Bacterial Pathogens and Disease I-Exotoxins Flashcards
What is the difference between :
- Pathogen
- Pathogenicity
- Virulence
- Toxigenicity
Pathogen = A microorganism capable of causing disease
Pathogenicity = The ability of an infectious agent to cause disease
Virulence = The quantitative ability of an agent to cause disease
Toxigenicity = The ability of a microorganism to produce a toxin that contributes to the development of disease
- What are some factors/mechanisms that affect the virulence of an agent?
Adherence Factors, Biofilms,
Invasion of host cells and tissues,
Toxins (endotoxins and exotoxins).
- What are exotoxins?
- Heterogeneous group of proteins produced and secreted by living bacterial cells
- Cause disease symptoms in host during disease.
- Act via a variety of diverse mechanisms.
- What type of bacteria produce exotoxins
- gram negative, gram positive or both?
Both gram + and gram -
- Exotoxins give bacteria a selective advantage?
o They Cause disease – may help transmission of disease, however in severe disease host may be a literal and evolutionary dead end.
o However, with many toxins the disease causing activity may be not be the primary function.
- What do exotoxins do and how does this result in an evolutionary adavantage?
o Evade immune response o Enable biofilm formation o Enable attachment to host cells. o Escape from phagosomes • All allowing for colonisation, niche establishment and carriage - Evolutionary advantage.
- What happens when a person gets infected with Staphylococcus aureus?
- Entry
- Produce haemolytic toxins:
- α,β,δ, toxins ,Panton Valentine Leukocidin (PVL), LukAB, LukED, LukMF
- Phenol soluble modulins PSM (S.aureus codes for 8) (toxins, biofilm production) - Cause cells to lyse by forming pores
- Aggregate the lipid bilayer of host cells - lysis
- Majority of S. aureus in humans is asymptomatic carriage in the nose.
• So what are those toxins doing in the nose?
•These bacteria are spread by having direct contact with an infected person, by using a contaminated object, or by inhaling infected droplets dispersed by sneezing or coughing.
- What genes encode exotoxins?
Can be encoded by chromosomal genes Shiga toxin in Shigella dysenteriae, TcdA & TcdB in C. difficile
- Many toxins are encoded by extrachromosomal genes
Give examples of the toxins coded in plasmids and Lysogenic Bacteriophage?
o Plasmids – Bacillus anthracis toxin, tetanus toxin
o Lysogenic bacteriophage – e.g. streptococcal pyrogenic exotoxins in Scarlet Fever, Diphtheria toxin.
- One way of classifying exotoxins is through their toxin activity, how would they be classified then?
- Membrane Acting Toxins – Type I
- Membrane Damaging Toxins – Type II
- Intracellular Toxins – Type III
- What is the problem with classifying exotoxins based off toxin activity
o Many toxins may have more than one type activity.
o As mechanisms better understood (how the toxin works) this classification tends to break down.
- How do the “ Membrane acting Type 1 Toxins “
- Act
- interfere
- whats their target
• Act from without the cell.
• Interfere with host cell signaling by inappropriate activation of host cell receptors.
•Target receptors including: –Guanylyl cyclase → ↑ intracellular cGMP
-Adenyl cyclase → ↑
intracellular cAMP
-Rho proteins
-Ras proteins
- One example of the Membrane acting Type 1 Toxins is E.Coli Stable heat toxins - how does this toxin specifically work?
Sta binds to the GCR to increase the formation of cGMP.
• This activates many intracellular signal transduction pathways e.g. cGMP.
• This changes the permeability of ion pores e.g. increased Cl- excretion (water follows it and causes diarrhoea).
- How do the Membrane Damaging Type 2 Toxins work?
Cause damage to the host cell membrane:
•Insert channels into the host cell membrane.
eg : β sheet toxins e.g. S.aureus α – toxin, δ toxin, PVL
oα helix toxins – e.g. diphtheria toxin
•Enzymatical damage e.g. S. aureus β- haemolysin, PSM
- What are the two types of Membrane Damaging Type 2 Toxins?
