Medical Microbiology: Bacterial Pathogens and Diseases I (Exotoxins) Flashcards
1
Q
What is a pathogen?
A
- A microorganism capable of causing disease.
2
Q
What is meant by the term “pathogenicity?”
A
- The ability of an infectious agent to cause disease
3
Q
What is meant by the term “virulence?”
A
- The quantitative ability of an infectious agent to cause disease
4
Q
What is meant by the term “Toxigenicity?”
A
- The ability of a microorganism to produce a toxin that contributes to the development of disease.
5
Q
What are the different mechanisms of virulence?
A
- Adherence factors - Allows bacteria to adhere to host cell
- Biofilms - Group of microorganisms that stick to each other and to a surface and become embedded within an extracellulr matrix
- Invasion of host cells and tissues
- Toxins - Endotoxins and Exotoxins
6
Q
What are exotoxins?
A
- Heterogeneous group of proteins produced and secreted by living bacterial cells
- Produced by both gram negative and gram positive bacteria
- Cause disease symptoms in host cell during disease
- Act via a variety of diverse mechanisms
7
Q
What are the advantages to the bacteria of producing exotoxins?
A
- Evade immune response
- Enable biofilm formation
- Enable attachment to host cells
- Escape from phagosomes
- All of these allow for colonisation, niche establishment and carriage
8
Q
What are some of the exotoxins produced by Staphylococcus aureus and why does it produce them?
A
-
Haemolytic toxins
- Cause mainly blood cells to lyse by forming pores
- Important cause of features of S. aureus disease
-
Phenol soluble modulins (PSM)
- Aggregate the lipid bilayer of host cells leading to lysis
9
Q
Describe the actions of Staphylococcus aureus on the human body
A
- Staphylococcus aureus is a gram positive bacteria and usually sits within the nose and doesn’t cause disease - It’s a commensal organism
1. Alpha haemolytic toxin and phenol-soluble modulin (PSM) toxins inhibit fusion of phagosome to lysosome. This enables bacteria to escape from phagosome into the cytoplasm, allowing intracellular niche establishment and replication
2. PSM toxins target cohabiting bacterial species giving Staphylococcus aureus an advantage in this particular niche
3. PSM toxins allow Staphylococcus aureus to slide across surfaces
4. Exotoxins allow for Staphylococcus aureus to produce biofilm - involved in each step - Alpha-toxin involved in initial attachment phase
- Beta-toxin covalently links to itself allowing for formation of complex biofilm
- PSM toxins involved in detachment of mature biofilm allowing for dispersal to new sites of infection
10
Q
Describe the genetics of exotoxins
A
- Some exotoxins can be encoded by chromosomal genes e.g.
- Shiga toxin in Shigella dysenteriae
- TcdA & TcdB in C. difficile
- Many exotoxins are encoded by extrachromosomal genes
- Plasmids – Bacillus anthracis toxin, tetanus toxin
- Lysogenic bacteriophage – e.g. streptococcal pyrogenic exotoxins in Scarlet Fever
11
Q
What are the different classes of exotoxins?
A
- Membrane Acting Toxins – Type I
- Membrane Damaging Toxins – Type II
- Intracellular Toxins – Type III
12
Q
What is the problem with the classification of exotoxins?
A
- Many exotoxins may have more than one type activity
13
Q
Briefly describe the activity of the membrane acting exotoxins (type I)
A
- They act either on the outiside of cells or on cell membranes
- They interfere with host cell signalling by causing inappropriate activation of host cell receptors
- Target receptors include:
- Guanylyl cyclase - Increases intracellular cGMP
- Adenylyl cyclase - Increases intracellular cAMP
- Rho proteins
- Ras proteins
14
Q
Give an example of a membrane acting exotoxin and explain its mechanism of action
A
- Example: E. coli Stable Heat Toxin
- This binds to guanylyl cyclase receptor which casues an increase in intracellular cGMP
- This leads to increased release of Cl- and HCO3<strong>- </strong> from the cell via the CFTR channel
- This also leads to increased release of H+ and increased uptake of Na+ via the NHE3 channel
- Increased release of Cl- from cell causes water to follow osmotically resulting in diarrhoea
15
Q
Briefly describe the activity of membrane damaging exotoxins (type II)
A
- They cause damage to the host cell membrane
- There are two types:
-
Receptor mediated - Insert channels or pores into host cell membrane
- β sheet toxins e.g. S.aureus α – toxin
- α helix toxins – e.g. diphtheria toxin
-
Receptor independent - Enzymatical damage (attaches to membrane causing it to breakdown)
- S. aureus β - haemolysin
- PSM
16
Q
Describe in more detail how the different types of membrane-damaging exotoxins exert their effects
A
- Receptor mediated
- Exotoxin binds to a specific receptor on the cell membrane
- When enough exotoxins are bound to toxin-specific receptor they form hexa, hepta or octametic defined pores through the membrane
- Receptor independent
- Exotoxin binds/attaches to membrane
- Binding causes membrane disintergration
- This allows exotoxin to form short-lived pores within membrane
17
Q
Breifely describe the activity of the intracellular exotoxins (Type III)
A
- Active within the cell – must gain access to the cell
- Usually 2 components - AB toxins
- Receptor binding and translocation function - B
- Toxigenic (enzymatic) activity - A
- AB toxins may have single or multiple B units e.g. Cholera toxin AB5
18
Q
Give some examples of the different types of enzymatic activity that the Enzymatic component A of an AB exotoxin may have
A
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ADP – ribosyl transferases: Covalently modify adenylyl cyclase molecules by adding ADP-ribosyl groups
- E.g. Exotoxin A of Pseudomonas aeruginosa, pertussis toxin.
