Lecture 9 - Journal Club

Question 1

1) What is endocytosis?

2) What is intracellular trafficking?

3) What are the possible intracellular fates of endosomes?

4) Are there only a certain type of endosomes?

Answer 1

1) a process by which proteins, molecules and pathogens are INTERNALIZED into cells. uptake into compartments called ENDOSOMES.

2) the process by which intracellular compartments move around the cell.

3)
- degradation by lysosomes
- sent to Golgi
- recycled back to plasma membrane

4) No, there are many different types of endosomes.

Question 2

Each different compartment has a unique composition of:

Why is this important?

Answer 2

1) Proteins on the membranes (including regulatory proteins like small GTPases)

2) membrane LIPIDS (ex, phosphoinositides)

*helps researchers determine COMPARTMENT IDENTITY

Question 3

What are the three important trafficking pathways in host?

Answer 3

1) endocytic pathway: vesicles internalized from the PM traffic toward early endosomes, late endosomes, and ultimately LYSOSOMES for degradation

2) secretory pathway: vesicle trafficking from ER to either PM or lysosomes

3) vesicle trafficking from endosomes to PM or golgi

*1 is the focus of the journal club paper

Question 4

Fill in the blanks regarding what was already known in the field already:

Salmonella must evade the _____ pathway in order to survive in host cells. It inhibits the ______ of ______ compartments to ______. This phenotype is _____-driven and is controlled by the ___ T3SS.

Answer 4

Lysosomal; trafficking; endocytic; lysosomes; effector; SPI-2

Question 5

What was not known yet in the field prior to this paper?

Answer 5

• what effector was responsible for inhibiting endocytic trafficking to lysosomes
• what host protein the effector targets in the host cell
• what the effector’s mechanism of action is

Question 6

What is the DQ-BSA assay?

Why is it used?

How does it work?

Answer 6

DQ-BSA: fluorescent dye (fluorophore) covalently linked to the protein BSA (bovine albumin).

It was used to label degradative compartments (lysosomes).

The DQ-BSA is internalized into cells by endocytosis in a QUENCHED (non-fluorescent) form. When it undergoes interactions with endocytic pathway and encounters lysosomes, BSA backbone is cleaved to release fluorescent peptides! This tells us that trafficking is occurring and there are active lysosomes.

If due cannot make it to lysosomes (trafficking is suppressed), little/no fluorescence!

Question 7

______ is a membrane bound organelle that contains DEGRADATIVE enzymes (break down cell matter/microbes). Many degradative enzymes are activated in an _____ environment (__ pH).

Answer 7

Lysosome; acidic; low

Question 8

What are the two parts of the DQ-BSA assay for a salmonella infection?

Answer 8

1) GFP-expressing S. Typhimurium infection: to allow time for Salmonella to INHIBIT host endocytic pathway

2) DQ-BSA: treat cells with dye (“pulse”), wash odd, and incubate in a regular media without dye (“chase”) to allow dye to traffic to lysosomes. PULSE and CHASE.

Question 9

To determine which salmonella effectors impair lysosome trafficking, the DQ-BSA assay was used. Before making use of this assay, researchers had to validate the assay.

How did they do this?

Answer 9

Salmonella is labeled green (GFP)
Red signal: delivery of DQ-BSA to lysosomes.

WT infection (should inhibit trafficking): WT infected cells have lower intensity of red signal (dye cannot make it to lysosome).

SPI-2 T3SS mutant infection (should not inhibit trafficking): High intensity of red signal (dye makes it to lysosome)!

Question 10

1) Which effectors were knocked out to assess which one impairs lysosome trafficking?

2) What were the controls used in the assay?

3) Which effector is necessary to inhibit lysosome trafficking?

Answer 10

1) sopB, sopD, and sopD2

2) They used WT (which should inhibit trafficking; decreased intensity of red signal) and SPI-2 T3SS mutant (should not inhibit trafficking; increased intensity of red signal).

3) SopD2!

SopB and SopD knockout yielded in WT results (decreased intensity of red signal), while SopD2 knockout yielded in high intensity of the red signal (comparable trafficking to non-infected cells), concluding that knocking it out reverses the trafficking inhibition.

Question 11

SopD2 is secreted by the ___ T3SS at the same time when the trafficking _____ occurs.

It is important for ______ (mouse infection model).

It localized to ____ and ___ endocytic compartments (the sites of trafficking inhibition)

Answer 11

SPI-2; inhibition

Virulence

SCVs; late

*therefore it was a candidate of interest
*unknown: host target and mechanism of action

Question 12

1) What are SopD2 and SopD? In which strains are they found?

2) When and from what are they secreted by?

3) What do they both contribute to?

4) Do they share the same roles in the host?

Answer 12

1) SopD2 and SopD are PARALOGS!! They are encoding genes thought to have arise from GENE DUPLICATION from a common ancestor.

sopD: in all salmonella.
sopD2: in S. enterica only; pseudogenes in S. Typhi.

2)
SopD: secreted by SPI1 T3SS during EARLY infection.
SopD2: secreted by SPI2 T3SS during MID-LATE infection.

