MIC 4124 9a - Journal Club III Salmonella

Goal of paper

Investigating SopD2 effector's mechanism of action of disrupting host endocytic trafficking by inhibiting Rab7

Why is the paper useful?

It is an example of how researchers discover an an effector protein's mechanism of action and why understanding host-pathogen interactions is important in pathogenesis research

Salmonella pathogenesis

Intracellular pathogen that resides in vacuoles

Effectors are important for mediating infection:

- >30 effectors produced

- secreted by two type III secretion systems (T3SSs)

Which serovar of Salmonella is the focus of the paper?

The non-typhoid serovar S. typhimurium

How many T3SS does salmonella use to secrete effectors into non-phagocytic cells? What are they?

Two T3SSs

- SPI-1 T3SS

- SPI-2 T3SS

SPI-1 T3SS role

turned on before infection; predominantly involved in inducing pathogen uptake into SCV

SPI-2 T3SS role

turned on after uptake into SCV; predominantly involved in promoting bacterial survival, replication and spread

Which stage of infection does the paper focus on?

mid-late stages of infection, which are controlled by SPI-2 T3SS effectors

Salmonella infection steps (7)

1. SPI-1 T3SS effectors induce invasion ruffle → bacterial uptake into SCV

2. Endocytic pathway begins to target SCVs

3. SPI-2 T3SS effectors secreted to promote bacterial survival & replication

4. Salmonella inhibits lysosomal pathway

5. SCVs migrate to the perinuclear region and multiply near Golgi

6. Effectors induce the formation of tubules (Salmonella-induced tubules (SITs)) that extend to the cell periphery

7. SCVs migrate along microtubules to cell periphery (needed for cell-to-cell spread)

Which step of salmonella infection is the topic of the journal club paper?

Salmonella inhibits lysosomal pathway

Endocytosis

a process by which proteins, molecules and pathogens are internalized into cells

- uptake into compartments called endosomes

Intracellular trafficking

the process by which intracellular compartments move around the cell

The possible fates of intracellular endosomes (3)

- degradation by lysosomes

- sent to Golgi

- recycled back to plasma membrane

Are there many different types of endosomes? If so, how are they differentiated?

Yes, each different compartment has a unique composition of membrane proteins (ex: small GTPases) and membrane lipids (ex: PIs)

- helps researchers determine compartment identity

Three 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 in the field already?

- In order to survive in the cell, Salmonella inhibits trafficking of endocytic compartments to lysosomes (but we don't know how)

- this phenotype is effector-driven, and is controlled by the SPI-2 T3SS

What was not known in the field yet?

- Which effector(s) are responsible for endocytic inhibition

- What host protein does the effector(s) target in host cells?

- What is the effector's mechanism of action?

Major Questions of the paper (4)

- Which Salmonella effector targets trafficking to lysosomes?

- What region of the effector protein is responsible for this phenotype

- What host protein does the Salmonella effector target?

- How does this host-pathogen interaction affect the host target's function?

DQ-BSA Assay

Fluorescence-based assay used to label degradative compartments (Lysosomes)

DQ-BSA

fluorescent dye (fluorophore) covalently linked to the protein BSA (bovine albumin)

Lysosome

a membrane-bound organelle that contains degradative enzymes; break down cell matter or invading microbes

Many degradative enzymes are activated in what type of environment?

an acidic environment, hence why mature lysosomes have low pH

As early endosomes mature...?

their internal pH drops

- Early endosome (pH ~6.3) → Mature lysosome (pH ~4.7)

Fluorophore

molecule that can emit light in the form of fluorescence after excitation

How does the DQ-BSA Assay work?

