounts for the potentially lethal consequences attributable to LPS. As a species, humans are particularly susceptible to the immunostimulant capacity of LPS; even minute doses may be lethal.
LPS is a constituent of the outer membrane of Gram-negative bacteria cells. Endotoxin consists of a polysaccharide part attached to a lipid (lipid A). The polysaccharide part facilitates the solubility of the molecule in water. It is made up of two parts, an oligosaccharide with a composition varying with bacterial species (accounts for the specificity of the different lipopolysaccharides) and a rather invariable core section, which is located between the oligosaccharide and the lipid A. The inner core region linking the lipid, together with the lipid A, provides the immunomodulating and toxic effects induced by LPS.
What is the primary receptor that recognises LPS?
TLR4
What are the three receptors for LPS in human cells?
- soluble or membrane-bound CD14-MD2-TLR4 molecules
- CD11/CD18 molecules (β2 integrins)
- scavenger receptors for lipid molecules
- In human plasma and other body fluids, LPS trafficking is facilitated by a hepatically derived, acute-phase plasma protein known as ___-_______ _______ (LBP).
- LBP performs a shuttle service picking up polymeric LPS aggregates and transferring LPS monomers to ____. LPS competes with another neutrophil-derived LPS-binding molecule known as ____________/____________-__________ _______ (BPI).
- LBP ______ in the delivery of LPS to immune effector cells while BPI ________ LPS delivery to ____. The relative concentrations of these two LPS-binding proteins primarily determine the net effect of LPS release.
- After docking to membrane-bound ____, LPS is delivered to an essential extracellular adaptor protein known as MD2. This LPS–MD2 complex is then presented to the extracellular leucine-rich domain of ____ where multimers of this complex aggregate on lipid rafts on the cell surface. This series of events then triggers a signal to the intracellular space, subsequently activating LPS-responsive _____.
- Once ____ binds to its LPS ligand two possible pathways of cellular activation can occur: through either the myeloid differentiation factor 88 (MyD88) or the TLR domain adaptor inducing interferon (INF)-beta (TRIF) pathway.
- A series of signaling events occur with sequential activation of specific tyrosine and threonine/serine _______. This signaling cascade ultimately leads to phosphorylation, ubiquitination, and degradation of inhibitory κB (IκB) along with other transcriptional __________. IκB degradation releases _______ ______ __ (NFκB) to translocate into the nucleus.
- Clotting elements, complement, other acute phase proteins, cytokines, chemokines, and nitric oxide synthase genes have NFκB-binding sites at their promoter regions. The outpouring of inflammatory _________ and other inflammatory mediators after LPS exposure contributes to generalized _____________, procoagulant activity, tissue injury, and _______ _____.
- In human plasma and other body fluids, LPS trafficking is facilitated by a hepatically derived, acute-phase plasma protein known as LPS-binding protein (LBP).
- LBP performs a shuttle service picking up polymeric LPS aggregates and transferring LPS monomers to CD14. LPS competes with another neutrophil-derived LPS-binding molecule known as bactericidal/permeability-increasing protein (BPI).
- LBP assists in the delivery of LPS to immune effector cells while BPI inhibits LPS delivery to CD14. The relative concentrations of these two LPS-binding proteins primarily determine the net effect of LPS release.
- After docking to membrane-bound CD14, LPS is delivered to an essential extracellular adaptor protein known as MD2. This LPS–MD2 complex is then presented to the extracellular leucine-rich domain of TLR4 where multimers of this complex aggregate on lipid rafts on the cell surface. This series of events then triggers a signal to the intracellular space, subsequently activating LPS-responsive genes.
- Once TLR4 binds to its LPS ligand two possible pathways of cellular activation can occur: through either the myeloid differentiation factor 88 (MyD88) or the TLR domain adaptor inducing interferon (INF)-beta (TRIF) pathway.
- A series of signaling events occur with sequential activation of specific tyrosine and threonine/serine kinases. This signaling cascade ultimately leads to phosphorylation, ubiquitination, and degradation of inhibitory κB (IκB) along with other transcriptional activators. IκB degradation releases nuclear factor κB (NFκB) to translocate into the nucleus.
- Clotting elements, complement, other acute phase proteins, cytokines, chemokines, and nitric oxide synthase genes have NFκB-binding sites at their promoter regions. The outpouring of inflammatory cytokines and other inflammatory mediators after LPS exposure contributes to generalized inflammation, procoagulant activity, tissue injury, and septic shock.