Receptor mediated: use toxin-specific receptor to form defined ion pores = alpha toxin, Luk-(AB,GH,DE), PVL
Receptor Independent: attach to membrane and disintegrate it to form short-lived pores.(mainly alpha type PSMs)
- How do the “Intracellular Type 3 Toxins” work?
• Active within the cell – must gain access to the cell
• Usually 2 components – AB Toxins
o Receptor binding and translocation function – B
oToxigenic (enzymatic) – A
o May be single or multiple B units e.g. Cholera toxin AB5
- The enzymatic component A of intracellular type 3 toxins has a wide array of activities- list some
o ADP – ribosyl transferases - e.g. Exotoxin A of Pseudomonas aeruginosa, pertussis toxin.
o Glucosyltransferases – e.g. TcdA and TcdB of Clostridium difficile
o Deamidase – e.g. dermonecrotic toxin of Bordetella pertussis.
o Protease – e.g. Clostridial neurotoxins: botulism & tetanus
o Adenylcyclase - e.g. EF toxin of Bacillus anthracis.
- How does YopE inject its toxin into the host cell
o Type III secretion and toxin injection e.g. YopE in Yersinia species – membrane produces a needle structure to inject the toxin in.
- What are some cytokines that exotoxins are able to release via an inflammatory cytokine release?
IL1, IL1β, TNF, IL 6,δ interferon, IL18.
- What is the mechanism as to HOW the exotoxin causes an inflammatory cytokine release?
- Superantigen – non-specific bridging of the MHC Class II 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 inflammasome leading to release IL1 β and IL18 e.g. S. aureus toxin A, PVL.
- What can inactive a toxin?
• Toxins can be inactivated using formaldehyde or glutaraldehyde → toxoids
Toxoids = inactive proteins but still highly immunogenic – form the basis for vaccines
- What are three examples of vaccines that Toxoids form the basis of?
o Tetanus Vaccine, Diphtheria, Pertussis (acellular).*
- Treatment of toxin mediated disease can be affected by administering preformed antibodies to the toxin - what antibodies where used to treat Tetanus, Botulism and Diphtheria antitoxin?
o Diphtheria antitoxin – horse antibodies.
o Tetanus – pooled human immunoglobulin. Specific or normal.
o Botulism – horse antibodies
- What type of antibodies do we use in experiments and research?
monoclonal antibodies.
- What is the microbiology of Clostridium Difficile?
o Anaerobic, gram-positive bacillus that forms spores and produces 3 types of toxins.
o It can be carried asymptomatically in the gut.
- What is the Epidemiology of Clostridium Difficile?
- How is it spread
- Where in the body does it colonise
o Common hospital acquired infection worldwide.
o Spread by ingestion of spores – remain dormant in environment.
o Coloniser of the human gut up to 5% in adults.
- What are some risk factors that increase your chances of Clostridium Difficile infection?
antibiotic use, age, antacids & prolonged hospital stay
- How can antibiotic use cause Clostridium Difficile?
- Antibiotics provide a competitive advantage to spore forming anaerobes (which c.difficle are) over non spore forming anaerobes.
- This means they disrupt the microbial ecosystem of the gut to allow C. difficile colonisation and growth. All antibiotics have potential for causing disease.
- Which antibiotics are worse (for clostridium difficile) and which ones are less likely to cause clostridium difficile?
•Some antibiotics worse than others
o 2nd and 3rd generation cephalosporins, Quinolones, Clindamycin?
•Others less likely
o Aminoglycosides, Trimethoprim, Vancomycin
- What is a biofilm?
Densely packed communities off pathogen cells that adhere to a surface using toxins eg PSM
- What three toxins does C.Difficile produce and what genes code for them
Cytotoxin A - coded by TcdA gene
Cytotoxin B - coded by TcdB gene
Binary Toxin - CDT- minor role in the disease
33 . What type of toxins are A and B ( that c.difficle produces) and what is the A component?
Type III AB toxins
A component = glycosolating enzyme
Specifically glucosyltransferase domain
34. The AB toxin has the components : GTD CPD DD RBD what do they stand for?
GTD = Glucosyltransferase domain ( A component)
CPD= Cysteine protease domain
DD= Delivery hydrophobic domain
RBD= Receptor binding domain (B component)
- How does the toxins in c.difficle cause disease?