3) Both contribute to virulence!

4) Evolved unique roles in the host!!

*host targets/mechanism of action unknown at the time of the paper

Question 13

Can SopD2 alone inhibit lysosome trafficking?

How was this determined?

Answer 13

Yes! SopD2 is sufficient to inhibit lysosome trafficking.

Researchers expressed SopD2 through transfection of a GFP-tagged mammalian expression plasmid in host cells. They did not infect the cells with the bacteria to prevent complication of observations by other effectors!

Then, they did the DQ-BSA pulse and chase assay.

The red fluorescence significantly decreased in cells infected by SopD2 compared to control plasmid with just GFP (meaning less trafficking than non-transfected cells!).

This trend was not observed with another SPI-2 T3SS effector, SifA.

Question 14

Briefly answer the following questions of SopD2 regarding its primary sequence:

1) What are the first 75 aa important for?

2) What are the first 150 aa important for?

Answer 14

1) Localization (membrane targeting domain)

2) Effector secretion T3SS (translocation domain)

Question 15

(T/F) SopD2 and SopD share extensive structural homology through all of the 3D structure.

Answer 15

False!

The structures are different at the N-termini.

However, they share extensive structural homology through MAJORITY of the 3D structure.

Question 16

How was it determined what region of SopD2 is responsible for the phenotype? What region is it?

Answer 16

SopD2 truncation expression!

They transfected mammalian expression plasmids with different parts of SopD2 (1-75 aa, 1-150aa, 31-64aa, 1-125aa, etc) and did the DQ-BSA assay.

They found truncations of 1-150, 1-125 aa to have significantly decreased red signal (meaning lysosome trafficking was not occurring) compared to non-transfected cells!

Therefore, the N-terminal region of SopD2 is important for inhibiting trafficking to lysosomes.

Question 17

(T/F) Compartments use regulatory proteins (ex. Rab GTPases) to control trafficking. Host binding proteins called adapter proteins bind to molecular motors to conjugate compartments to microtubules.

Answer 17

True!

Question 18

1) What is Rab7? What does it control?

2) Why is it an ideal target for a pathogen to evade lysosomal function?

Answer 18

1) Rab7 is a critical lysosome regulator that controls:
a) endosome maturation (into lysosomes)
b) trafficking of late endosomes and lysosomes
c) fusion of endocytic compartments

2) Lysosomes cannot mature or function properly without Rab7!! All vacuolar pathogens need to find a way to evade lysosome function in order to survive inside host cells.

Question 19

What are FYCO1 and RILP?

Answer 19

These are adapter proteins (host binding proteins) of Rab7 that bind to molecular motors to conjugate compartments to the microtubules!

FYCO1 binds kinsein (movement towards cell periphery)

RILP binds dynein (movement towards nucelus)

Question 20

To know if SopD2 is targeting Rab7, researchers first looked to see if the localize to the same place in host cells.

How did they do this?

What did they find?

Answer 20

They expressed SopD2 and Rab7 inside host cells, each with an epitope tag for detection.

They found that SopD2 and Rab7 do colocalize to the same compartments inside host cells!

Question 21

1) When do adapter proteins bind to GTPases?

2) How can we asses a GTPase function?

3) What are GTPase activity probes?

4) What were the Rab7 activity probes used in the paper?

Answer 21

1) Adapter proteins for GTPases are host binding partners that PREFERENTIALLY bind ACTIVE GTPase. If a GTPase is active on a membrane, its host binding partner should localize to the membrane. If it is not active, its host binding partner should not localize.

2) Take advantage of adapter proteins to probe GTPase function.

3) Fluorescent tagged versions of the adapter protein that can be used to visualize GTPase function.

4) GFP-tagged RILP (dynein adapter) and mCHERRY-tagged FYCO1 (kinsein adapter). This was to assess Rab7 function.

Question 22

1) What is LAMP1?

2) What can we know about Rab7 if there is strong localization of RILP to LAMP1 compartments?

Answer 22

1) LAMP1 is the marker for compartments where Rab7 localizes (late endosomes/lysosomes)

2) That would tell us that Rab7 is active!! RILP (adapter protein) only bind their GTPases when the GTPases are active.

Question 23

Does SopD2 manipulate Rab7 function?

How do we know?

Answer 23

Yes! SopD2 can suppress Rab7 trafficking function!

In control cells, RILP localizes strongly to LAMP1 compartments (meaning Rab7 is active).

In cells expressing SopD2, RILP does not localize strongly (meaning Rab7 is inactive).

Question 24

We know that when we transfect cells with SopD2 plasmids, there is a manipulation of Rab7 functions. However, does SopD2 manipulate Rab7 function DURING INFECTION (natural levels of SopD2)?

Answer 24

Yes! SopD2 can suppress Rab7 trafficking function during infection.

There was salmonella WT strain infected cells and sopD2 knockout strain infected cells. These were transfected with mCherry-FyCO1.

