Bacterial Pathogens and Diseases I (Exotoxins)

Question 1

Define pathogen

Answer 1

Pathogen: A microorganism capable of causing disease.

Question 2

Define Pathogenicity

Answer 2

Pathogenicity: The ability of an infectious agent to cause disease.

Question 3

Define Virulence:

Answer 3

Virulence: The quantitative ability of an agent to cause disease.

Question 4

Define Toxigenicity

Answer 4

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

Question 5

Describe the mechanism of virulence

Answer 5

• Adherence Factors – molecules in surface of cells that allow for attachment and start colonisation
• Biofilms – complex structure of macromolecu les
• Invasion of Host Cells and Tissues
• Toxins – endotoxins and exotoxins

Question 6

What are exotoxins?

Answer 6

• Heterogeneous group of proteins produced and secreted by living bacterial cells. they are actively secreted by bacteria.
• Produced by both gram negative and gram-positive bacteria.
• Cause disease symptoms in host during disease.
• Act via a variety of diverse mechanisms.

Question 7

Why have exotoxins?

Answer 7

What selective advantages do exotoxins give to the bacteria?
• Cause disease? – 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.

Question 8

What are other activities of exotoxins?

Answer 8

• 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 9

What exotoxins are produced by SA?

Answer 9

Haemolytic toxins
cause cells to lyse by forming pores which damage the membrane
Important cause of features of S. aureus disease.
α,β,γ, toxins ,Panton Valentine Leukocidin (PVL), LukAB, LukED, LukMF
Phenol soluble modulins PSM
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?

Question 10

Describe The host cell membrane attacking toxins of Staphylococcus aureus and their roles beyond host cell lysis

Answer 10

Phagocytosis of invading
• bacteria is followed by fusing of the phagosome to the lysosome, resulting in destruction of the bacteria. S. aureus alpha (α) and phenol-soluble modulin
• (PSM) toxins inhibit fusing of the lysosome. This enables the bacteria to escape from the phagosome into the cytoplasm, allowing intracellular niche
• establishment and replication. (B) PSM toxins target cohabiting bacterial species within established niches, aiding in competition for resources and
• competitive exclusion of nonkin isolates. (C) PSM toxins have surfactant properties in vitro, enabling sliding movement across agar surfaces in the
• absence of traditional mobility structures such as flagella and pili. (D) Pore-forming toxins are involved at each step of S. aureus biofilm formation. During
• the initial cell attachment phase, alpha-toxin is involved in establishing cell-to-cell contacts, enabling the formation of secondary biofilm structures. In the
• later stages of the biofilm lifestyle, extracellular matrices develop, surrounding the cells within the biofilm. In the presence of extracellular DNA (eDNA),
• beta-toxin covalently cross-links with itself, adding to this extracellular nucleoprotein biofilm matrix and contributing to the formation of complex biofilm
• secondary structuring. Detachment from the mature biofilm allows for dispersal to new sites of infection. PSM toxins are involved in this stage of the biofilm
• lifestyle, aiding release of cell clusters from the main body of the biofilm.

Question 11

Describe the genetics of exotoxins

Answer 11

Most encoded in chromosomal genes.
 Many encoded by extrachromosomal genes in plasmids and lysogenic
bacteriophages (via transduction)
 Can be transferred between bacteria through plasmids via conjugation, transduction
and transfection. Therefore, virulence factors are mobiles.
 Scarlett fever- caused by streptococcus (some strains only cause sore throat) which
has been lysogenically converted- infected by a bacteriophage that has carried a
gene encoding a haemolytic toxin- now causes Scarlett fever. Therefore, not all
strains but certain toxigenic strains.
 Diphtheria- also cause by bacteriophage introducing new virulence factors.

Question 12

How can exotoxins be classified?

What are the problems with classifying toxins?

Answer 12

1. Membrane Acting Toxins – Type I
2. Membrane Damaging Toxins – Type II
3. Intracellular Toxins – Type III
   • This classification has its problems –
   o Many toxins may have more than one type activity.
   o As mechanisms better understood this classification tends to break down.

Question 13

How do membrane acting toxins 1 act, interfere and target?

Answer 13

• Act from without the cell by interfering with signalling receptors on cell surface to producing binding ligand.
• Ligand-receptor complex activates intracellular second-messenger cascades that results in the breakdown of metabolic processes in the cell.
• Many receptors available- guanyl cyclase (GTPase) receptor is a common one. Many hormones bind. Activating cAMP/cGMP and act on Rho and Ras proteins, involved in microfilament and actin filament processing- endosome trafficking. Dysregulates processes.
• E. coli Stable heat toxin is an example- Sta toxin binds to receptor, increasing cGMP and changes activity of chloride secretion pathway (affects pores). Increase in secretion of chloride and fluid follows osmotically, leading to watery diarrhoea.
• Causes little direct tissue damage but through metabolic changes.

Question 14

How do Membrane Damaging Toxins – Type II work?

Answer 14

Damage membrane like pores. Disrupts homeostasis in cells.
Cause damage to the host cell membrane.
1. Insert channels into host cell membrane.
1. β sheet toxins e.g. S.aureus α – toxin, δ toxin, PVL
2. α helix toxins – e.g. diphtheria toxin
2. Secrete enzymes to break down membrane. e.g. S. aureus β- haemolysin, PSM
OR
1. Receptor mediated
2. Receptor Independent

Damage membrane by inserting channels in the membrane (receptor mediated).
There are beta sheet toxins and alpha helix toxins.
 Secrete enzymes to break down membrane.
 Can be receptor mediated or receptor independent.