- The toxin binds to the cell surface membrane using the RBD and is internalised in an endosome.
- The endosome undergoes acidification which produced pores in its membrane.
- This allows the GTD (A component) to be released into cytoplasm.
- It results in the inactivation of Rho GTPases by glycosylation which causes downstream effects.
• Cytopathic effects: loss of cell-cell contacts, cytoskeleton breakdown, increased permeability.
• Cytotoxic effects: inflammasome activation, ROS production and induction of apoptosis.
- What are some clinical signs and symptoms of C.difficle?
Patchy necrosis with neutrophil infiltration
Epithelial ulcers
Pseudomembranes – leucocytes, fibrin, mucous, cell debris.
- Explain how the signs and symptoms of c.difficile can get progressively worse?
Asymptomatic Watery Diarrhoea Dysentery Pseudomembranous Colitis Toxic Megacolon and Peritonitis
- What are some ways to diagnose C.difficle?
- > The clinical signs and symptoms
- > Raised white cell count in blood
- > 2 phase test
- > Detection of tcdA and tcdB genes using PCR
- > Colonoscopy - look for pseudomembranous colitis
- What is the different treatment options for C.difficle
Treatment dependent on severity and presence of surgical complications
Ideally removal of offending antibiotic – not always possible
Antibiotics fidaxomicin or metronidazole or vancomycin
Surgery – partial, total colectomy
Recurrent – faecal transplant.
- What is VTEC/ STEC?
VTEC (Verocytotoxin E.coli ) , or Shiga-toxin (Stx) producing E. coli (STEC) can cause disease mild to life threatening disease.
Stx carried by some E. coli – most commonly O157:H7
- How can we positively identify E.coli 0157:H7?
Identified usually by growth on sorbitol MacConkey agar (SMac) – does not ferment sorbitol and hence is clear.
Other less common types not identified using SMac.
- What is the epidemiology of E.coli 0157:h7?
E. coli O157:H7 naturally colonizes the gastrointestinal tracts of cattle who are generally asymptomatic.
- What are the three main ways of transmission of E.Coli 0157:H7?
Predominantly via consumption of contaminated food and water
Person to person, particularly in child day-care facilities
Animal to person. E.g. in petting zoos, dairy farms, or camp grounds.
*VERY LOW INFECTIOUS DOSE
- Why do we get many different shiga like toxins?
Have many variations in the aa sequence
- What type of bacteria is the gene for the toxin carried on?
Gene carried on lysogenic bacteria.
- What type of toxin is the shiga like toxin?
Type III exotoxin – AB5
- What is the A components of the shiga like toxin?
N-Glycosidase
- What is the mechanism of how the shiga toxin causes disease?
Bind to receptor globotriaosylceramide Gb3-> present on kidneys or globotetraosylceramide (Gb4) on host cell membrane
Bound toxin internalised by 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 disassociate
A1 binds to 28S RNA subunit – blocks protein synthesis.
- What is the pathogenesis of STEC?
STEC closely adheres to the epithelial cells of the gut mucosa.
The route by which Stx is transported from the intestine to the kidney/ tissues is possibly polymorphonuclear neutrophils (PMNs)
Bind to glomerular endothelial cells of kidney, cardiovascular and central nervous system.
Very high levels of Gb3 in kidney so kidneys most affected.
Thought that Stx favours inflammation resulting in microvascular thrombosis and inhibition of fibrinolysis.
- What are some clinical signs and symptoms of STEC?
Can be severe and life threatening
Abdominal cramps, watery or bloody diarrhoea – may not be present
Haemolytic uraemic syndrome:
Anaemia
Renal Failure
Thrombocytopaenia
Less common are neurological symptoms : lethargy, severe headache, convulsions, encephalopathy.
- What ways can we diagnose STEC?
Clinical signs and symptoms
Haematological and biochemical evidence.
Stool culture – Growth on SMac
PCR for Stx genes
- What is the treatment for STEC?
Supportive including renal dialysis and blood product transfusion
Antibiotics have little to no role