In WT, FYCO1 cannot bind membrane to mediate trafficking (low level of colocalizing to LAMP1 compartments)

In knockout strain, FYCO1’s ability to bind membrane is restored (high levels of colocalizing to LAMP1 compartments)

Question 25

Now that scientists knew that SopD2 localizes with Rab7 and suppresses its function, they wanted to know if these two proteins interact.

What was the first assay they did to test that?

Answer 25

Co-immunoprecipitation assay!

Expressed SopD2 and Rab inside host cells each with an epitope tag for detection (transfect with 2 plasmids). Flag-tagged SopD2 and RFP-tagged Rab7.

Then, they lysed the cells and performed an antibody purification via the epitope tag (if you purify one of the proteins, is the other one stuck to it?)

They purified SopD2 using its flag-tag and looked to see if Rab7 is stuck to it by a Western Blot.

Question 26

1) In the Western Blot analysis performed to see if SopD2 interacts with Rab7, what is the difference between bound and lysates?

2) What is the difference between CA and DN Rab7? Why were they used in the WB?

Answer 26

1) Lysates: what went into the experiment. Bound: what came out of the experiment.

2)
CA: constitutively active (locked into active form)
DN: dominant negative (locked into inactive form).

If we compare it to the WT, the binding preference of the effector can provide clues to its function!

Question 27

Briefly describe the WB analysis used to assess if SopD2 interacts with Rab7 and what was concluded from it.

Answer 27

Cells were lysed and SopD2 was purified using its FLAG tag. Researches wanted to know if Rab7 was stuck to it.

Detection was done using the antibody against RFP (which is bound to Rab7). This tells us if Rab7 is stuck to the SopD2.

No bands in the RFP only lane (control) for the bound; SopD2 can interact Rab7.

No nucleotide binding preference with Rab7 (equally bound to all versions of it: WT, CA, and DN).

Question 28

1) What is a disadvantage of Co-immunoprecipitation assay?

2) Why was this needed to be overcome?

3) How was this overcome in the paper?

Answer 28

1) Co-immunoprecipitation can’t differentiate between direct (proteins directly contact each other) and indirect interactions (a protein in between).

2) In the study of host-pathogen interactions, it is important to know whether the effector is DIRECTLY MANIPULATING the candidate host target.

3) Express and purify both proteins of interest in NON-EUKARYOTIC cells before setting up interaction experiment. This is called in vitro binding experiment.

Question 29

1) Briefly describe the steps of in vitro binding assay.

2) Why must you reload the GTPase with GTP/GDP during the assay? What is the difference between GTPyS loaded and GDP loaded?

3) Why is 2 important?

Answer 29

1) In vitro binding assay:
- Bacterial strain expressing tagged protein —> culture bacteria —> purify each protein independently (His-HA-tagged effector and GST-tagged Rab7) —> in vitro binding —> affinity purification.

2) GTPases bind GTP/GDP, but during the purification step, they can fall off. Therefore, before the in vitro binding experiment, you must reload the GTPase with GTP/GDP.

GTPyS loaded = active GTPase
GDP loaded = inactive GTPase

3) The binding preference of the effector can provide clues to its function.

Question 30

What was determined through the WB analysis of in vitro binding assay?

Answer 30

SopD2 DIRECTLY interacts with Rab7 via its N-terminus (unlike SopD).

Question 31

1) What is Mant-GDP?

2) What are the two steps to study nucleotide exchange using purified GTPase (in vitro nucleotide exchange assay)?

3) Why was this assay done in the paper?

Answer 31

1) A fluorescent analog: modified GDP used to study protein-NT interactions. Different properties upon binding protein: not bound - minimal fluorescence, bound - fluorescent.

2)
a) load Mant-GDP to GTPase, purified from bacteria (demonstrates fluorescence)

b) Nucleotide exchange in presence of GTP: Mant-GDP is removed, causing decreased fluorescence.

3) To determine the effect of SopD2 on Rab7 GTPase function.

Question 32

1) In an in vitro nucleotide exchange assay, the fluorescence is measured by __________.

2) What was seen in the presence of SopD2?

3) What was seen in control?

4) What does this tell us?

Answer 32

1) Spectrophotometry

2) Nucleotide exchange slows, suppressing GTPase activation (increased fluorescence)!

3) Fluorescence decreases as nucleotide exchange occurs.

4) SopD2 INHIBITS NUCLEOTIDE EXCHANGE, suppressing Rab7 activation!

Question 33

Fill in the blanks regarding the summary of the paper:

1) Effector SopD2 inhibits the delivery of ______ cargo (including _____) to _______.

2) SopD2 directly targets the host GTPase _____.

3) The __-terminus of SopD2 mediates the phenotype with ____.

4) SopD2 inhibits ___ _______ exchange, this suppresses GTPase _______ and impairs the ability of the GTPase to bind its adapter proteins for trafficking (_____ and ______).

Answer 33

1) delivery; SCVs; lysosomes

2) Rab7

3) N; Rab7

4) Rab7 nucleotide; activation; RILP; FYCO1

Question 34

How does SopD2 promote tubule formation (SITs)?

Answer 34

SopD2 dislodges endogenous trafficking apparatus controlled by Rab7, allowing other effectors to hijack and control it!