Virulence Factors (regulation)

Question 1

List all the regulatory mechanisms discussed in S.A.

Answer 1

• SpaA (agr independent) control of proteases
• agrBCDA locus controlling VF switch
• Bacterial interference
• MecA regulation

Question 2

What is the main environmental change is detected by SpaA and Agr. Why is this important

Answer 2

Growth phase change

Required for the shift from colonising to creating damage (when system has enough cells and not enough nutrients - starts causing damage)

Question 3

What is growth phase dependant VF production an example of?

Answer 3

Quorum sensing

Question 4

How does SpaA act independently of Agr?

Answer 4

Down regulates proteases that could cleave important VFs
Increases virulence and toxin production

Question 5

How does SpaA affect Agr - how does Agr respond

Answer 5

Upregulates Agr activity
Agr upregulates toxins and down regulates surface proteins

Question 6

Describe the components of the AgrBCDA locus

Answer 6

Genes:
AgrA = activator of P2 and P3
AgrC = sensor of AIP
Form a 2-component system together
AgrD = forms an 8 a.a. signalling molecule: AIP (pheromone)
AgrB = cleaves and cyclises AgrD alongside MroQ
MroQ = Cleaves AgrD’s N-terminal end (forming AIP and N-AgrD)

Divergent promotors: P2 and P3
P3 encodes toxins and RNAIII
P2 facilitates the transcription of the agr locus

Both require RNAII/RNAIII to function

Question 7

How does the AgrBCDA locus promote VF switch

Answer 7

AgrD is produced and cyclised into an AIP by AgrB and MroQ - creating a thioester linkage allowing it to be recognised

P2 is constituently produced so low levels of AIP are being made. It reaches a threshold level once cells are at late exponential phase

AgrC detects AIP at threshold level. Auto-phosphorylates itself and then phosphorylates AgrA. AgrA becomes activated and binds P2 to increase its expression and P3

= density dependent signalling

Creates auto-inducing (+) feedback cycle of upregulation
P3 creates RNAIII and this allows it to express toxins like Hla

Question 8

How does RNAIII control translation of surface proteins and toxins

Answer 8

• (+) regulates toxins by binding its mRNA as an antisense mRNA and removing the stem loop, freeing up the RBS
• (-) regulates surface proteins by binding as antisense mRNA which blocks the RBS and maintains the stem loop

Question 9

What is bacterial interference

Answer 9

S.A. creates 3 different groups of AIP with different order of amino acids

Question 10

How does bacterial interference help S.A.

Answer 10

Helps in mixed infections as the AIP can act on different strains of S.A. and have an inhibitory effect on toxin production if they are not within the same AIP group

Question 11

List all the regulatory mechanisms discussed in C.diff

Answer 11

• PaLoc for TcdA and TcdB (TcdD,E,C control) and CDT
• Spo0A cascade for sporulation
• Germination factors

Question 12

Describe the C.diff PaLoc

Answer 12

CdtD
CdtA
CdtE
CdtB
CdtC

Question 13

How does the C.diff PaLoc regulate toxin expression

Answer 13

CdtD -> (+) regulator of function - acts as a sigma factor to facilitate RNA Polymerase binding
CtdE -> acts like a holin from phages (forms pores and helps secretion of toxin A and B)
CtdC -> (-) regulator of function - acts as an anti sigma factor that binds and sequesters CdtD

Question 14

How have modifications in the C.diff PaLoc impacted its function

Answer 14

The 2003 Canada/USA outbreak saw an 18bp deletion in the CdtC gene
Caused hyper-virulence as this increased toxin function by removing any repression

Question 15

How does Spo0A function as a VF

Answer 15

It is the master regulator for sporulation which allows transmission

Question 16

How does Spo0A cause sporulation

Answer 16

Activated itself through phosphorylation by around 5 characterised sensor Histidine Kinases

Spo0A activation sets off a sporulation cascade that results in spores forming (endospore/mother cell)

Question 17

Why is a sequential cascade neccessary?

