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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

Analysis of relative fluorescence of EHEC effector-phiLOV fusion proteins under T3SS-inducing conditions. (a) EHEC expressing either Tir-phiLOV or Map-phiLOV was grown under T3SS-inducing conditions to determine whether expression of each effector could be measured using a simple plate reader assay. Each effector-phiLOV fusion was measured in triplicate, and the mean fluorescence was monitored over exponential growth. (b) Fluorescence of EHEC expressing either Tir-GFP or Tir-phiLOV was monitored to determine how the Tir-phiLOV fusion would perform directly compared to Tir-GFP.
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Figure 1: Analysis of relative fluorescence of EHEC effector-phiLOV fusion proteins under T3SS-inducing conditions. (a) EHEC expressing either Tir-phiLOV or Map-phiLOV was grown under T3SS-inducing conditions to determine whether expression of each effector could be measured using a simple plate reader assay. Each effector-phiLOV fusion was measured in triplicate, and the mean fluorescence was monitored over exponential growth. (b) Fluorescence of EHEC expressing either Tir-GFP or Tir-phiLOV was monitored to determine how the Tir-phiLOV fusion would perform directly compared to Tir-GFP.

Mentions: To explore the properties of Tir-phiLOV and Map-phiLOV, we evaluated their relative fluorescence when expressed in EHEC under the control of their native promoters. Since it was possible that phiLOV fusions might be secreted, expression studies were performed in an EHEC T3SS-deficient strain that lacks the ATPase (EscN) ensuring that all the fusion protein was retained in the bacterial cytoplasm. This allowed for a more direct comparison between different reporters and constructs without the possibility of protein secretion. Bacteria were cultured in MEM-HEPES media to induce the expression of the T3SS and the level of fluorescence for both phiLOV reporters monitored throughout the growth phase. Fluorescence readings showed that Tir-phiLOV was approximately three times brighter than Map-phiLOV (Fig. 1a). Indeed, we concluded that the expression of Map-phiLOV was too low to warrant further study, a limitation we discuss further below. To determine the relative expression of Tir-phiLOV compared to existing GFP reporters, we also compared the Tir-phiLOV reporter with a Tir-GFP reporter (pAJR75) (25). Both plasmids comprised the same promoter sequence and plasmid backbone, ensuring that the only variable was the fluorescent reporter being tested. The GFP reporter displayed fluorescence that accumulated over time. In comparison, the phiLOV reporter was less fluorescent, around 2.5 to 3 times less than the GFP through the entire exponential phase (Fig. 1b). However, despite being less fluorescent compared to GFP, the Tir-phiLOV reporter was detectable on a simple fluorescence plate reader at similar growth stages to Tir-GFP, suggesting that it would be readily imaged within single cells.


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)

Analysis of relative fluorescence of EHEC effector-phiLOV fusion proteins under T3SS-inducing conditions. (a) EHEC expressing either Tir-phiLOV or Map-phiLOV was grown under T3SS-inducing conditions to determine whether expression of each effector could be measured using a simple plate reader assay. Each effector-phiLOV fusion was measured in triplicate, and the mean fluorescence was monitored over exponential growth. (b) Fluorescence of EHEC expressing either Tir-GFP or Tir-phiLOV was monitored to determine how the Tir-phiLOV fusion would perform directly compared to Tir-GFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Analysis of relative fluorescence of EHEC effector-phiLOV fusion proteins under T3SS-inducing conditions. (a) EHEC expressing either Tir-phiLOV or Map-phiLOV was grown under T3SS-inducing conditions to determine whether expression of each effector could be measured using a simple plate reader assay. Each effector-phiLOV fusion was measured in triplicate, and the mean fluorescence was monitored over exponential growth. (b) Fluorescence of EHEC expressing either Tir-GFP or Tir-phiLOV was monitored to determine how the Tir-phiLOV fusion would perform directly compared to Tir-GFP.
Mentions: To explore the properties of Tir-phiLOV and Map-phiLOV, we evaluated their relative fluorescence when expressed in EHEC under the control of their native promoters. Since it was possible that phiLOV fusions might be secreted, expression studies were performed in an EHEC T3SS-deficient strain that lacks the ATPase (EscN) ensuring that all the fusion protein was retained in the bacterial cytoplasm. This allowed for a more direct comparison between different reporters and constructs without the possibility of protein secretion. Bacteria were cultured in MEM-HEPES media to induce the expression of the T3SS and the level of fluorescence for both phiLOV reporters monitored throughout the growth phase. Fluorescence readings showed that Tir-phiLOV was approximately three times brighter than Map-phiLOV (Fig. 1a). Indeed, we concluded that the expression of Map-phiLOV was too low to warrant further study, a limitation we discuss further below. To determine the relative expression of Tir-phiLOV compared to existing GFP reporters, we also compared the Tir-phiLOV reporter with a Tir-GFP reporter (pAJR75) (25). Both plasmids comprised the same promoter sequence and plasmid backbone, ensuring that the only variable was the fluorescent reporter being tested. The GFP reporter displayed fluorescence that accumulated over time. In comparison, the phiLOV reporter was less fluorescent, around 2.5 to 3 times less than the GFP through the entire exponential phase (Fig. 1b). However, despite being less fluorescent compared to GFP, the Tir-phiLOV reporter was detectable on a simple fluorescence plate reader at similar growth stages to Tir-GFP, suggesting that it would be readily imaged within single cells.

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