Bacterial Pathogens and Disease - EXOTOXINS

Question 1

What is a pathogen?

Answer 1

A microorganism capable of causing disease

Question 2

What is pathogenicity?

Answer 2

The abillity of an infectious agent to cause disease

Question 3

What is virulence?

Answer 3

The quantitative abillity of an agent to cause disease

Question 4

What is toxigenicity?

Answer 4

The abillity of a microorganism to produce a toxin which contributes towards the development of diseases

Question 5

List some virulence mechanisms

Answer 5

• Adherance factors
• Biofilms
• Invasion of host cells and tissues
• Toxins - endotoxins and exotoxins

Question 6

What are exotoxins?

Answer 6

Exotoxins are a group of hetergeneous proteins which are produced and secreted by gram + and gram - living bacterial cells

• Cause disease symptoms in the host
• Act via a variety of diverse mechanisms

Question 7

What is the purpose of bacteria having exotoxins?

Answer 7

• Exotoxins will cause disease → However in a severe disease may cause death meaning bacteria will not be able to replicate and cause an evolutionary dead end
• However, with many toxins the disease causing activity may not be the primary function others include:
  
  • Evade immune response of host
  • Enable biofilm formation
  • Enable attachment of host cells
  • Escape from phagosomes

Question 8

What can functions such as evading immune response, biofilm formation, attachment of host cells and escape of phagosomes result in?

Answer 8

• Allow for colonisation, niche establishment and carriage = Evolutionary advantage

Question 9

Looking at Staphylococcus aureus what toxin does it produce and what are their functions?

Answer 9

• HAEMOLYTIC TOXINS = Causes cells to lyse by forming pores
  
  • Important feature of S.aureus disease
    
    • α,β,δ, toxins ,Panton Valentine Leukocidin (PVL), LukAB, LukED, LukMF
• PHENOL SOLUBLE MODULINS (PSM) = Causes lipid bilyaer of host cells to break down through aggregation - lysis

Question 10

What happens when these toxins are released in the nose?

Answer 10

• Alpha toxins and PSMs prevent phagolysosome formation by blocking fusion of the lysosome with the phagosome
• PSMs will kill any cohabiting bacteria in the nose giving S.aureus an advantage by reducing competition
• PSMs will have surfactant properties and allow S.aureus to slide across surfaces (as its not too motile on its own)
• Biofilm formation → Alpha toxins enable bacteria to attach to a surface and grow, beta toxins aid in secondary structure formation, PSMs allow for further growth and detachment so bacteria can go and disperse to new sites of infection

Question 11

Expand on the genetics of exotoxins

Answer 11

• Can be encoded by chromosomal geens
  
  • Shinga toxin in Shingella Dysenteriae, TcdA and TcdB in C.dificile
• Many toxins are also encoded by extrachromosomal genes
  
  • Plasmids – Bacillus anthracis toxin, tetanus toxin
  • Lysogenic bacteriophage e.g. streptococcal pyrogenic exotoxins in Scarlet Fever, Diphtheria toxin

Question 12

How can exotoxins be classified?

Answer 12

1. Membrane Acting Toxins = Type I
2. Membrane Damaging Toxins = Type II
3. Intracellular Toxins = Type III

Question 13

Describe Type I Exotoxins

Answer 13

These are MEMBRANE ACTING

They act from without the cell and interfere with host signalling by inappropriate activation of host cell receptors on membrane

Question 14

Give some examples of target receptors that type I exotoxins receptors activate

Answer 14

Target receptors include:

• Guanylyl cyclase increase intracellular cGMP
• Adenyl cyclase increase intracellular cAMP
• Rho proteins
• Ras proteins

See notes for E.Coli heat stable toxin

Heat stable toxin binds to the GC-C receptor and producing cGMP acts on CFTR which increases electrolyte secretion (Cl-,HCO3-) = secretory diarrhoea

