Bacterial Pathogens and Disease I - Exotoxins

Question 1

What are the definitions of:
Pathogen
Pathogenicity
Virulence
Toxigenicity ?

Answer 1

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

Question 2

What are exotoxins?

Answer 2

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 during disease. Act via a variety of diverse mechanisms.

Question 3

What mechanisms can determine virulence ?

Answer 3

Adherence Factors
Biofilms
Invasion of Host Cells and Tissues
Toxins – endotoxins and exotoxins

Question 4

Why have exotoxins?

Answer 4

What selective advantages do exotoxins give to the bacteria?
•Cause disease? – but actually this may help transmission of disease, however in severe disease host may be a literal and evolutionary dead end. •However with many toxins the disease causing activity may be not be the primary function. Other activities •Evade immune response•Enable biofilm formation •Enable attachment to host cells. •Escape from phagosomes •All allowing for colonisation, niche establishment and carriage - Evolutionary advantage.

Question 5

How can exotoxins be classified? Why does this create problems?

Answer 5

As very diverse group of proteins and many ways to classify.•Classification can be by the toxins activity .1.Membrane Acting Toxins – Type I

2. Membrane Damaging Toxins – Type II
3. Intracellular Toxins – Type III•

This classification has its problems – •Many toxins may have more than one type activity.•As mechanisms better understood this classification tends to break down.

Question 6

What do we know about Type I- membrane acting toxins?

Answer 6

Act from without the cell.
Interfere with host cell signaling by inappropriate activation of host cell receptors.
Target receptors include •Guanylyl cyclase → ↑ intracellular cGMP•Adenyl cyclase → ↑ intracellular cAMP•Rho proteins•Ras proteins

Question 7

What do we know about Type II- Membrane Damaging Toxins?

Answer 7

Cause damage to the host cell membrane.
1.Insert channels into host cell membrane. •β sheet toxins e.g. S.aureus α – toxin, δ toxin, PVL•α helix toxins – e.g. diphtheria toxin
2.Enzymatical damage e.g. S. aureus β- haemolysin, PSM
OR
1.Receptor mediated- not interfering with receptor function just interacting with receptor to allow for formation of pore(which can go on to damage ,membrane)
2.Receptor Independent - may attach to membrane and damage by altering phospholipid configuration

Question 8

What do we know about Intracellular toxins – type III?

Answer 8

Active within the cell – must gain access to the cell
•Usually 2 components – AB Toxins.
•Receptor binding and translocation function – B
•Toxigenic (enzymatic) – A
•May be single or multiple B units e.g. Cholera toxin AB5 (have electrostatic interactions)

AB toxins tend to be covalently linked

Question 9

Exotoxins are able to induce what inflammatory cytokines for release? (name examples) and what mechanisms are involved in this?

Answer 9

examples of inflammatory cytokines:
IL1, IL1β, TNF, IL 6,δ interferon, IL18

Mechanisms:
•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 βand IL18 e.g. S. aureus toxin A, PVL.

Question 10

How can toxins be inactivated?

Answer 10

oxins can be inactivated using formaldehyde or glutaraldehyde → toxoids

•Toxoids are inactive proteins but still highly immunogenic – form the basis for 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.
• Tetanus – pooled human immunoglobulin. Specific or normal.
• Botulism – horse antibodies

•Experimental and research – monoclonal antibodies

Question 11

What is Clostridium difficile? (Microbiology and Epidemiology)

Answer 11

Microbiology:
•gram-positive bacillus.
•anaerobic.
•spore-forming.
•toxin-producing
.•can be carried asymptomatically in the gut. 
•has 3 potent toxins.

carried in GI tract as a commensal

Epidemiology:
•Common hospital acquired infection worldwide.
•Spread by ingestion of spores – remain dormant in environment.
•Coloniser of the human gut up to 5% in adults.
•Risk factors – antibiotic use, age, antacids & prolonged hospital stay.

Question 12

What do we know about antibiotics impacts on c Difficile?

Answer 12

Thought to act by disrupting the microbial ecosystem within the gut. Antibiotics impacts microbiota

• Antibiotics provide a competitive advantage to spore forming anaerobes over non spore forming anaerobes.
• Allows C. difficile colonisation and growth.
• All antibiotics have potential for causing disease.
• Some antibiotics worse than others
• 2nd and 3rd generation cephalosporins
• Quinolones
• Clindamycin?
• Others less likely:
• Aminoglycosides
• Trimethoprim
• vancomycin

Question 13

How does toxin interact with membrane? check ppt slide for summary (dont need to know too much detail)

Answer 13

Cytotoxin A - TcdA coded by tcdA gene
Cytotoxin B – TcdB coded by 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.

toxin interacts gets internalised
forms pore mediated by hydrophobic domain, allow for release of active component A of toxin, interacts with raw proteins, leading to inactivation by glycosylation
proteins abilities are affected leads to changes in structure of cell eg cytoskeleton breakdown can also lead to apoptosis etc

overall briefly:

1. Toxins binding to specific host cell receptors
2. Toxins internalisation
3. endosome acidifcation
4. pore formation in the endosome
5. GTD release from the endosome to the host cell cytoplasm
6. RHO GTPases inactivation by glycosylation
7. Downstream effects within host cells

Question 14

What is used to diagnose C Difficile?

Answer 14

Clinical signs and symptoms•Raised white cell count in blood. •Detection of organisms and toxins in stool•2 phase test1.Glutamate dehydrogenase – detects if C. difficile organism present. 2.Toxin enzyme linked immunosorbent assay (ELISA) for TcdA and TcdB toxins.•Detection of tcdA and tcdb genes – PCR •Colonoscopy – pseudomembranous colitis

Question 15

What options are there for C Difficile treatment?

Answer 15

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.

Question 16

What is Verocytotoxin Escherichia coli (VTEC) disease?

Answer 16

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: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.

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 camp grounds

.•Very low infectious dose

Question 17

What does the mechanism in (VTEC) involve?

Answer 17

Bind to receptor globotriaosylceramide Gb3 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.

Question 18

What do we know about the pathogenesis of STEC?

Answer 18

STEC closely adheres to the epithelial cells of the gut mucosa. •The route by which Stx is transported from the intestine to the kidney and other tissues is debated, 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.

Question 19

What do we know about STEC disease?

Answer 19

Can be severe and life threatening
•Children < 5 years  greatest risk
•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.

Question 20

What does diagnosis and treatment of STEC involve?

Answer 20

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