-
Glucosyltransferases: Modify rRNA preventing protein synthesis from occuring
- E.g. TcdA and TcdB of Clostridium difficile
-
Deamidase
- E.g. dermonecrotic toxin of Bordetella pertussis
-
Protease
- E.g. Clostridial neurotoxins: botulism & tetanus - damage neuromuscular junctions or presynaptic vesicles
-
Adenylcyclase: Increases levels of cAMP
- E.g. EF toxin of Bacillus anthracis
- ALL OF THESE LEAD TO CELL DYSFUNCTION AND TISSUE DAMAGE
19
Q
Apart from the AB toxin mechanism what are some other mechanisms of intracellular exotoxins (type III)?
A
-
Type III secretion and toxin injection
1. Bacteria binds to the surface of a cell
2. Proteins assemble within the bacterial membrane to form a hypodermic needle
3. Hypodermic needle penetrates membrane of host cell
4. Through this hypodermic needle bacteria injcets cell with effcetor proteins
20
Q
Apart form their enzymatic activity what other effects can exotoxins have on the host cell?
A
- Exotoxins are able to induce inflammatory cytokine release
- These include cytokines such as: IL1, IL1β, TNF, IL 6,δ interferon, IL18
21
Q
How are exotoxins able to induce the release of inflammatory cytokines?
A
-
Superantigen – Causes non specific bridging of the MHC Class II and T- cell receptor leading to massive cytokine production
- This occurs without MHC class II presenting specific antigen to T-lymphocyte receptor as superantigen covalently cross-links MHC class II to T-lymphocyte receptor
- Increase in cytokine production leads to septic shock
- E.g. Staphylococcal Exfoliative Toxin A, Toxic Shock Syndrome Toxin 1 (TSST1)
-
Activation of the different inflammasome leading to release of IL1 β and IL18
- E.g. S. aureus toxin A, PVL.
22
Q
What are toxoids and how are they produced?
A
- Toxoids are inactive proteins that are still highly immunogenic
- They form the basis of vaccines against toxins e.g.
- Tetanus Vaccine
- Diphtheria
- Pertussis (acellular)
- Toxoids are formed by using using formaldehyde or glutaraldehyde to inactivate a specific toxin
23
Q
Give examples of toxin mediated diseases that can be treated using antibodies and the types of antibodies used to treat them
A
-
Diphtheria
- Diphtheria toxin injected into horse which carries out immune response and produces antibodies
- Antibodies then purified and used as part of Diphtheria antitoxin
-
Tetanus
- Treated using pooled human immunoglobulin. Specific or normal
-
Botulism
- Treated using horse antibodies
- This form of treatment is called passive immune treatment
24
Q
Describe the microbiology of the bacteria Clostridium difficile
A
- Gram-positive bacteria
- Anaerobic
- Spore-forming
- Toxin-producing
- Can be carried asymptomatically in the gut.
- Produces 3 toxins
25
Describe the epidemiology of the bacteria *Clostridium difficile*
* Common hosptial acquired infection
* Spread via ingestion of spores - remain dormant in environment
* Coloniser of the human gut up to 5% in adults
26
What are some risk factors of *Clostridium difficile* infection
* **Antibiotic use**
* **Age**
* **Antacids** - Decreases acidity of stomach meaning it doesn't kill *Clostridium difficile* spore allowing it to get into intestines
* **Prolonged hospital stay**
27
What effect can antibiotics have on *Clostridium difficile*
* Antibiotics can disrupt the microbial ecosystem within the gut
* This provides a competitive advantage to spore forming anaerobes over non spore forming anaerobes.
* This Allows C. difficile colonisation and growth within the gut
28
What are the 3 main toxins produced by *Clostridium difficile*?