Question 15

Describe the action of Intracellular Toxins – Type III

Answer 15

Go inside cells and damage from inside.

2. A- activity. B – binding.
3. Bind to receptors and get internalised into cell – degradation – active component – enzymatically biologically .
4. Diagram shows it being cleaved and …
5. Vulnerable to heat.
6. Other toxins are heat stable.
7. Simple heating does destroy heating of heating.
8. Active within the cell – must gain access to the cell
9. Usually 2 components – AB Toxins
10. Receptor binding and translocation function – B
11. Toxigenic (enzymatic) – A
12. May be single or multiple B units e.g. Cholera toxin AB5

Question 16

Describe the actions of enzymatic component A

Answer 16

ADP-ribosyl transferases- covalently modify adenyl cyclase to disrupt levels of ADP in
cell.
 Glucosyltransferases (gtd)- modify RNA, affecting protein synthesis.
 Proteases- e.g. clostridial neurotoxins produced in botulism and tetanus.
 Adenylcylases- e.g. in anthrax. Affects levels of adenyl cyclase in cells.
 Other actions- Proteins assemble creating needle to penetrate host cell and
secreting toxins.

Question 17

How do exotoxins cause dysregulation of the immune system?

How do exotoxins act as super antigens

Answer 17

• Creates inflammatory cytokine cascade.
• Overproduction of inflammatory cytokines and immunopathology.
• Exotoxin acts as super antigens.
• Antigen presenting cells take in antigens, process them and present them in the context of MHCII. Bacterial superantigens create same result without any antigen presentation.
• Covalently cross-links MHCII to the T cell receptor, thus activating T cells- not specific as no antigen presentation so 30-40% of all T cells activated.. They produce loads of cytokines leading to septic shock and tissue damage.
• Scarlett fever action via superantigen. As does toxin produced by staphylococcus, known as the toxic shock syndrome toxin 1 (TSST1).

Question 18

What are toxoids, vaccines and antobodies

Answer 18

• Toxins can be inactivated using formaldehyde or glutaraldehyde → toxoids (proteins which induce antibodies)
• Toxoids are inactive proteins but still highly immunogenic – form the basis for vaccines.
o Tetanus Vaccine
o Diphtheria
o Pertussis (acellular).*
• Treatment of toxin mediated disease can be affected by administering preformed antibodies to the toxin
o Diphtheria antitoxin – horse antibodies.
o Tetanus – pooled human immunoglobulin. Specific or normal.
o Botulism – horse antibodies
• Experimental and research – monoclonal antibodies

Question 19

Describe Clostridium difficile:

What is it?

How many toxins does it produce?

What are the risk factors?

What type of toxin does it produce?

Answer 19

• Spore-forming and can survive for months and can’t kill easily- soap and water- not alcohol wipes.
• Gram-positive and can be carried asymptomatically in the gut (up to 5% of biome). Supressed by commensal gut bacteria.
• 3 toxins produced.
• Common hospital acquired infection.
• Risk factors- antibiotics, age, antacids, prolonged hospital stay.
• C. difficile cytotoxins- receptor binding domain, hydrophobic domain and GTD and protease domain. Therefore, they are type III AB toxins.
• Receptor mediated endocytosis and internalised, acidification of endosome and release of active component by forming pore in endosome. Disruption of cAMP/cGMP and Rho/Ras proteins.
• Cytotoxic and cytopathic effects.
• Effects- patchy necrosis with neutrophil infiltration (Pseudomembranous colitis). Plaques with dead cells, pus and bacteria.
• Causes acute water diarrhoea.

Question 20

Describe Verocytotoxin Escherichia coli (VTEC):

What type of toxin does it encode?

Answer 20

• E. coli that has acquired a gene that encodes a Shiga-toxin (Stx).
• Also known as STEC- Shiga-toxin producing E. coli.
• Very damaging and causes haemorrhagic, cute water diarrhoea (similar to dysentery).
• Can also cause haemolytic uraemic syndrome due to pathology.
• Most common STEC is O157:H7.
• Can identify the difference between normal E. coli and O157:H7 by growing sample on sorbitol MacConkey agar (SMac) and normal E. coli will show up red, as it ferments sorbitol but the other doesn’t.
• Not all identified this way- only more common ones.
• Kids can catch this by petting farm animals- kidney disease.
• Potent and potentially fatal.
• Very common in animals- contaminated food can cause haemolytic-uremic syndrome.
• Stx is a type III AB5 exotoxin. Enzymatic activity is N-Glycosidase (damages ribosomal RNA and inhibits protein synthesis).
• B binding domains gives specificity and the ability to bind endothelial and epithelial cells.
• Enters body via tissue damage of gut and migrates and binds to endothelial receptors in kidney cells- haemolytic uraemic syndrome.
• Inflammatory response- disseminated thrombosis of bacteria. This blocks kidney glomeruli.

Question 21

Describe the Pathogenic mechanism of the shiga toxin

Answer 21

Pathogenic mechanism:

1. Toxin binds receptor.
2. Endocytosis.
3. Transported through Golgi into RER.
4. Active subunit cleaved off and glycosylates to 28S ribosomal RNA.
5. Prevents protein synthesis.