Bacterial pathogens and disease 1 (exotoxins)

Question 1

Define pathogen

Answer 1

A microorganism capable of causing disease

Question 2

Define pathogenicity

Answer 2

The ability of an infectious agent to cause disease

Question 3

Define virulence

Answer 3

The quantitative ability of an agent to cause disease

Question 4

Define toxigenicity

Answer 4

The ability of a microorganism to produce a toxin that contributes to the development of disease

Question 5

What are exotoxins?

Answer 5

→ 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 6

What mechanisms can determine virulence?

Answer 6

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

Question 7

Why have exotoxins? What selective advantages do exotoxins give to the bacteria?

Answer 7

→ 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

1. Evade immune response
2. Enable biofilm formation
3. Enable attachment to host cells.
4. Escape from phagosomes
5. All allowing for colonisation, niche establishment and carriage - Evolutionary advantage.

Question 8

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

Answer 8

→ 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 9

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

Answer 9

→ Act from without the cell
→ Interfere with host cell signalling by inappropriate activation of host cell receptors.

→ Target receptors include:
1. Guanylyl cyclase → ↑ intracellular cGMP
2. Adenyl cyclase → ↑ intracellular cAMP
3. Rho proteins*Ras proteins

Question 10

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

Answer 10

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. Enzymatic 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 11

What do we know about Intracellular toxins – type III?

Answer 11

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 12

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

Answer 12

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 13

How can toxins be inactivated?

Answer 13

Toxins 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 14

What is Clostridium difficile? (Microbiology and Epidemiology)

Answer 14

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 15

What do we know about antibiotics impacts on c Difficile?

Answer 15

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 16

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

Answer 16

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:

→ Toxins binding to specific host cell receptors
→ Toxins internalisation
→ Endosome acidifcation
→ Pore formation in the endosome
→ GTD release from the endosome to the host cell cytoplasm
→ RHO GTPases inactivation by glycosylation
→ Downstream effects within host cells

Question 17

What is used to diagnose C Difficile?

Answer 17

Clinical signs and symptoms:

→ Raised white cell count in blood.
→ Detection of organisms and toxins in stool
→ 2 phase test
1.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 18

What options are there for C Difficile treatment?

Answer 18

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 19

What is Verocytotoxin Escherichia coli (VTEC) disease?

Answer 19

→ 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 20

What does the mechanism in (VTEC) involve?

Answer 20

→ 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 21

What do we know about the pathogenesis of STEC?

Answer 21

→ 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 22

What do we know about STEC disease?

Answer 22

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 23

What does diagnosis and treatment of STEC involve?

Answer 23

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