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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 Tir-phiLOV secretion and translocation. EHEC strains were cultured in T3SS-inducing conditions and harvested at an OD600 of 0.6. Samples were centrifuged to yield the supernatant fraction (sn), and the bacterial pellet was lysed with Bugbuster (whole cell [wc]). (a) EHEC and EHECΔescN transformed with a pTir-phiLOV probed with α-iLOV antibodies. An α-σ70 immunoblot acted as a control for bacterial lysis. (b) Comparison of Tir-phiLOV and Tir secretion from EHEC transformed with empty vector (−) or pJAG13 (+) after probing with an α-Tir antibody. (c) EHEC transformed with a pTAC-phiLOV plasmid probed with α-iLOV antibodies shows no secretion into the supernatant. To test translocation, a bacterium-host cell adhesion assay was performed using EHEC with either no plasmid (−) or pTir-phiLOV (+) on EBL eukaryotic cells. Four hours after addition of the bacteria, the EBL cells were treated with gentamicin, harvested, and fractionated into cytoplasmic (cy), membranes (me), and lysed bacteria (wc). The fractions were probed with α-iLOV antibodies (d), α-calnexin antibodies (e), and α-σ70 antibodies (f), with the lysed bacteria acting as a positive control (wc). (g) To evaluate whether Tir-phiLOV was functional, WT EHEC, a Δtir mutant, and the Δtir/Tir-phiLOV strain were added to HeLa cells and fixed at various time points after the infection. Condensation of host cell actin was visualized by use of Alexa Fluor-labeled phalloidin. Bacteria were detected by addition of α-O157 antibodies. Deletion of Tir reduces the ability of bacteria to attach to host cells and prevents condensation of host cell actin, a trait that was restored by transformation with the plasmid expression Tir-phiLOV.
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Figure 2: Analysis of Tir-phiLOV secretion and translocation. EHEC strains were cultured in T3SS-inducing conditions and harvested at an OD600 of 0.6. Samples were centrifuged to yield the supernatant fraction (sn), and the bacterial pellet was lysed with Bugbuster (whole cell [wc]). (a) EHEC and EHECΔescN transformed with a pTir-phiLOV probed with α-iLOV antibodies. An α-σ70 immunoblot acted as a control for bacterial lysis. (b) Comparison of Tir-phiLOV and Tir secretion from EHEC transformed with empty vector (−) or pJAG13 (+) after probing with an α-Tir antibody. (c) EHEC transformed with a pTAC-phiLOV plasmid probed with α-iLOV antibodies shows no secretion into the supernatant. To test translocation, a bacterium-host cell adhesion assay was performed using EHEC with either no plasmid (−) or pTir-phiLOV (+) on EBL eukaryotic cells. Four hours after addition of the bacteria, the EBL cells were treated with gentamicin, harvested, and fractionated into cytoplasmic (cy), membranes (me), and lysed bacteria (wc). The fractions were probed with α-iLOV antibodies (d), α-calnexin antibodies (e), and α-σ70 antibodies (f), with the lysed bacteria acting as a positive control (wc). (g) To evaluate whether Tir-phiLOV was functional, WT EHEC, a Δtir mutant, and the Δtir/Tir-phiLOV strain were added to HeLa cells and fixed at various time points after the infection. Condensation of host cell actin was visualized by use of Alexa Fluor-labeled phalloidin. Bacteria were detected by addition of α-O157 antibodies. Deletion of Tir reduces the ability of bacteria to attach to host cells and prevents condensation of host cell actin, a trait that was restored by transformation with the plasmid expression Tir-phiLOV.

Mentions: To determine whether the Tir-phiLOV fusion could indeed be secreted via the T3SS, wild-type EHEC was transformed with pJAG13 and cultured in a MEM-HEPES media that induces expression of the T3SS. Immunoblotting showed that the Tir-phiLOV fusion protein was detectible in both whole-cell and secreted fractions (Fig. 2a). Monitoring the levels of σ70, a bacterial cytoplasmic protein, confirmed that Tir-phiLOV in the secreted fraction did not result from bacterial cell lysis (Fig. 2a). Moreover, deletion of EscN (ΔescN), the ATPase required for T3SS activity, prevented export of the Tir-phiLOV fusion protein, allowing detection only in the whole-cell fraction (Fig. 2a). To compare the secretion of native Tir and Tir-phiLOV, the secreted protein fractions from WT EHEC and the WT transformed with pAJG13 were probed using antibodies for Tir. This revealed both native Tir and Tir-phiLOV in the supernatant of the transformed strain in equal amounts (Fig. 2b). However, when phiLOV was expressed independently of Tir, phiLOV was only detected in bacterial whole-cell fractions, indicating that the fluorescent tag itself is not secreted independently of the effector (Fig. 2c). These data supported the notion that Tir-phiLOV could be secreted via the T3SS, a key advantage of the phiLOV reporter system over GFP.


