Journal Club Salmonella Final

Endocytosis

a process by which proteins, molecules and

pathogens are internalized into cells from the outside in endosomes

Intracellular Trafficking:

the process by which

intracellular compartments move around the cell

Many possible intracellular fates of endosomes

- degradation by lysosomes

- sent to Golgi

- recycled back to plasma membrane

why are there so many intracellular fates of endosomes

there are many different types of endosomes and it depends what is internalized by them

Characteristics of different types of host trafficking pathways

- proteins on the membrane (including

regulatory proteins like small GTPases)

- membrane lipids

Important Trafficking Pathways

- Endocytic Pathway

- Secretory Pathway

- Retrieval Pathway

Endocytic Pathway

vesicles

internalized from the plasma

membrane traffic toward early

endosomes, late endosomes, and

ultimately lysosomes for degradation

Secretory Pathway

vesicle trafficking from ER to either plasma membrane or lysosomes

Retrieval Pathway

vesicle trafficking

from endosomes to plasma membrane

or Golgi

What was known already in the field in the scope of the journal paper

- Salmonella must evade the lysosomal pathway

in order to survive in host cells

- Salmonella inhibits the lysosomal pathway;

more specifically, it inhibits trafficking of

endocytic compartments to lysosomes

- This phenotype is effector-driven, and is

controlled by the SPI-2 T3SS

DQ-BSA Assay

Labelling of Degradative Compartments (Lysosomes)

DQ-BSA

fluorescent dye covalently linked to the protein BSA

How does DQ-BSA detect lysosomes

Many degradative enzymes are activated in an acidic

environment, so able to see based on the activity status of the lysosome

How does DQ-BSA work

- internalized into cells by endocytosis in a quenched (non-fluorescent) form

- undergoes interactions with endocytic pathway; when

encounters lysosomes, BSA backbone is cleaved to release

fluorescent peptides

what happens if the dyes don't make it to the lysosomes

there will be little to no fluorescence

DQ-BSA Assay: Salmonella Infection

1. Salmonella Infection: to allow time for Salmonella to inhibit host endocytic trafficking

2. DQ-BSA: treat ("pulse"), wash off, and incubate ("chase") to allow dye to traffic to lysosomes

What is the purpose of the fluorescence-based trafficking assay?

To test whether Salmonella effectors impair lysosome trafficking

What does red fluorescence indicate in this assay?

Delivery of DQ-BSA to lysosomes

What does green fluorescence indicate?

Presence of GFP-labeled Salmonella

What is the effect of wild-type Salmonella on lysosome trafficking?

It suppresses trafficking, reducing red signal

What is the effect of the ΔssaR mutant on lysosome trafficking?

It does not suppress trafficking, red signal remains normal

What does the quantification show about WT vs ΔssaR infection?

WT-infected cells have significantly lower red signal than ΔssaR-infected or non-infected cells

What does the ΔsopD2 mutant show in the trafficking assay?

Normal trafficking similar to non-infected cells, red signal is restored.

What does the ΔsopD mutant show in the trafficking assay?

No effect on trafficking, red signal remains suppressed

What does this experiment conclude about SopD2?

SopD2 is necessary for inhibiting lysosome trafficking

What is the purpose of using effector knockouts (ΔsopB, ΔsopD, ΔsopD2)?

To identify which effector is responsible for trafficking inhibition

What is SopD2?

An SPI-2 T3SS effector secreted during lysosome trafficking inhibition

Why is SopD2 important?

It is required for virulence in mouse infection models

Where does SopD2 localize?

To Salmonella-containing vacuoles (SCVs) and late endocytic compartments

What is still unknown about SopD2?

Its host target and mechanism of action

What is the relationship between SopD and SopD2?

They are paralogs from a gene duplication event

Which Salmonella species have sopD and sopD2?

sopD is in all Salmonella; sopD2 is in S. enterica only

When is SopD expressed and by which system?

During early infection via SPI-1 T3SS

When is SopD2 expressed and by which system?

During mid-late infection via SPI-2 T3SS

Do SopD and SopD2 both contribute to virulence?

Yes, both contribute to virulence in models like mice

What was unknown about SopD and SopD2 at the time?

Their host targets and mechanisms of action

How can the role of SopD2 in trafficking be tested independently?

By expressing GFP-tagged SopD2 in host cells without infection

Why is transfection used instead of infection?

To isolate the effect of SopD2 without interference from other effectors

What does the assay show when SopD2 is expressed alone?

SopD2 alone can suppress lysosome trafficking, seen as reduced red DQ-BSA signal

What does GFP-SopD2 expression in host cells demonstrate?

It is sufficient to inhibit lysosome trafficking

How does GFP-SopD2 affect DQ-BSA trafficking?

It reduces red signal compared to non-transfected and GFP-only cells

What control confirms SopD2 specificity?

GFP-SifA does not inhibit trafficking, showing SopD2-specific effect

What does the quantification show about SopD2-transfected cells?

They have significantly lower DQ-BSA fluorescence than controls

Which domains are present in SopD2?

A translocation domain for T3SS secretion and a membrane-targeting domain for localization

Where do SopD and SopD2 differ structurally?

At the N-terminus

What technique helped reveal differences between SopD and SopD2?

Crystal structure analysis

How similar are SopD and SopD2 overall?

They show extensive structural homology through most of the protein

What was the goal of the SopD2 truncation experiment?

To identify which region of SopD2 is required for trafficking inhibition

Which SopD2 truncations inhibit lysosome trafficking?

Truncations including residues 1-150 or smaller still inhibit trafficking

What happens when the N-terminal region (1-150) is removed?

