Bacterial Pathogens and Diseases 1 (Exotoxins) Flashcards
Define exotoxins, compare and contrast with endotoxin
Exotoxins are a heterogeneous group of proteins produced and secreted by both gram-negative and gram-positive bacteria
Endotoxins are mostly produced by gram-negative bacteria
Endotoxins are toxic components of the outer membrane of certain bacteria that are released when the bacteria die.
Exotoxins are generally more potent and specific in their effects.
Exotoxins aid in transmission and allow for colonisation, niche establishment and carriage - Evolutionary advantage:
- Evade immune response
- Enable biofilm formation
- Enable attachment to host cells
- Escape from phagosomes
Why have exotoxins? In case of Staphylococcus aureus
Haemolytic toxins (α and β) - cause cells to lyse by forming pores
α can block the fusion with phagosomes and aid in the attachment of tissues
β aid in the accumulation and gives structure (secondary structure with eDNA)
Phenol soluble modulins (PSM) toxin - aggregate the lipid bilayer of host cells - induce lysis
PSMs can aid in the escape from phagosomes as well as the movement of S. Aureus
What is bacterial genetics of exotoxin and how does it mediated disease
Toxins can be encoded by chromosomal genes (Shiga toxin in Shigella dysenteriae, TcdA & B in C.difficile)
Many toxins are coded by extrachromosomal genes; mobile:
1) Plasmids:
- Can be easily transferred between organisms of the same species (allowing for rapid spread of the toxin-producing trait) e.g. bascillus anthracis toxins, tetanus toxin
- Transfer beneficial genes such as resistance
2) Chromosomal:
- Genes for exotoxin production may be integrated into the bacterial chromosome and passed down through generations of bacteria
- Genes of toxisn can be exchanged between microorganisms through lysogenic bacteriophage - e.g. streptococcal pyrogenic exotoxins in Scarlet Fever, Diphtheria toxin
What are the classification of Exotoxins
Loosely classified by mode of action and function of the toxin, many toxins may have more than one type of activity
Type 1: Membrane Acting Toxins, Act from without the cell, interferes with host cell signalling by inappropriate activation of host cell receptors.
Target receptors include:
- Guanylyl cyclase = ↑ intracellular cGMP e.g. E.coli Stable Heat Toxin
- Adenyl cyclase = ↑ intracellular cAMP
- Rho & Ras proteins
Type 2: Membrane Damaging Toxins, 2 mechanisms:
1) receptor-mediated (many binds to receptor to form hexato-octameric pores)
2) receptor-independent (attachment to membrane, disintegration of membrane then formation of short-lived pores):
- Insert channels into host cell membrane e.g. β sheet toxins of S.aureus
- Enzymatical damage e.g. S.aureus β-haemolysin and α type PSMs
Type 3: Intracellular Toxins, usually 2 components - AB toxins
- receptor binding & translocation = B (receptor-mediated endocytosis)
- Toxigenic (enzymatic) = A
- May be single or multiple B units e.g. Cholera toxin AB₅
- Type 3 secretion and toxin injection - bacterial produce proteins that produce as needles to inject toxins into other cells
- Type 4 pump & secretion systems, mostly act to pump toxins out in a large scale
Enzymatic Component A:
- ADP - ribosyl transferases - e.g. Exotoxin A of Pseudomonas aeruginosa, pertussis toxin
- Glucosyltransferases - e.g. TcdA and TcdB of Clostridium difficile
- Deamidase - e.g. dermonecrotic toxin of Bodetella pertussis
- Protease - e.g. Clostridial neurotoxins: botulism & tetanus
- Adenylcyclase - e.g. EF (Edema factor) toxin of Bacillus anthracis
What is the role of Superantigens in the regulation of inflammation
Exotoxins are able to induce inflammatory cytokine release e.g. IL1, IL1β, TNF, IL 6, interferon γ, IL18
Mechanisms:
- non-specific bridging of the MHC Class 2 and T-cell receptor leading to cytokine production e.g. Staphylococcal Exfoliative Toxin A, Toxic Shock Syndrome Toxin 1 (TSST1)
- Via activation of the different inflammasomes leading to the release of IL1β and IL18 e.g. S.aureus toxin A, PVL
