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Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System.

Gawthorne JA, Audry L, McQuitty C, Dean P, Christie JM, Enninga J, Roe AJ - Appl. Environ. Microbiol. (2016)

Bottom Line: Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors.We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference.Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of the Shigella flexneri effector IpaB.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Translocation of IpaB-LOV in Shigella flexneri. WT S. flexneri and an isogenic T3SS-deficient strain (ΔmxiD) were transformed with the IpaB-phiLOV construct. Bacterial DNA and host nuclei were stained with DAPI (blue), and actin focus formation was tracked by using phalloidin-rhodamine (red). Before host cell contact, IpaB-phiLOV localizes to the bacterial poles (upper panel and second panel). After translocation, it is located at the forming entry foci in the vicinity of the bacterium (lower two panels).
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Figure 5: Translocation of IpaB-LOV in Shigella flexneri. WT S. flexneri and an isogenic T3SS-deficient strain (ΔmxiD) were transformed with the IpaB-phiLOV construct. Bacterial DNA and host nuclei were stained with DAPI (blue), and actin focus formation was tracked by using phalloidin-rhodamine (red). Before host cell contact, IpaB-phiLOV localizes to the bacterial poles (upper panel and second panel). After translocation, it is located at the forming entry foci in the vicinity of the bacterium (lower two panels).

Mentions: Fluorescence imaging, such as testing time points for optimum expression and translocation, was performed using a Zeiss AxioImager M1 widefield fluorescence microscope equipped with a Hamamatsu Orca CCD camera and appropriate fluorescence filter sets. Imaging of the precise localization of the Tir-phiLOV fusion during the translocation process, such as those shown in Fig. 3d to h and Fig. 4a to d, were obtained using a DeltaVision RT epifluorescence imaging system (Applied Precision) and SoftWoRx software. Rapid three-dimensional time-lapse imaging of Tir-phiLOV (Fig. 4e to h) and IpaB-phiLOV (Fig. 5) were obtained using a spinning disk confocal microscope using the 488-nm laser for phiLOV excitation (Perkin-Elmer). Data were captured and analyzed using Volocity Suite software (Perkin-Elmer), allowing quantification of two-dimensional (2D) images (pixels) or 3D images (voxels).


Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System.

Gawthorne JA, Audry L, McQuitty C, Dean P, Christie JM, Enninga J, Roe AJ - Appl. Environ. Microbiol. (2016)

Translocation of IpaB-LOV in Shigella flexneri. WT S. flexneri and an isogenic T3SS-deficient strain (ΔmxiD) were transformed with the IpaB-phiLOV construct. Bacterial DNA and host nuclei were stained with DAPI (blue), and actin focus formation was tracked by using phalloidin-rhodamine (red). Before host cell contact, IpaB-phiLOV localizes to the bacterial poles (upper panel and second panel). After translocation, it is located at the forming entry foci in the vicinity of the bacterium (lower two panels).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836418&req=5

Figure 5: Translocation of IpaB-LOV in Shigella flexneri. WT S. flexneri and an isogenic T3SS-deficient strain (ΔmxiD) were transformed with the IpaB-phiLOV construct. Bacterial DNA and host nuclei were stained with DAPI (blue), and actin focus formation was tracked by using phalloidin-rhodamine (red). Before host cell contact, IpaB-phiLOV localizes to the bacterial poles (upper panel and second panel). After translocation, it is located at the forming entry foci in the vicinity of the bacterium (lower two panels).
Mentions: Fluorescence imaging, such as testing time points for optimum expression and translocation, was performed using a Zeiss AxioImager M1 widefield fluorescence microscope equipped with a Hamamatsu Orca CCD camera and appropriate fluorescence filter sets. Imaging of the precise localization of the Tir-phiLOV fusion during the translocation process, such as those shown in Fig. 3d to h and Fig. 4a to d, were obtained using a DeltaVision RT epifluorescence imaging system (Applied Precision) and SoftWoRx software. Rapid three-dimensional time-lapse imaging of Tir-phiLOV (Fig. 4e to h) and IpaB-phiLOV (Fig. 5) were obtained using a spinning disk confocal microscope using the 488-nm laser for phiLOV excitation (Perkin-Elmer). Data were captured and analyzed using Volocity Suite software (Perkin-Elmer), allowing quantification of two-dimensional (2D) images (pixels) or 3D images (voxels).

Bottom Line: Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors.We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference.Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of the Shigella flexneri effector IpaB.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom.

No MeSH data available.


Related in: MedlinePlus