* **Cytotoxin A** - TcdA encoded by tcdA gene
* **Cytotoxin B** - TcdB encoded by tcdB gene
* **Binary toxin** - C. diff transferase (CDT) – minor role in disease
29
Describe the structure of TcdA and TcdB
* They have 4 main domains:
* GTD - Glucosyltransferase domain
* CPD - Cysteine protease domain
* DD - Delivery hydrophobic domain
* RBD - Receptor binding domain
30
Briefly describe how *Clostridium difficile* exerts its effects on the host cell
* TcdA and TcdB enter the cell via receptor mediated endocytosis
* Release of active component of toxin into the cytoplasm
* Disruption of cGMP and cAMP via disruption of Ras and Rho proteins
31
What are some of the effects of *Clostridium difficile* infection on the host cell?
* Cy**topathic effects**
* Cytoskeletion breakdown
* Loss of cell-cell contacts
* Increased epithelial permeability
* **Cytotoxic effects**
* Activation of inflammasome
* Increase in ROS levels
* Induction of programmed cell death
32
What are some of the effects of *Clostridium difficile* infection on the body?
* Patchy necrosis with neutrophil infiltration
* Epithelial ulcers
* Pseudomembranous colitis - Swelling/inflammation of colon
* Pseudomembranes include: leucocytes, fibrin, mucous, cell debris
33
How can you diagnose *Clostridium difficile* disease?
* Clinical signs and symptoms
* Raised white cell count in blood.
* Detection of organisms and toxins in stool (2 phase test)
* Glutamate dehydrogenase – detects if C. difficile organism present.
* Toxin enzyme linked immunosorbent assay (ELISA) for TcdA and TcdB toxins.
* Detection of tcdA and tcdb genes – PCR
* Colonoscopy – pseudomembranous colitis
34
How can you treat *Clostridium difficile* disease?
* Treatment dependent on severity and presence of surgical complications
* Ideally removal of offending antibiotic
* Use of antibiotics fidaxomicin or metronidazole or vancomycin
* Surgery – partial, total colectomy
* If recurrent – faecal transplant
35
Describe the microbiology of Verocytotoxin Escherichia coli (VTEC)
* VTEC can cause disease mild to life threatening disease
* Shiga-toxin (Stx) carried by some E.coli
36
Describe the epidemiology of Verocytotoxin Escherichia coli
* E. coli naturally colonizes the GI tracts of cattle who are generally asymptomatic
* Transmission:
* Predominantly via consumption of contaminated food and water
* Person to person, particularly in child day-care facilities, and from
* Animal to person. E.g. in petting zoos, dairy farms, or camp grounds.
* Very low infectious dose
37
Describe some of the features of the toxin that causes Verocytotoxin Escherichia coli (VTEC)/(STEC) disease
* Shiga like toxin (SLT) = shigatoxin (Stx) = verocytotoxin (VTEC)
* Various different forms of this toxin which have slight variations in amino acid sequence, e.g. Stx and Stx1
* Gene the encodes toxin is carried on lysogenic bacteria
38
Describe the structure of the toxin that causes Verocytotoxin Escherichia coli (VTEC)/(STEC) disease
* Type III exotoxin – AB5
* Enzymatic component A = N-Glycosidase - damages rRNA
* Bound to 5 B subunits
39
Describe the mechanism of action of the shigatoxin
1. Toxin binds to receptor globotriaosylceramide Gb3 or globotetraosylceramide (Gb4) on host cell membrane
2. Bound toxin internalised by receptor mediated endocytosis
3. Carried by retrograde trafficking via the Golgi apparatus to the endoplasmic reticulum.
4. The A subunit is cleaved off by membrane bound proteases
5. Once in the cytoplasm A1 and A2 disassociate
6. A1 binds to 28S RNA subunit of ribosome which blocks protein synthesis
40
Describe the pathogenesis of STEC/VTEC
* STEC closely adheres to the epithelial cells of the gut mucosa
* Route by which Shigatoxin (Stx) is transported from the intestine to the kidney and other tissues unclear - possibly via polymorphonuclear neutrophils (PMNs)
* Stx can bind to glomerular endothelial cells of kidney, cardiovascular and central nervous system.
* Very high levels of Gb3 in kidney so kidneys most affected.
* Stx favours inflammation resulting in microvascular thrombosis and inhibition of fibrinolysis
41
What are some of the symptoms of STEC disease?
* Abdominal cramps, watery or bloody diarrhoea (may not be present)
* **Haemolytic uraemic syndrome**
* Anaemia
* Renal Failure
* Thrombocytopaenia (low platelet level)
* Other symptoms may include:
* Lethargy
* Severe headache
* Convulsions
* Encephalopathy - damage to brain
42
How can you diagnose STEC disease?
* Clinical signs and symptoms
* Haematological and biochemical evidence.
* Stool culture – Growth on SMac
* PCR for Stx genes
43
How can you treat STEC disease?
* Supportive including renal dialysis and blood product transfusion
* Antibiotics have little to no role