Explain toxins in context of vaccination (Toxoids)
Toxins can be inactivated using formaldehyde or glutaraldehyde → toxoids
Toxoids are inactive proteins but still highly immunogenic - vaccines
- Tetanus Vaccine, Diphtheria, Pertussis
Treatment of toxin-mediated disease can be affected by administering preformed antibodies to the toxin e.g. horse antibodies in Diphtheria antitoxin and Botulism
What is the structure and role of exotoxin in Clostridium difficile disease
C.difficile is a gram-positive bacillus, anaerobic, spore-forming, toxin-producing 3 toxins
- Spread by ingestion of spores - remain dormant in environment (resistant)
- Act by disrupting the microbial ecosystem within the gut
- Antibiotics provide a competitive advantage to spore-forming anaerobes allowing C.difficile colonisation and growth
AB toxin:
- Cytotoxin A - TcdA coded by tcdA gene - glycosylating enzymes - stimulates the release of inflammatory cytokines, which can cause damage to the lining of the intestine
- Cytotoxin B - TcdB coded by tcdB gene - disrupts the actin cytoskeleton of host cells, leading to cell death
- Binary toxin - C.diff transferase (CDT) - minor role in disease
Structure:
- GTD (glucosyltransferase domain),
- CPD (cysteine protease domain),
- DD (delivery hydrophobic domain)
- RBD (receptor binding domain)
MOA:
1) Toxin binding to specific host cell receptors via RBD
2) Toxin internalisation
3) Endosome acidification
4) Pore formation in the endosome via DD
5) GTD release from the endosome to the host cell cytoplasm
6) Rho GTPases inactivation by glucosylation
7) Downstream effects within the cell, cytopathic effects (cytoskeleton breakdown, loss of cell-cell contacts, increased epithelial permeability), cytotoxic effects (activation of the inflammasome, increase in ROS levels & induction of programmed cell death)
Cytopathic & cytotoxic:
- Patchy necrosis with neutrophil infiltration
- Epithelial ulcers
- Pseudomembranes - leucocytes, fibrin, mucous, cell debris
Diagnosis:
- clinical signs and symptoms, raised WBC in blood
- Detection via 2 phase test 1) Glutamate dehydrogenase - detects if C.difficle organism is present. 2) ELISA for TcdA and TcdB toxins
- Detection of tcdA and tcdB genes via PCR
- Colonoscopy - pseudomembranous colitis
Treatment:
- ideally removal of offending antibiotics - not always possible
- Antibiotics fidaxomicin or metronidazole or vancomycin
- surgery - partial or total colectomy
- recurrent - faecal transplant
What is the structure and role of exotoxin in Verocytotoxin Escherichia coli (VTEC)/(STEC) disease
In Escherichia coli (VTEC) disease, the exotoxin produced by the bacteria is called Shiga-toxin (Stx), which can cause severe diarrhoea and kidney damage. The toxin works by inhibiting protein synthesis in host cells, leading to cell death and tissue damage.
Stx carried by some E.coli, identified usually by growth on sorbitol MacConkey agar (SMac) - does not ferment sorbitol and hence is clear
E.coli naturally colonises GI tracts of cattle and transmits through contaminated food and water
Type 3 exotoxin, MOA:
- Bind to receptor globotriaosylceramide (Gb3) or globotetraosylceramide (GB4) on host cell membrane
- Bound toxin internalised via receptor-mediated endocytosis
- Carried by retrograde trafficking via the Golgi apparatus to the endoplasmic reticulum
- The A subunit is cleaved off by membrane-bound proteases
- Once in the cytoplasm A1 and A2 dissociate
- A1 bind to 28S RNA subunit - blocks protein synthesis
Pathogenesis:
- STEC closely adheres to the epithelial cells of the gut mucosa
- Bind to glomerular endothelial cells of kidney, CVS & CNS via possible polymorphonuclear nutrophils (PMNs)
Diagnosis:
- Clinical signs and symptoms
- Haematological and biochemical evidence
- Stool culture - Growth on SMac
- PCR for Stx genes
Treatment:
- Supportive including renal dialysis and blood product transfusion
- Antibiotics have little to no role
