Journal paper

Question 1

What does this paper focus on

Answer 1

Mid-late stages of infection, using T3SS SPI2
How does the pathogen invade the endocytic pathway?

Question 2

Is the vacuole SCV a type of endosome? What gives its identity?

Answer 2

Yes. lipid composition and proteins on the membrane

Question 3

What does the pathogen inhibit

Answer 3

Trafficling towards lysosomes

Question 4

What fluorophore is used to dye lysosomes?
Is the fluorophore introduced before or after salmonella infection?

Answer 4

DQ-BSA will be cleaved in lysosomes and emit fluorescence
After: through ““pulse and chase”

Question 5

How does the fluorescence based assay work?

Answer 5

Salmonella = green
DQ-BSA = red
If in same spot = trafficking lysosomes weren’t inhibited by effectors = bad

Question 6

What was known about effector SopD2 before?

Answer 6

SPI2 effector localises to SCVs and late endosomes/lysosomes but mechanism of action was unkown

Question 7

Why do both SopD and SopD2 exist?

Answer 7

gene duplication

Question 8

How did we see the SopD2 inhibits lysosome traffiking?

Answer 8

Wt cell inhibits trafficking (no fluorescence in same spot)
When SopD2 was taken off, there is no more trafficking (was comparable to non-infeccted cells)
y axis = % of control

Question 9

Difference between SopD and SopD2 in S. typhimurium (stages)?

Answer 9

SopD2 is for late stages, SopD is for early stage
They have different N-terminals

Question 10

How do we test if SopD2 works alone to inhibit trafficking?

Answer 10

Don’t infect cells with bacteria
Only with GFP tagged SopD2 plasmid

If there is inhibition of trafficking, DQ-BSA and SopD2 won’t be at the same place

Question 11

In the intensity graphs, high % means

Answer 11

NO INHIBITION OF TRAFFICKING

Question 12

What did we see in the SOPD2 works alone assay?

Answer 12

There is no fluorescence merge, there is a decrease in %, meaning that SopD2 INHIBITS lysosome trafficking on its own

Question 13

How did we find out that the N-terminal of SopD2 inhibited trafficking?

Answer 13

Did truncations of SopD2. Only the ones with N-terminals present inhibit (% was lower = inhibition)

Question 14

What is SopD2 targeting to inhibit trafficking?

Answer 14

Rab7 (GTPase that controls movement in both directions of compartments)
Rab7 is required for lysosome maturation. Rab7 is found ON SCVs

Question 15

Do SopD2 and Rab7 localise to the same compartments?

Answer 15

UYes

Question 16

What are FYCO1 and RILP?

Answer 16

Adaptor proteins for
Kinesin (UP)
Dyenin (DOWN)
There are fluoresced

Question 17

What does LAMP1 do?

Answer 17

Marker for SCV membrane
This is a marker for where Rab7 localises

Question 18

What does RILP do?

Answer 18

It localises itself into LAMP1 compartments that also contain Rab7

Question 19

What does SopD2 do to RILP’s localization?

Answer 19

RILP dosen’t localise strongly to LAMP1 compartments anymore. The RILPS are found in the cytosol instead. This also happens to FYCO.

So, SopD2 suppresses Rab7 trafficking function by somehow interacting with Rab7 during infection.

Question 20

Does SopD2 interact direcly with Rab7 GTPase?

Answer 20

Yes

Question 21

How does the co-immunoprecipitation assay work for SopD2 and Rab7?

Answer 21

Tranfect cells with tagged SopD2 and Rab7
Lyse the cells
Fish the tagged SopD2. Look at what is abound to it through western blot
What is boud will give a line

Question 22

What did we see in the co-immunoprecipitation assay?

Answer 22

Rab7 attaches to SopD2. But we don;’t know if it’s direct or not.
The version of Rab7 (GTP/GDP) dosen’t matter

Question 23

What is the purpose of an in-vitro binding assay?

Answer 23

See if the proteins Rab7 and SopD2 are directly connecting with eachother, and how

Question 24

How does in-vitro binding assay work?

Answer 24

Produce the tagged proteins Rab7 and SopD2 in bacteria
Isolate the proteins
Readd the GTP (as it falls off)
Add the proteins in solution in vitro with antibodies
Do western blot

Question 25

What did the in vitro study show?

Answer 25

SopD2 interacts with the Rab7 N-terminal, no matter if it’s GDP or GTP bound

SopD dosen’t interact with the Rab7 N-terminal

Question 26

What does SopD2 do exactly to Rab7? (proven by nucleotide exchange assay)

Answer 26

It inhibits nucleotide (GTP/GDP) exchange! Supressess Rab7 activation

Question 27

How does nucletide (GTP/GDP) exchange assay work?

Answer 27

Use Mant-GDP that fluoresceces
If GTP is present, ManGDP will be removed, leading to decrease in fluorescence (spectrophotometry)

If no decrease is seen, nucleotide exchange is inhibited by something

Question 28

To conclude, how does SopD2 promote tubule formation?

Answer 28

SopD2 dislodges Rab7 (responsible for ENDOGENOUS microtubule migration to happen).
This allows other effectors to use microtubules for SCV’s purposes