Virulence Factors (in disease process)

Question 1

Name the key VFs of C.difficile

Answer 1

• TcdA
• TcdB
• CDT (binary toxin)
• Spo0A
• CspC germinant receptor

Question 2

Describe the elements of the TcdA/B PaLoc

Answer 2

TcdD = (+) regulator = sigma factor that mediates recognition of RNA polymerase to the promotors

TcdE = holin like protein from phages that forms pores allowing export of TcdA/B through cell wall

TcdC = (-) regulator anti-sigma factor that sequesters TcdD

Question 3

How do TcdB and TcdA cause damage

Answer 3

Bind and enter epithelial cells on the GI tract
Enter through endocytosis
Monoglucosylate small GTPases like Rho and CDC4 that polymerise actin
Actin depolymerises and causes cytoskeletal damage to cells
Makes them rounder and destroys tight junctions between them
Allows tcdB to enter between them and penetrate deeper tissue

Question 4

How do TcdB and TcdA escape vesicles

Answer 4

Theory:
The pH change during acidification induces a conformational change
Translocation domain forms a pore in the membrane
Allows the enzymatic domain to be released and act on Rho GTPases

Autoproteolytic

BUT:
Requires Insp6 which is in the cytosol = mechanism unknown

Question 5

How does CDT cause damage

Answer 5

Coded by its own PaLoc
Also inhibits actin polymerisation causing breakdown of cell tight junctions

Question 6

What role does Spo0A play in virulence?

Answer 6

Sporulation master regulator so aids in transmission of disease

Cannot form spores or transmit disease without Spo0A

Question 7

How does CspC aid in virulence?

Answer 7

Detects germinants - initiates ability to cause disease

Question 8

Name the key VFs of S.aureus

Answer 8

Blocking neutrophil
- Recruitment (neutrophil recognition factors E.g. cytokines)
- Opsonisation (block complement pathway with matching VFs)
- Killing (resist ROS)

Question 9

Give 4 methods, aided by VFs, that S.A. uses to evade neutrophil killing

Answer 9

• Creates electrostatically charged membrane to repulse antimicrobials
• Creates a coat of polysaccharides
• Produces dismutases that break down ROS species in to molecular oxygen (SodM, SodA)
• Excretes nucleases to escape NETosis

Question 10

Give 2 genetic diseases resulting in decreased neutrophil killing

Answer 10

Chronic Granulatomous Disease -> Hereditary disease:
- Mutated gene
-> Neutrophils cannot make H2O2 (oxidative agent). More open to S.A. infections as they are caspase (+)
-> Form granulomas as well = clumps of cells that block urinary tract

Congenital neutrophina -> not enough neutrophils produced

Question 11

How can the carotenoid pigment play a role as a VF?

Answer 11

Helps protect against reactive oxidising species (seen it do this with photosynthesis as well)

Question 12

Give the VF S.A. uses for immune evasion

Answer 12

SpA -> binds IgG in inactive state

Question 13

Give another role of SpA

Answer 13

Binds TNFR-1 and induces inflammatory response in the airways
Inhibits neutrophil recruitment there

Question 14

Give 3 VFs S.A. uses for adhesion and nutrient acquisition

Answer 14

• Isd -> Fe acquisition (kills blood cells)
• Surface Protein A (Spa) -> prevents opsonisation (binds IgG)
• ClfA (binds fibrinogen), FnBP (binds to fibronectin) -> adhesion (There are many more adhesins)

Question 15

Give the 4 main exotoxins produced by S.A.

Answer 15

• Haemolysin-a
• Leukocidins
• Exfoliative toxins A and B
• Phenol soluble Modulins

Question 16

Describe how Haemolysin Alpha (Hlsa) causes damage

Answer 16

Binds the metalloprotease ADAM10 and upregulates its activity. Cleaves more Cadherin-E and destroys adheren junctions between cells allowing deeper penetration into tissue

Question 17

Describe how leukocidins cause damage. give an example

Answer 17

Form pores in leukocytes, mainly neutrophils causing lysis
E.g. PVL is associated with community acquired MRSA

Question 18

Why do these VFs target neutrophils?

Answer 18

Make up 60% of leukocytes

Question 19

How do exfoliative toxins A and B cause disease

Answer 19

They are proteases that destroy the epithelial layer, causing separation of skin (sloughing) = causes scalded skin syndrome

Question 20

How many enterotoxins does S.A. have

Answer 20

9

Question 21

Give 2 examples of enterotoxins

Answer 21

TSST-1
Coagulase

Question 22

How does coagulase help S.A.

Answer 22

Produces 2 coagulases: Coa and vWbp
- Hijacks our blood clotting mechanism to PROTECT us against us
- Binds prothrombin and forms staphylothrombin
- This causes fibrinogen to form more fibrin
- Creates a clot/abscess around the cells so neutrophils cannot enter (physical barrier)

Question 23

What do superantigens do?

Answer 23

Cause an overreaction of the immune system leading to cytokine storm and toxic shock

Question 24

Give an example of damage caused by superantigens

Answer 24

TSST-1 in TSS
- Forms a bridge between MCHII antigen presenting cells and T-lymphocytes
- Causes crosslinking
- Non-specific T-cell activation
- Release of proinflammatory cytokines
- Cytokine storm
- Toxic shock

Question 25

What disease do the S. aureus enterotoxins cause?

Answer 25

Food poisoning -> interactions of the enterotoxins with the immune system following ingestion of contaminated food leads to food poisoning symptoms as the body attempts to expel the bacteria.

Question 26

How many types of Haemolysin are produced by Staphylococcus aureus?

Answer 26

3 types of Haemolysin

Question 27

Does S. aureus use all of its possible virulence determinants?

Answer 27

No, not all strains carry all virulence determinants present in the species -> this means that different strains have varying capability of causing different disease states, associated with different disease.

Question 28

What are Phenol Soluble Modulins? - S. aureus

Answer 28

- These are small peptides exotoxins associated with community acquired MRSA

Question 29

What is MRSA?

Answer 29

Methicillin Resistant Staphylococcus Aureus strains

Question 30

What is the ROLE of Phenol Soluble Modulins? - S. aureus

Answer 30

- Pro-inflammatory -> interact with the immune system to release a variety of cytokines + neutrophil motility - Associated with more severe Osteomyelitis (inflammation of bone marrow) than in hospital acquired MRSA - Aggregation of alpha type PSMs into fibrils aids biofilm formation / integrity

Question 31

What are the mechanisms of methicillin resistance by S. aureus?

Answer 31

S. aureus alters the target of beta-lactams, the expression of PBP2A (MecA) -> PBP2A is a transpeptidase with low affinity for b-lactams and its presence increases the redundancy of cell wall synthesis systems -> causing the inhibition of PBP1a and PBP1b to no longer be lethal.

Question 32

How is MecA thought to have developed in MRSA?

Answer 32

Horizontal gene transfer

Question 33

Organisation of Mec locus:

Answer 33

Inverted repeats flank the mec A gene complex (mecA mecRI mecI) and the ccr gene complex -> its structure suggests that it is spread by transduction.

Question 34

What are the major virulence factors of:

Answer 34

Polysaccharide capsule