Lecture 3 host factors

Question 1

Why do we need new antibiotics?

Answer 1

• (Multi) drug-resistant pathogens are increasing in resistance against antibiotics
• Less antibiotics created in the clinic because it becomes more difficult to find new ones
• New antibiotics are not well bought
• Bacteria have found resistance against a lot of known pathways

Question 2

What are ways the bacteria have adapted? What makes gram- bact. more resistant than gram+?

Answer 2

1) Loss of porins
2) Beta-lactamases in periplasmic space (Beta-lactamase: breaks down beta-lactam antibiotics, e.g. ampicillin)
3) Overexpression transmembrane efflux pump
4) Antibiotic-modifying enzymes
5) Target mutations
6) Ribosomal mutation or modification
7) Bypass targets
 Due to their distinctive structure, Gram-negative bacteria are more resistant than Gram-positive bacteria

Question 3

Maraviroc & Plerixafor are viral entry inhibitors. What do they do?

Answer 3

They limit entry of the virus by blocking the receptor
- CXCR4 inhibitor: Plerixafor: lacking oral availability (brought in intravenous) and cardiac disturbances
- CCR5 inhibitor: Maraviroc  succesful in combination with other viral inhibitors
(Receptor antagonists)

Question 4

Why target intracellular pathogens?

Answer 4

• Intracellular pathogens rely to various extends on the host for their success. By blocking key host proteins, Replication could be limited
• Certain bacteria are obligated to use intracellular life and therefore easy to target
• Bacteria that can survive outside are more difficult to target since they do not necessarily rely on intracellular life

Question 5

What kind of cell behavior can salmonella modify once inside?

Answer 5

• transport

- transcription

Question 6

What is a way to prevent salmonella growth?

Answer 6

Interfering with the acidification could inhibit growth of Salmonella.

Question 7

What is the kinome of an organism?

Answer 7

kinome of an organism = the complete set of protein kinases encoded in its genome.
A protein kinase = a kinase which selectively modifies other proteins by covalently adding phosphates to them.

Question 8

What protein is essential for salmonella maturation? How was this concluded?

Answer 8

Rab7. SiRNA with CTRL: still able to mature. SiRNA with Rab7: unable to mature.

Question 9

What do Rab7, T35S and LAMP do?

Answer 9

Rab7 = regulates transport. 
LAMP = marker.
T35S = mediates vacuole creation.

Question 10

Why should SiRNA experiment be validated? How do you do that?

Answer 10

Validation is important, as siRNA can have off-target effects. Knockdown confirmed by qPCR, but best is Western Blot, since this is when you measure actual protein levels.

Question 11

The experiment concerning chemical inhibitors revealed that there was no correlation between literature and screen results. The only inhibitor that worked was..

Answer 11

H-89: een PKA en AKT/PKB (PKB = andere naam voor AKT) inhibitor (protein kinase A, betrokken bij intracellulaire overleving)

Question 12

Do the inhibitors have efficacy in vivo?

Answer 12

Mice were infected with Salmonella (SL1344) at day 0
When mice showed symptoms of disease, they were injected twice with 4 mg AKT inhibitor
- Addition of kinase inhibitor lead to more survival. However, more lethality due to cancer. Therefore it cannot be said that the inhibitors were effective.

Question 13

How does H-89 (AKT inhibitor) block salmonella proliferation?

Answer 13

It inhibits the acidification and therefore replication. Step is blocked in Salmonella replication, remaining in the early stage and therefore no spreading

Question 14

What can be concluded when silencing SopB regarding AKT?

Answer 14

o Delta SopB (effector secreted by Salmonella) is needed to activate AKT. When delta SopB is silenced. It can be seen that Salmonella cannot mature. Therefore, it can be said that Salmonella activates AKT during infection.

Question 15

What is one of the most modified GTPases in cancer?

Answer 15

Ras. Active mutant Ras-like Rho proteins: stress cell (stripes through the cell) Active mutant Rac: border effect

Question 16

What is the general switch mechanism for Ras-like proteins?

Answer 16

Signal -> GDP -> GEF: on -> GTP -> Effector on.

GTP -> GAP: off -> GDP -> effector off.

• GAP is the off switch for GTP
  o Inhibition of GAP therefore means effector remaining active
• GEF is the on switch for GTP
  o Inhibition of GEF therefore means effector remaining inactive

Question 17

+- how many GTP-binding proteins are there in 1383 genomes and what makes a difference between them?

Answer 17

• There are more than 38000 GTP-binding proteins in 1383 genomes
• All have a very similar structure/folding, only the interacting surface makes the difference
  o E.g. difference in charge

Question 18

What does the loaded spring mechanism do?

Answer 18

Basic switch structure that explains switch from GDP to GTP-bound state. Grey part does not change at all, but 2 elements contain 2 amino-acids that are bound to the phosphate in the middle (‘springs that are loaded’). They change the structure when switching.

Question 19

What do RAB GTPases do regarding salmonella survival?

Answer 19

Intracellular transport is regulated by RAB GTPases. Intracellular transport is essential for Salmonella survival.

Question 20

How does salmonella use AKT, Rab14, AS160 & SopB for maturation?

Answer 20

Secretion SopB -> Akt
Akt->inactivation AS160 (GAP)
Inactive AS160 (GAP) -> active Rab14
Rab14, maturation into an acidified rab positive replication compartment , the SCV

Rab14 is a GTPase that is essential for intracellular transport of salmonella. Rab14 is turned on and off by GAP & GEF. The GAP = AS160.
There is also AKT, secreted by salmonella, which is able to phosphorylate and thus turn off GAP. This results in Rab14 remaining active. -> salmonella can mature further -> beneficial for survival.

• Rab14 recruitment is also sopB dependent: when sopB cannot activate PKB/AKT, no recruitment seen
• Phosphorylation of PKB/AKT inactivates its GAP activity, keeping the Rab14 active

Question 21

Rab14 is not present on the salmonella containing vacuole (SCV). Maturation most likely regulated by a rab14 effector. How do we identify the effector? Explain.

Answer 21

Two hybrid system ( First attempt: Mass spectrometry, however this failed)

o 1 bait, fused to DNA binding domain
o 1 prey, fused to activator domain
o When they bind, protein is activated and therefore protein-protein interaction can be observed
- Rabs need to be active to interact. By making active locked mutants, they can be used in a Y2H

Question 22

What other Rab proteins does AS160(GAP) regulate?

Answer 22

AS160 regulates Rab2A, Rab8A and Rab14

Question 23

Explain the Rab14 Y2H interaction with nischarin and its binding partners.

Answer 23

1) Nischarin = prey (dual effector) which interacts with GTP-bound Rab14 and PI(3)P (strengthens the bond) to direct the maturation of early endosomes to Rab9-containing late endosomes.

1) vesicle + Rab14 + nischarin
2) Loss of Rab14 and accumulation of Rab9
3) Eventually Rab9 accumulates and nischarin is lost

Question 24

What happens when nischarin is blocked? /Rab9/Rab14?

Answer 24

o Experiments show that Nischarin blockage leads to prevention of acidification. Which is essential in Salmonella survival
o Knockdown of Nischarin and Rab9 and Rab14 affect survival. Suggesting they are important for creating a replicative niche