Answer 17

To create order in the process, so each spore layer forms correctly

Question 18

What factors affect sporulation

Answer 18

Nutrient starvation (C/N/P)

Question 19

How does C.diff ensure survival

Answer 19

Bet-hedging strategy -> keeps a small proportion of the population constantly in spore form.

Question 20

How does C.diff sense sporulation

Answer 20

Detecting germinants like primary and secondary bile salts
Using proteins like CspC

Question 21

How does S.aureus regulate resitance

Answer 21

Through regulation of MecA by MecI and MecR

Question 22

Describe the process of MecA expression

Answer 22

MecR sits in the membrane and detects levels of b-lactams
Metalloprotease domain in the cytosol which becomes activated
Hydrolyses MecI dimer sitting on the operator region
Frees MecA transcription

Creates PBP2A

Question 23

Why does PBP2A show resistance to b-lactams

Answer 23

Binds them with lower affinity
They are acetylated slower - this is the covalent bond that forms between b-lactams and PBP2A

This slows the rate of binding and creates a less stable complex

Question 24

How was the Mec element gained

Answer 24

From the environment by transduction (phage incorporation)

Question 25

What virulence factors are down/upregulated in stationary phase?

Answer 25

Downregulated surface proteins (e.g. adhesins and 'immuno-evasion proteins') and upregulated exoproteins (Dispersal

Question 26

Coordination organisation of mediated regulators in Staph. aureus:

Answer 26

There are 194 regulatory components in use by S. aureus; these are organised into a hierarchal network to better fine tune and control its response to the environment.

Question 27

What is Agrs?

Answer 27

Accessory gene regulators -> involved in the switch between surface protein and exoprotein expression given changes to the environment

Question 28

What is Sar?

Answer 28

Staphyloccocus accessory regulator-> DNA binding protein that binds to agr promoter region -> upregulates Agr expression

Question 29

What term is used to categorise the modifications of AgrD in the regulation of AIP?

Answer 29

Post-translational modifications

Question 30

How does the growth phase relate to the expression of the agr locus?

Answer 30

As the density of bacteria increases, the AIP concentration increased in the environment -> these signals are recognised by surrounding bacteria (process of quorum sensing) -> until reaching threshold activation -> gene switching

Question 31

AgrC activation:

Answer 31

AgrC binds to an octapeptide, this change in conformation allows it to autophosphorylate into a histidine protein kinase (AgrC-P).

Question 32

What does AgrC-P act on?

Answer 32

AgrC-P acts on AgrA

Question 33

What is AgrA?

Answer 33

AgrA is a regulator DNA binding protein -> activates the transcription of promoter 1 and 2

Question 34

Why is the AgrA activation circuit described as autoregulatory?

Answer 34

Increased AgrA -> Increased P2 and P3 -> increased AIP -> increased AgrA

Question 35

Example of RNAIII negative regulation of SPA - Staph

Answer 35

- RNAIII binds to the spa mRNA, binding over the shine Dalgarno sequence, blocking the binding site, inhibiting translation, downregulating spa production. - Additionally the binding to the mRNA forms a termination stem loop (change in secondary structure) activating the destruction of the mRNA.

Question 36

Example of RNAIII positive regulation of Haemolysin alpha - Staph

Answer 36

- In the absence of RNAIII -> the secondary structure of the 5’ of the mRNA transcript have complementary bases, leading to the formation of a stem loop and subsequent destruction, additionally this also blocks the RNA binding site. - With RNAIII, the RNAIII binds to the hla transcript, prevents the stem loop formation, freeing the ribosome binding site, allowing translation and production of the protein.

Question 37

SarA: Staph

Answer 37

- Repressor of proteases and up-regulator of AgrR - ^function dependent on position of binding which is dependent on environment - Rapid response to environment -Controls virulence determinants at all levels of expression