Question 15

Describe type II Exotoxins

Answer 15

• These are MEMBRANE DAMAGING
  1) Insert channels into host cell membrane
• β sheet toxins e.g S.aureus α-toxin, γ toxin, PVL
• α helix toxins à e.g diptheria toxin

2) Enzymatic damage e,g S. aureus β-haemolysin, PSM

These can either be receptor mediated or receptor independant

Question 16

Describe type III Exotoxins

Answer 16

• These are INTRACELLULAR TOXINS

There are usually two components one with allows toxin entry and one which causes damage

• Receptor binding and translocation function - B
• Toxigenic (enzymatic) – A
• May be single or multiple B units e.g. Cholera toxin AB5

Some bacteria will also contain a needle like system through which they can directly insert the toxin inside the cell without receptor interaction e.g CagA in H.Pylori

Question 17

In intracellular toxins, the enzymatic component A has a variety of activities.

List some of them

Answer 17

• 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 Bordetella pertussis.
• Protease – e.g. Clostridial neurotoxins: botulism & tetanus
• Adenylcyclase - e.g. EF toxin of Bacillus anthracis

Question 18

Expand on superantigens and cytokines

Answer 18

Exotoxins are able to induce inflammatory cytokine release IL1, IL1β, TNF, IL 6,δ interferon, IL18

MECHANSIMS:

1) Superantigen – nonspecific 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)

2) Inflammasome which will detect damage to cells and results in activation of the different inflammasome leading to the release of IL1β and IL18 e.g. S. aureus toxin A, PVL.

Question 19

Expand on vaccines, toxoid and antibodies

Answer 19

• Toxins can be inactivated using formaldehyde or glutaraldehyde ⇒ Toxoids
  
  • Toxoids are inactive proteins but are still highly immunogenic and form the basis of vaccines
    
    • Tetanus Vaccine
    • Diphtheria
    • Pertussis (acellular)
• Treatment of toxin mediated disease can be affected by administering preformed antibodies to the toxin
  
  • Diphtheria antitoxin ⇒ Horse antibodies
• Diphtheria toxin injected into horses and antibodies produced are taken and administered to humans
  
  • Tetanus ⇒ Pooled human immunoglobulin. Specific or Normal
  • Botulism ⇒ Horse antibodies
• Experimental and research – monoclonal antibodies

Question 20

Provide two examples of toxin mediated diseases

Answer 20

1. Clostridium difficile
2. Verocytotoxin Escherichia Coli (VTEC) (STEC) disease

Question 21

Describe the microbiology of clostridium difficle

Answer 21

• Gram positive bacillus
• Anaerobic (only grows in absence of oxygen)
• Spore-forming
• Toxin-producing
• Can be carried asymptomatically in the gut
• Produces 3 toxins

Question 22

What is the epidemiology of Clostridium difficle?

Answer 22

• Common hospital acquired infection
• Spread by ingestion of spores ⇒ Remains dormant in environment
• Its a coloniser of human gut up to 5% in adults
• RISK FACTORS
  
  • Antibiotic use
  • Age
  • Antacids
  • Prolonged hospital stay

Question 23

How do antibiotics cause C.difficle?

Answer 23

• Disrupt the microbial ecosystem within the gut
• Antibiotics provide a competitive advantage to spore forming anaerobes in comparison to non-spore forming anaerobes
  
  • Allows C.difficle colonisation and growth

Question 24

What antibiotics are more likely to cause diseases like C.difficle?

Answer 24

• 2nd and 3rd generation cephalosporins
• Quinolones (ciprofloxacin)
• Clindamycin

Question 25

What antibiotics are less like to cause disease?

Answer 25

* Aminoglycosides * Trimethoprim * Vancomycin

Question 26

What are the toxins in C.difficle?

