Journal paper Flashcards
What does this paper focus on
Mid-late stages of infection, using T3SS SPI2
How does the pathogen invade the endocytic pathway?
Is the vacuole SCV a type of endosome? What gives its identity?
Yes. lipid composition and proteins on the membrane
What does the pathogen inhibit
Trafficling towards lysosomes
What fluorophore is used to dye lysosomes?
Is the fluorophore introduced before or after salmonella infection?
DQ-BSA will be cleaved in lysosomes and emit fluorescence
After: through ““pulse and chase”
How does the fluorescence based assay work?
Salmonella = green
DQ-BSA = red
If in same spot = trafficking lysosomes weren’t inhibited by effectors = bad
What was known about effector SopD2 before?
SPI2 effector localises to SCVs and late endosomes/lysosomes but mechanism of action was unkown
Why do both SopD and SopD2 exist?
gene duplication
How did we see the SopD2 inhibits lysosome traffiking?
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
Difference between SopD and SopD2 in S. typhimurium (stages)?
SopD2 is for late stages, SopD is for early stage
They have different N-terminals
How do we test if SopD2 works alone to inhibit trafficking?
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
In the intensity graphs, high % means
NO INHIBITION OF TRAFFICKING
What did we see in the SOPD2 works alone assay?
There is no fluorescence merge, there is a decrease in %, meaning that SopD2 INHIBITS lysosome trafficking on its own
How did we find out that the N-terminal of SopD2 inhibited trafficking?
Did truncations of SopD2. Only the ones with N-terminals present inhibit (% was lower = inhibition)
What is SopD2 targeting to inhibit trafficking?
Rab7 (GTPase that controls movement in both directions of compartments)
Rab7 is required for lysosome maturation. Rab7 is found ON SCVs
Do SopD2 and Rab7 localise to the same compartments?
UYes
What are FYCO1 and RILP?
Adaptor proteins for
Kinesin (UP)
Dyenin (DOWN)
There are fluoresced
What does LAMP1 do?
Marker for SCV membrane
This is a marker for where Rab7 localises
What does RILP do?
It localises itself into LAMP1 compartments that also contain Rab7
What does SopD2 do to RILP’s localization?
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.
Does SopD2 interact direcly with Rab7 GTPase?
Yes
How does the co-immunoprecipitation assay work for SopD2 and Rab7?
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
What did we see in the co-immunoprecipitation assay?
Rab7 attaches to SopD2. But we don;’t know if it’s direct or not.
The version of Rab7 (GTP/GDP) dosen’t matter
What is the purpose of an in-vitro binding assay?
See if the proteins Rab7 and SopD2 are directly connecting with eachother, and how
How does in-vitro binding assay work?
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
What did the in vitro study show?
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
What does SopD2 do exactly to Rab7? (proven by nucleotide exchange assay)
It inhibits nucleotide (GTP/GDP) exchange! Supressess Rab7 activation
How does nucletide (GTP/GDP) exchange assay work?
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
To conclude, how does SopD2 promote tubule formation?
SopD2 dislodges Rab7 (responsible for ENDOGENOUS microtubule migration to happen).
This allows other effectors to use microtubules for SCV’s purposes
