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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

Imaging of Tir-phiLOV translocation. EHEC strains were transformed with pTAC-phiLOV or Tir-phiLOV and added to EBL eukaryotic cells. After 2 h, the bacteria were fixed, and images were obtained and quantified. (a) WT EHEC transformed with Tir-phiLOV showed expression of the reporter after cell contact. The bacterial cytoplasm was marked using the chromosomal RFP reporter and host cell actin stained using labeled phalloidin. (b and c) Expression and localization of phiLOV (b) and Tir-phiLOV (c) of bacteria attached to host cells. The images show Z-slices from the “top” (slice 1) to the “bottom” (slice 9) of attached bacteria. Areas of correlation between phiLOV and RFP are colored yellow, whereas pink shows areas where no correlation was measured. (d) Quantification of the number of pixels associated with each Z-slice for the Tir-phiLOV, phiLOV, and bacterial RFP cytoplasmic channels (ZAP193A and -B). (e to h) 3D, false-colored projections of the bacteria show that Tir-phiLOV (green) is spatially distinct from the bacterial cytoplasm (red). Areas of correlation between phiLOV and RFP are highlighted using yellow with pink, showing areas where no correlation was measured. Representative images are shown; a minimum of 10 individual bacteria were analyzed per experiment.
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Figure 3: Imaging of Tir-phiLOV translocation. EHEC strains were transformed with pTAC-phiLOV or Tir-phiLOV and added to EBL eukaryotic cells. After 2 h, the bacteria were fixed, and images were obtained and quantified. (a) WT EHEC transformed with Tir-phiLOV showed expression of the reporter after cell contact. The bacterial cytoplasm was marked using the chromosomal RFP reporter and host cell actin stained using labeled phalloidin. (b and c) Expression and localization of phiLOV (b) and Tir-phiLOV (c) of bacteria attached to host cells. The images show Z-slices from the “top” (slice 1) to the “bottom” (slice 9) of attached bacteria. Areas of correlation between phiLOV and RFP are colored yellow, whereas pink shows areas where no correlation was measured. (d) Quantification of the number of pixels associated with each Z-slice for the Tir-phiLOV, phiLOV, and bacterial RFP cytoplasmic channels (ZAP193A and -B). (e to h) 3D, false-colored projections of the bacteria show that Tir-phiLOV (green) is spatially distinct from the bacterial cytoplasm (red). Areas of correlation between phiLOV and RFP are highlighted using yellow with pink, showing areas where no correlation was measured. Representative images are shown; a minimum of 10 individual bacteria were analyzed per experiment.

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)

Imaging of Tir-phiLOV translocation. EHEC strains were transformed with pTAC-phiLOV or Tir-phiLOV and added to EBL eukaryotic cells. After 2 h, the bacteria were fixed, and images were obtained and quantified. (a) WT EHEC transformed with Tir-phiLOV showed expression of the reporter after cell contact. The bacterial cytoplasm was marked using the chromosomal RFP reporter and host cell actin stained using labeled phalloidin. (b and c) Expression and localization of phiLOV (b) and Tir-phiLOV (c) of bacteria attached to host cells. The images show Z-slices from the “top” (slice 1) to the “bottom” (slice 9) of attached bacteria. Areas of correlation between phiLOV and RFP are colored yellow, whereas pink shows areas where no correlation was measured. (d) Quantification of the number of pixels associated with each Z-slice for the Tir-phiLOV, phiLOV, and bacterial RFP cytoplasmic channels (ZAP193A and -B). (e to h) 3D, false-colored projections of the bacteria show that Tir-phiLOV (green) is spatially distinct from the bacterial cytoplasm (red). Areas of correlation between phiLOV and RFP are highlighted using yellow with pink, showing areas where no correlation was measured. Representative images are shown; a minimum of 10 individual bacteria were analyzed per experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Imaging of Tir-phiLOV translocation. EHEC strains were transformed with pTAC-phiLOV or Tir-phiLOV and added to EBL eukaryotic cells. After 2 h, the bacteria were fixed, and images were obtained and quantified. (a) WT EHEC transformed with Tir-phiLOV showed expression of the reporter after cell contact. The bacterial cytoplasm was marked using the chromosomal RFP reporter and host cell actin stained using labeled phalloidin. (b and c) Expression and localization of phiLOV (b) and Tir-phiLOV (c) of bacteria attached to host cells. The images show Z-slices from the “top” (slice 1) to the “bottom” (slice 9) of attached bacteria. Areas of correlation between phiLOV and RFP are colored yellow, whereas pink shows areas where no correlation was measured. (d) Quantification of the number of pixels associated with each Z-slice for the Tir-phiLOV, phiLOV, and bacterial RFP cytoplasmic channels (ZAP193A and -B). (e to h) 3D, false-colored projections of the bacteria show that Tir-phiLOV (green) is spatially distinct from the bacterial cytoplasm (red). Areas of correlation between phiLOV and RFP are highlighted using yellow with pink, showing areas where no correlation was measured. Representative images are shown; a minimum of 10 individual bacteria were analyzed per experiment.
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