MIC 4124 9b - Journal Club III Salmonella

Trafficking

Intracellular compartments moving along microtubules using molecular motors

Which molecular motors are used for trafficking?

Dynein and kinesin

Dynein direction of movement

movement toward nucleus

Kinesin direction of movement

movement toward cell periphery

What do compartments use to control trafficking?

Regulatory proteins such as

- Rab GTPases

- Adapter proteins (host binding partners)

Which RabGTPase could SopD2 be targeting?

Rab7

Rab7 role

A critical lysosome regulator that controls:

- Endosome maturation (into lysosomes)

- Trafficking of late endosomes and lysosomes

- Fusion of endocytic compartments

Importance of Rab7 to lysosomes

Lysosomes cannot mature or function properly without Rab7

Therefore Rab7 is an ideal target for a pathogen to evade lysosomal function

Do SopD2 and Rab7 localize to the same place in host cells?

Yes, SopD2 and Rab7 colocalize to the same compartments

- SopD2 and Rab7 were visualized in a host cell using an epitope tag. They were visualized in the same compartments

GTPase adapter proteins

host binding partners that preferentially bind active GTPase

- if GTPase is active on a membrane, its host binding protein should localize to the membrane

- if the GTPase is inactive, its host binding partner should not localize

Kinesin binding adapter protein

FYCO1

Dynein binding adapter protein

RILP

GTPase activity probes

fluorescent tagged versions of the adapter protein that can be used to visualize GTPase state (active or inactive)

- allows us to assess Rab7 function

Rab7 activity probes used in paper

- GFP-tagged RILP: dynein adapter

- mCherry-tagged FYCO1: kinesin adapter

GTPase activity probe control experiment

In non-infected human epithelial cells, The RILP channel localizes strongly with the LAMP1

- tells the that Rab7 on these compartments are active

LAMP1

host marker that tells us where Rab7 localizes

What happens to RILP in cell expressing SopD2?

RILP does not localize strongly

- The expression of SopD2 reduces the extent of colocalization of LAMP1 (Rab7) and RILP colocalization

- SopD2 can suppress Rab7 trafficking function

Can FYCO1 mediate trafficking?

No, FYCO1 cannot bind membrane to mediate trafficking

Does SopD2 manipulate Rab7 function during infection?

Yes, SopD2 can suppress Rab7 trafficking function during infection

- In human epithelial cells infected with ΔsopD2 mutant S. typhimurium are able to colocalize LAMP1 and FYCO1

After determining that SopD2 and Rab7 colocalize, what was the authors' next question?

Do SopD2 and Rab7 interact?

What assay did the authors use to determine Rab7 and SopD2 interaction?

Co-immunoprecipitaton Assay

What is the co-immunoprecipitation assay?

- Transfect host cells with 2 plasmids with flag-tagged SopD2 and RFP-tagged Rab7

- Lyse the cells

- Use an antibody targeted at one protein, purify the antibody and what it binds and check if other proteins are stuck to it

- Follow up with western blot to determine which proteins are bound

CA GTPase

constitutively active GTP (locked in active form)

DN GTPase

dominant negative GDP (locked in inactive form)

Co-immunoprecipitaton Assay results

In antibodies targeted at SopD2, WT Rab7, CA Rab7, and DN Rab7 were all also 'picked up' as they were bound to SopD2

- SopD2 can interact with Rab7

- There is no nucleotide binding preference with Rab7 (SopD2 binds Rab7 regardless of status)

Can co-immunoprecipitation differentiate between direct and indirect binding?

No, just tells us if the molecules bind

What assay was used to determine is the interaction between SopD2 and Rab7 was direct or indirect?

In vitro binding (IVB) assay

In vitro binding (IVB) assay protocol

- Express & purify both proteins of interest in non-eukaryotic cells

- Mix them together in a tube

- Using an antibody for one protein purify the sample

- Perform a Western blot to see whether the other protein stuck

Controls for the in vitro binding assay

- SopD: (-) control, we know that SopD does not do anything

- GTPγS: active GTPase (cannot be hydrolyzed)

- GDP: inactive GTPase

To see whether SopD2 preferentially binds a form of GTPase

In vitro binding assay results

1-150 SopD2 N-terminus and 150-319 SopD2 C-terminus both incubated with Rab7.

- Rab7 only binds the 1-150aa sequence of SopD2

- SopD2 directly interacts with Rab7 via its N-terminus

What assay did the authors use to determine the effect of SopD2 on Rab7 GTPase function?

In Vitro Nucleotide Exchange Assay

Mant-GDP

modified GDP used to study protein-nucleotide interactions

When Mant-GDP is bound what happens to its fluoresence? Unbound?

- not bound: minimal fluorescence

- bound: fluorescent (inactive GTPase)

Fluorescence measured by spectrophotometry (excitation 335nm, emission 448nm)

In Vitro Nucleotide Exchange Assay purpose

To study nucleotide exchange using purified GTPase

- GTPase is purified from bacteria

- Mant-GDP is loaded onto to GTPase→ demonstrates fluorescence

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

In Vitro Nucleotide Exchange Assay results

Presence of SopD2: nucleotide exchange slows, suppressing GTPase activation

Control: fluorescence decreases as nucleotide exchange occurs (Naturally)

Therefore, SopD2 inhibits nucleotide exchange, suppressing Rab7 activation

Review: Journal Club Paper findings (4)

- Effector SopD2 inhibits the delivery of endocytic cargo (including SCVs) to lysosomes

- SopD2 directly targets the host GTPase Rab7

- The N-terminus of SopD2 mediates the phenotype with Rab7

- SopD2 inhibits Rab7 nucleotide exchange; this suppresses GTPase activation and impairs the ability of Rab7 to bind its adapter proteins for trafficking (RILP and FYCO1)

How does SopD2 promote tubule formation?

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

Future directions of the paper

- Do other effectors contribute to suppressing lysosome function?

- Do other intracellular bacterial pathogens use similar mechanisms?

- Does SopD2 use this mechanism to target other GTPases?

- Could SopD2 be a possible therapeutic target?