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 Tir-phiLOV secretion and translocation. EHEC strains were cultured in T3SS-inducing conditions and harvested at an OD600 of 0.6. Samples were centrifuged to yield the supernatant fraction (sn), and the bacterial pellet was lysed with Bugbuster (whole cell [wc]). (a) EHEC and EHECΔescN transformed with a pTir-phiLOV probed with α-iLOV antibodies. An α-σ70 immunoblot acted as a control for bacterial lysis. (b) Comparison of Tir-phiLOV and Tir secretion from EHEC transformed with empty vector (−) or pJAG13 (+) after probing with an α-Tir antibody. (c) EHEC transformed with a pTAC-phiLOV plasmid probed with α-iLOV antibodies shows no secretion into the supernatant. To test translocation, a bacterium-host cell adhesion assay was performed using EHEC with either no plasmid (−) or pTir-phiLOV (+) on EBL eukaryotic cells. Four hours after addition of the bacteria, the EBL cells were treated with gentamicin, harvested, and fractionated into cytoplasmic (cy), membranes (me), and lysed bacteria (wc). The fractions were probed with α-iLOV antibodies (d), α-calnexin antibodies (e), and α-σ70 antibodies (f), with the lysed bacteria acting as a positive control (wc). (g) To evaluate whether Tir-phiLOV was functional, WT EHEC, a Δtir mutant, and the Δtir/Tir-phiLOV strain were added to HeLa cells and fixed at various time points after the infection. Condensation of host cell actin was visualized by use of Alexa Fluor-labeled phalloidin. Bacteria were detected by addition of α-O157 antibodies. Deletion of Tir reduces the ability of bacteria to attach to host cells and prevents condensation of host cell actin, a trait that was restored by transformation with the plasmid expression Tir-phiLOV.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4836418&req=5

Figure 2: Analysis of Tir-phiLOV secretion and translocation. EHEC strains were cultured in T3SS-inducing conditions and harvested at an OD600 of 0.6. Samples were centrifuged to yield the supernatant fraction (sn), and the bacterial pellet was lysed with Bugbuster (whole cell [wc]). (a) EHEC and EHECΔescN transformed with a pTir-phiLOV probed with α-iLOV antibodies. An α-σ70 immunoblot acted as a control for bacterial lysis. (b) Comparison of Tir-phiLOV and Tir secretion from EHEC transformed with empty vector (−) or pJAG13 (+) after probing with an α-Tir antibody. (c) EHEC transformed with a pTAC-phiLOV plasmid probed with α-iLOV antibodies shows no secretion into the supernatant. To test translocation, a bacterium-host cell adhesion assay was performed using EHEC with either no plasmid (−) or pTir-phiLOV (+) on EBL eukaryotic cells. Four hours after addition of the bacteria, the EBL cells were treated with gentamicin, harvested, and fractionated into cytoplasmic (cy), membranes (me), and lysed bacteria (wc). The fractions were probed with α-iLOV antibodies (d), α-calnexin antibodies (e), and α-σ70 antibodies (f), with the lysed bacteria acting as a positive control (wc). (g) To evaluate whether Tir-phiLOV was functional, WT EHEC, a Δtir mutant, and the Δtir/Tir-phiLOV strain were added to HeLa cells and fixed at various time points after the infection. Condensation of host cell actin was visualized by use of Alexa Fluor-labeled phalloidin. Bacteria were detected by addition of α-O157 antibodies. Deletion of Tir reduces the ability of bacteria to attach to host cells and prevents condensation of host cell actin, a trait that was restored by transformation with the plasmid expression Tir-phiLOV.
Mentions: To determine whether the Tir-phiLOV fusion could indeed be secreted via the T3SS, wild-type EHEC was transformed with pJAG13 and cultured in a MEM-HEPES media that induces expression of the T3SS. Immunoblotting showed that the Tir-phiLOV fusion protein was detectible in both whole-cell and secreted fractions (Fig. 2a). Monitoring the levels of σ70, a bacterial cytoplasmic protein, confirmed that Tir-phiLOV in the secreted fraction did not result from bacterial cell lysis (Fig. 2a). Moreover, deletion of EscN (ΔescN), the ATPase required for T3SS activity, prevented export of the Tir-phiLOV fusion protein, allowing detection only in the whole-cell fraction (Fig. 2a). To compare the secretion of native Tir and Tir-phiLOV, the secreted protein fractions from WT EHEC and the WT transformed with pAJG13 were probed using antibodies for Tir. This revealed both native Tir and Tir-phiLOV in the supernatant of the transformed strain in equal amounts (Fig. 2b). However, when phiLOV was expressed independently of Tir, phiLOV was only detected in bacterial whole-cell fractions, indicating that the fluorescent tag itself is not secreted independently of the effector (Fig. 2c). These data supported the notion that Tir-phiLOV could be secreted via the T3SS, a key advantage of the phiLOV reporter system over GFP.

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