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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 colocalization with host cell actin and translocation in real-time. EHEC were transformed with Tir-phiLOV and added to HeLa cells. (a and b) 3D, false-colored projections of the attached bacteria to show bacteria with multiple Tir-phiLOV foci. (c and d) 3D, false-colored projections of the attached bacteria to show association of Tir-phiLOV with host cell actin (false-colored blue in panel d). (e) Real-time monitoring of Tir-phiLOV expression and translocation from a single bacterium. (f to h) 3D false-colored projections of a 20-min time course of EHEC attachment and Tir-phiLOV expression on HeLa cells. (f) EHEC (red) and Tir-phiLOV (green). (g) EHEC (red) with positive (yellow; overlap with green channel) and negative (purple; no overlap with green channel) correlation channels. (h) The positive (yellow) and negative (purple) correlation of the red channel (EHEC) and green (Tir-phiLOV). Note the negative correlation channel beside the area corresponding to the bacteria, indicating translocated Tir-phiLOV, and the positive channel indicates the overlap of the red and green channels, indicating Tir-phiLOV still inside the bacteria.
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Figure 4: Imaging of Tir-phiLOV colocalization with host cell actin and translocation in real-time. EHEC were transformed with Tir-phiLOV and added to HeLa cells. (a and b) 3D, false-colored projections of the attached bacteria to show bacteria with multiple Tir-phiLOV foci. (c and d) 3D, false-colored projections of the attached bacteria to show association of Tir-phiLOV with host cell actin (false-colored blue in panel d). (e) Real-time monitoring of Tir-phiLOV expression and translocation from a single bacterium. (f to h) 3D false-colored projections of a 20-min time course of EHEC attachment and Tir-phiLOV expression on HeLa cells. (f) EHEC (red) and Tir-phiLOV (green). (g) EHEC (red) with positive (yellow; overlap with green channel) and negative (purple; no overlap with green channel) correlation channels. (h) The positive (yellow) and negative (purple) correlation of the red channel (EHEC) and green (Tir-phiLOV). Note the negative correlation channel beside the area corresponding to the bacteria, indicating translocated Tir-phiLOV, and the positive channel indicates the overlap of the red and green channels, indicating Tir-phiLOV still inside the bacteria.

Mentions: HeLa cells were seeded into 96-well Nunc plates with ca. 60% confluence and incubated overnight at 37°C with 5% CO2. The following day the cells were labeled with CellTrace-DDAO to prepare them for imaging. Bacteria were cultured in 10 ml of MEM without phenol red (MEM-no phenol red; Sigma) supplemented with 50 mM HEPES and antibiotics as required. The cultures were incubated at 37°C and 200 rpm until an OD600 of 0.6 was reached. The bacteria were diluted with warm MEM, 50 mM HEPES, and added to cells at an approximate multiplicity of infection of 20. The 96-well plate was centrifuged at 400 × g to initiate bacterial contact with the HeLa cells. The plate was washed every 60 min to prevent overgrowth of unattached bacteria in media. The expression and translocation of individual bacteria was monitored (see Fig. 4e for a single example). Some 15 bacteria were measured in this manner; however, they attach to host cells at different rates, resulting in the population being nonsynchronous. As a technical point, we found that HeLa cells were more suitable for fluorescence imaging over extended periods since they were better able to tolerate repeated exposure to ultraviolet light compared to the EBLs. HeLa cells have been used successfully to study pedestal formation of both EPEC and EHEC (23).


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 colocalization with host cell actin and translocation in real-time. EHEC were transformed with Tir-phiLOV and added to HeLa cells. (a and b) 3D, false-colored projections of the attached bacteria to show bacteria with multiple Tir-phiLOV foci. (c and d) 3D, false-colored projections of the attached bacteria to show association of Tir-phiLOV with host cell actin (false-colored blue in panel d). (e) Real-time monitoring of Tir-phiLOV expression and translocation from a single bacterium. (f to h) 3D false-colored projections of a 20-min time course of EHEC attachment and Tir-phiLOV expression on HeLa cells. (f) EHEC (red) and Tir-phiLOV (green). (g) EHEC (red) with positive (yellow; overlap with green channel) and negative (purple; no overlap with green channel) correlation channels. (h) The positive (yellow) and negative (purple) correlation of the red channel (EHEC) and green (Tir-phiLOV). Note the negative correlation channel beside the area corresponding to the bacteria, indicating translocated Tir-phiLOV, and the positive channel indicates the overlap of the red and green channels, indicating Tir-phiLOV still inside the bacteria.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Imaging of Tir-phiLOV colocalization with host cell actin and translocation in real-time. EHEC were transformed with Tir-phiLOV and added to HeLa cells. (a and b) 3D, false-colored projections of the attached bacteria to show bacteria with multiple Tir-phiLOV foci. (c and d) 3D, false-colored projections of the attached bacteria to show association of Tir-phiLOV with host cell actin (false-colored blue in panel d). (e) Real-time monitoring of Tir-phiLOV expression and translocation from a single bacterium. (f to h) 3D false-colored projections of a 20-min time course of EHEC attachment and Tir-phiLOV expression on HeLa cells. (f) EHEC (red) and Tir-phiLOV (green). (g) EHEC (red) with positive (yellow; overlap with green channel) and negative (purple; no overlap with green channel) correlation channels. (h) The positive (yellow) and negative (purple) correlation of the red channel (EHEC) and green (Tir-phiLOV). Note the negative correlation channel beside the area corresponding to the bacteria, indicating translocated Tir-phiLOV, and the positive channel indicates the overlap of the red and green channels, indicating Tir-phiLOV still inside the bacteria.
Mentions: HeLa cells were seeded into 96-well Nunc plates with ca. 60% confluence and incubated overnight at 37°C with 5% CO2. The following day the cells were labeled with CellTrace-DDAO to prepare them for imaging. Bacteria were cultured in 10 ml of MEM without phenol red (MEM-no phenol red; Sigma) supplemented with 50 mM HEPES and antibiotics as required. The cultures were incubated at 37°C and 200 rpm until an OD600 of 0.6 was reached. The bacteria were diluted with warm MEM, 50 mM HEPES, and added to cells at an approximate multiplicity of infection of 20. The 96-well plate was centrifuged at 400 × g to initiate bacterial contact with the HeLa cells. The plate was washed every 60 min to prevent overgrowth of unattached bacteria in media. The expression and translocation of individual bacteria was monitored (see Fig. 4e for a single example). Some 15 bacteria were measured in this manner; however, they attach to host cells at different rates, resulting in the population being nonsynchronous. As a technical point, we found that HeLa cells were more suitable for fluorescence imaging over extended periods since they were better able to tolerate repeated exposure to ultraviolet light compared to the EBLs. HeLa cells have been used successfully to study pedestal formation of both EPEC and EHEC (23).

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