Volume 8 issue 10 October 2007
Intracellular trafficking of Pseudomonas ExoS, a Type III cytotoxin
Qing Deng, Yue Zhang, and Joseph T. Barbieri

Supplemental Figure 1: ExoS traffics to perinuclear region in a dynamin independent manner. HeLa cells were transfected with either GFP fused wild-type Dynamin or dominant negative Dynamin (Dynamin K44A). 24 h post transfection, HeLa cells were infected with PA103, ΔU, T::Tc carrying pUCP-ExoS (G-A-)-HA at an MOI of 8:1. Four h post infection, cells were washed, fixed and immuno-stained with anti-HA antibody to visualized Type III delivered ExoS. Alexa 594 conjugated transferrin was also added to the transfected HeLa cells 10 min before fixation as positive control. DAPI is used to stain the nuclei in the overlay image. Stars indicate cells transfected with dominant negative dymanin. While transferrin uptake was inhibited, ExoS still accumulated in the perinuclear region. Wild type dynamin transfected cells were used as negative control. Bar: 100 µM.

Figure 1 (.jpg)

Supplemental Figure 2: ExoS does not colocalize with either Tfn or lysosomes. HeLa cells were transfected with RFP-ExoS and then fixed18 h post transfection. Tfn was added 10 min before fixation. Lysosomes were visualized by immuno-staining of endogenous LAMP 1 proteins. Nuclei are shown by DAPI staining. Bar: 50 µM.

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Supplemental Figure 3: Type III delivered ExoS co-localizes with transfected ExoS. HeLa cells were transfected with GFP-ExoS, then infected with PA103, ΔU, T::Tc carrying pUCP-ExoS (G-A-)-HA at an MOI of 16:1 and incubated at 37°C for 2 h. Cell were washed and incubated further with antibiotics to kill extra-cellular bacteria. Cell were fixed every 30 min and ExoS was visualized by immuno-staining against HA epitope. Nuclei are shown by DAPI staining. Fluorescence intensity along the line in overlay image is shown. Green, transfected ExoS; Red, Type III delivered ExoS; Blue, DAPI. Note higher amount of Type III delivered ExoS in the periphery in the early time points. Trace lines were placed across both the peri nuclear and the periphery region to compare the ratio of transfected/infected ExoS. Bar: 50 µM.

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Supplemental Figure 4: Movement of ExoS positive vesicles along microtubules. A) HeLa cells were transfected with GFP-Tubulin and RFP-ExoS. 18 h post transfection, real time cell images were taken by spinning disk confocal microscopy. Bar: 50 µM. B) Montage of the boxed region are enlarged to show movement of a vesicle (arrow) along a microtubule track.

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Supplemental Figure 5: Neither Nocodazole nor MβCD inhibit Type III delivery of ExoS. HeLa cells were pretreated with nocodazole or MβCD, infected with PA103-β¢;U,T::Tc(pUCP-ExoS)-HA (G-A-)at an MOI of 16:1, and then incubated at 37°C. At the indicated time, cells were either fixed and probed for HA epitope (ExoS) or lysed, separated by SDS-PAGE, and probed for HA epitope (ExoS). Bar: 200 µM.

Figure 5 (.jpg)

Supplemental Movie 1 for Fig 3(A): Fast moving and stationary populations of ExoS. HeLa cells were transfected with GFP-Rab9 and RFP-ExoS. 18 h post transfection, real time cell imaging were taken under Spinning Disk Confocal microscopy. Green, Rab 9; Red, ExoS. Both dynamic- and static- ExoS were observed with both populations partially co-localized with Rab 9.

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Supplemental Movie 2 for Fig 3(B): Fast moving and stationary populations of ExoS. A dynamic vesicle positive for both Rab 9 and ExoS.

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