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

- It interacts with the endocytic pathway; when it encounters lysosomes, the BSA backbone is cleaved to release fluorescent peptides

DQ-BSA assay in normal host trafficking

DQ-BSA containing endosome is trafficked to lysosome. Host lysosome cleaves BSA so, red fluorescent compartments are active and easily visible

DQ-BSA assay when host trafficking is suppressed

The dye cannot make it to lysosomes so, there will be little/no fluorescence

DQ-BSA Assay: Salmonella Infection protocol (Pulse-chase experiment)

- Salmonella Infection: to allow time for Salmonella to inhibit host endocytic trafficking

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

ΔssaR mutant

Mutant lacking SPI-2 T3SS that should not inhibit host trafficking

Pulse-chase experiment setup (assay validation)

Invasion Salmonella 8h → DQ Red BSA 1h pulse → 4h chase → microscope + analysis

Epithelial cells (HeLa) infected with GFP-expressing S. typhimurium

- Salmonella labelled in green (GFP)

- Cleaved DG-BSA at lysosomes in red

Pulse-chase experiment on WT and ΔssaR mutant salmonella results

- Lysosomes in non-infected cells are bright red

- Lysosomes in WT infected cell is darker (expected because Salmonella inhibits trafficking to lysosomes)

- Lysosomes in ΔssaR infected cell is bright red

Quantification of pulse chase experiment

Wild-type infected cells have lower intensity of red signals

What experiment did the authors perform to determine which effector impairs lysosome trafficking?

DQ-BSA assays comparing Salmonella effector knockout lines

- ΔsopD2 mutant

Pulse-chase experiment results on ΔsopD2 mutant

trafficking is comparable to the non-infected cells, red signal is as bright as cells not infected with Salmonella

- Knocking out sopD2 reverses the trafficking inhbition of Salmonella

- SopD2 is necessary to inhibit lysosome trafficking

Which other effectors were knocked out? Did they reverse trafficking inhibition?

ΔsopB and ΔsopD mutants were made. No, red light intensity was still reduced in these infected cells indicating that Salmonella was still able to inhibit trafficking.

What do we know about SopD2?

- SopD2 secreted by SPI-2 T3SS at the same time when the trafficking inhibition occurs

- SopD2 is important for virulence (mouse infection model)

- SopD2 localizes to SCVs and late endocytic compartments (the sites of trafficking inhibition)

SopD2 and SopD relationship

they are paralogs

- SopD is found in all Salmonella species

- SopD is found in S. enterica only (as a pseudogene in some strains ex: S. typhi)

Paralogs

Two or more genes that are thought to have arisen from gene duplication in a common ancestor

Are SopD and SopD2 functionally similar?

Not really, they show functional divergence

- SopD: secreted by SPI-1 T3SS during early infection

- SopD2: secreted by SPI-2 T3SS during mid-late infection

But they both contribute to virulence

How did the authors investigate the sole effects of SopD2?

They expressed SopD2 inside host cells. Cells were transfected with a plasmid containing GFP and SopD2. No other effectors can complicate observations.

Can SopD2 alone inhibit lysosome trafficking?

Yes, in cells transfected with SopD2 the red signal (DQ-BSA) was much lower than the non-transfected cells. So trafficking was inhibited in the cells transfected with SopD2.

- SopD2 is sufficient to inhibit lysosome trafficking

Was trafficking inhibition observed in cell transfected with other effectors?

No, trafficking was not inhibited in cells transfected with SifA another effector.

Primary sequence of SopD2 protein

The membrane targeting domain (amino acids 1-75) help SopD2 reach late endosome lysosomes (where it carries out its function)

3D structure of SopD2 vs. SopD

Extensive structural homology through majority of structure, but the structures of SopD2 and SopD are different at the N-termini

How did the authors determine which region of SopD2 is responsible for the phenotype?

SopD2 Truncation Expression transfection experiment. Incomplete segments of SopD2 were used transfect host cells.

- To test whether the structural difference b/w SopD2 and SopD is necessary to inhibit lysosomal trafficking

What region of SopD2 is responsible for the phenotype?

The N-terminal region

- The SopD2 version with only the N-terminal side was still able to inhibit lysosomal trafficking.

- Amino acid segments 1-150 and 1-125 were able to inhibit trafficking comparable to the whole SopD2 protein