Trafficking inhibition is lost

What does the quantification show about residues 1-150?

They are sufficient for trafficking inhibition, similar to full-length SopD2

Which region is important for SopD2's inhibitory function?

The N-terminal region

What is Rab7?

A GTPase that regulates lysosome maturation, trafficking, and fusion

Why is Rab7 essential for lysosome function?

Without Rab7, lysosomes cannot mature or function properly

What does Rab7 control in the endocytic pathway?

Endosome maturation, late endosome/lysosome trafficking, and compartment fusion

Why is Rab7 a target for pathogens?

Disrupting Rab7 helps pathogens evade lysosomal degradation in host cells

How was the localization of SopD2 and Rab7 tested?

By co-expressing GFP-Rab7 and RFP-SopD2 in host cells

What does colocalization of SopD2 and Rab7 suggest?

SopD2 localizes to many of the same compartments as Rab7

What conclusion can be drawn from their colocalization?

SopD2 and Rab7 colocalize, supporting a potential interaction or targeting

How can Rab7 activity be assessed?

By using fluorescently tagged adapter proteins that bind active Rab7

What are adapter proteins for GTPases?

Host binding partners that localize only when the GTPase is active

What happens if a GTPase is inactive?

Its adapter protein should not localize to the membrane

Which Rab7 activity probes were used in this study?

GFP-tagged RILP (dynein adapter) and mCherry-tagged FYCO1 (kinesin adapter)

What is GFP-RILPC33 used for?

A fluorescent probe to visualize active Rab7 via RILP localization

What is LAMP1 used for in this assay?

A marker for compartments where Rab7 normally localizes

What does colocalization of RILP and LAMP1 indicate?

That Rab7 is active and recruiting its adapter protein

What does the merged image confirm about Rab7?

Strong colocalization of RILP and LAMP1 confirms Rab7 is active

What happens to RILP localization in control cells?

RILP strongly localizes to LAMP1-positive compartments

What happens to RILP localization when SopD2 is expressed?

RILP fails to localize to compartments and accumulates in the cytosol

How does SopD2 affect Rab7 activity?

SopD2 suppresses Rab7's ability to recruit its effectors (e.g., RILP, FYCO1)

What does the quantification show about SopD2 expression?

It significantly increases cytosolic localization of RILP and FYCO1

What is the overall conclusion about SopD2?

SopD2 can suppress Rab7 trafficking function in host cells

What is the effect of wild-type Salmonella on FYCO1 localization?

FYCO1 fails to bind SCV membranes, indicating Rab7 function is suppressed

What happens to FYCO1 localization in ΔsopD2 infection?

FYCO1 binding to membranes is restored, indicating Rab7 is active

What does the quantification show about FYCO1 and SopD2?

More FYCO1 colocalizes with SCVs in ΔsopD2 than in wild-type infection

What is the conclusion about SopD2 during infection?

SopD2 suppresses Rab7 trafficking function in host cells during infection

How was interaction between SopD2 and Rab7 tested?

Using a co-immunoprecipitation assay with tagged proteins in transfected cells

What does the Western blot show about SopD2-Rab7 interaction?

SopD2 binds to Rab7 regardless of its activation state (WT, CA, DN)

What do CA and DN Rab7 mutants represent?

CA = constitutively active (GTP-bound), DN = dominant negative (GDP-bound)

Does SopD2 prefer active or inactive Rab7?

No preference; it binds both forms equally

What does co-immunoprecipitation reveal about protein interactions?

It shows interaction but cannot distinguish if it is direct or indirect

Why is it important to test for direct interaction in host-pathogen studies?

To determine if the effector directly manipulates the host target

What experiment can distinguish direct from indirect protein interactions?

An in vitro binding experiment using purified proteins in non-eukaryotic systems

What does the in vitro binding assay test?

Whether SopD2 directly binds Rab7 outside of a cellular context

How were SopD2 and Rab7 produced for this assay?

Each was expressed and purified from bacteria, then incubated together

Which form of SopD2 binds Rab7 in vitro?

The N-terminal region (1-150) of SopD2 binds Rab7

What does the Western blot confirm?

SopD2 directly interacts with Rab7 via its N-terminal region, with no preference for GDP or GTPγS

Does SopD2 binding depend on Rab7 activation state?

No, SopD2 binds both GDP- and GTPγS-loaded Rab7

What is Mant-GDP?

A fluorescent GDP analog used to study protein-nucleotide interactions

What happens when Mant-GDP is bound to a GTPase?

It fluoresces

What happens when Mant-GDP is released from the GTPase?

Fluorescence decreases

How is nucleotide exchange studied using Mant-GDP?

Load GTPase with Mant-GDP, then add GTP; loss of fluorescence indicates exchange

How is Rab7 nucleotide exchange measured in vitro?

By tracking fluorescence loss of Mant-GDP over time via spectrophotometry

What does slower loss of fluorescence indicate in this assay?

Reduced nucleotide exchange, meaning Rab7 remains inactive

How does SopD2 affect Rab7 nucleotide exchange?

SopD2 slows nucleotide exchange, suppressing Rab7 activation

What is the control in this assay?

Rab7 with BSA, showing normal fluorescence decrease from active exchange

What is the main function of SopD2 during Salmonella infection?

It inhibits delivery of endocytic cargo, including SCVs, to lysosomes

What host protein does SopD2 directly target?

Rab7, a host GTPase involved in endocytic trafficking

Which region of SopD2 is responsible for Rab7 targeting?

The N-terminal region

What is the consequence of Rab7 suppression by SopD2?

Rab7 can't bind its adapter proteins RILP and FYCO1, impairing trafficking