Answer 26

* **Cytotoxin A** ⇔ TcdA coded by the tcdA gene * **Cytotoxin B** ⇔TcdB coded by the tcdB gene * **Binary toxin** ⇔ C.diff transferase (CDT) – minor role in disease * Tcd A and Tcd B →Type III AB toxins. * The A component of toxins are glycosylating enzymes

Question 27

Describe the mechanism of the TcdA and TcdB toxin

Answer 27

1. Toxin will bind to specific host cell receptors 2. The toxins are internalised by the cell 3. The endosome in which the toxin is in is acidified 4. There is pore formation on the endosome 5. GTD is released from endosome to the host cell cytoplasm 6. Rho GTPases are inactivated by glycosylation 7. Down stream effects (cytopathic and cytotoxic effects)

Question 28

What are some of the effects of inactivated Rho GTPases in the cell?

Answer 28

CYTOPATHIC * Cytoskeleton breakdown * Loss of cell-cell contacts * Increased epithelial cell permeabillity CYTOTOXIC * Activation of inflammasome * Increase in ROS levels * Induction of programmed cell death

Question 29

What are some symptoms of C.difficle disease?

Answer 29

* Asymptomatic * Water diarrhoea * Dysentry * Psuedomembranous colitis * Toxic megacolon and peritonitis

Question 30

How do we diagnose for C.difficle?

Answer 30

* 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

Question 31

How do you treat for C.difficle?

Answer 31

* Treatment depends on severity and presence of surgical complications * Ideally remove offending antibiotic * Surgery - partial/ total colectomy * Recurrent C.difficle is treated via a faecal transplant * Will restablish the microbiota

Question 32

What is the VTEC/STEC disease?

Answer 32

* VTEC, 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:H7Identified usually by growth on sorbitol MacConkey agar (SMac) * E.Coli does not ferment sorbitol and hence is clear. * Other less common types not identified using SMac.

Question 33

What is the epidemiology of VTEC/STEC?

Answer 33

* E. coli O157:H7 naturally colonizes the gastrointestinal 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 campgrounds. * Very low infectious dose

Question 34

What is the pathogenesis of the toxins of VTEC?

Answer 34

* Toxin – Shiga like toxin (SLT) = shigatoxin (Stx) = verocytotoxin (VTEC) * Stx, Stx1, Stx1a, 1c, 1d Stx2a, 2c, 2d – variations in a.a. sequence * Gene carried on lysogenic bacteria. * **Type III exotoxin – AB5** * Enzymatic component A = N-Glycosidase * Bound to 5 B subunits

Question 35

Describe the mechanism of the Shigatoxin

Answer 35

1. Toxin will bind to receptors on the surface Gb3 and Gb4 2. Bound toxin is internalised by receptor mediated endocytosis 3. Carried by retrograde trafficking via the Golgi apparatus to the ER 4. The A subunit (active component) is cleaved off by membrane bound proteases 5. Once in cytoplasm A1 and A2 dissociate 6. A1 will bind to the 28S RNA subunit → Blocking protein synthesis

Question 36

Describe the pathogenesis of STEC

Answer 36

* STEC will adhere to epithelial cells of gut mucosa * The route by which Stx is transported from the intestine to the kidney and other tissues is debated * Bind to glomerular endothelial cells of kidney, CVS and CNS * Very high levels of Gb3 in kidney so kidney mainy affected * Thought that Stz favours inflammation resulting in **microvascular thrombosis** and inhibition of fibrinolysis

Question 37

Describe the STEC disease

Answer 37

* Can be severe and life threatening * Children \< 5 years greatest risk * Abdominal cramps, watery or bloody diarrhoaea = may not be present * Haemolytic ureamic syndrome * Anaemia - clots cause RBC's to break down * Renal failure * Thromocytopenia * Less common are neurological symptoms * Lethargy * Severe headache * Convulsions * Encephalopathy

Question 38

What is the diganosis and treatment of STEC?

Answer 38

**Diagnosis** * Stool culture = Growth SMac * PCR for Stx genes **Treatment** * Supportive including renal dialysis and blood product transfusion * Antibiotics have little or no role