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TBK1 protects vacuolar integrity during intracellular bacterial infection.

Radtke AL, Delbridge LM, Balachandran S, Barber GN, O'Riordan MX - PLoS Pathog. (2007)

Bottom Line: TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function.In tbk1(-/-)cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles.Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
TANK-binding kinase-1 (TBK1) is an integral component of Type I interferon induction by microbial infection. The importance of TBK1 and Type I interferon in antiviral immunity is well established, but the function of TBK1 in bacterial infection is unclear. Upon infection of murine embryonic fibroblasts with Salmonella enterica serovar Typhimurium (Salmonella), more extensive bacterial proliferation was observed in tbk1(-/-) than tbk1(+/+) cells. TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function. In tbk1(-/-)cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles. Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types. Taken together, these data demonstrate a requirement for TBK1 in control of bacterial infection distinct from its established role in antiviral immunity.

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TBK1 Kinase Activity Suppresses Salmonella Intracellular Growth(A) MEFs were infected with Salmonella-GFP (green), fixed at 8 h p.i., counterstained with TRITC-phalloidin to visualize actin (red), and then analyzed by confocal immunofluorescence microscopy. Images were acquired using either a 100× or 40× objective.(B) Salmonella intracellular growth was measured over time in tbk1+/+ (gray triangles) and tbk1−/− (black squares) MEFs in the absence (solid lines) or presence (dashed lines) of Type I interferon. Where indicated, MEFs were treated with 100 U/ml rIFN-β prior to and during infection. The data are representative of five independent experiments.(C) Salmonella intracellular growth in irf3+/+ (gray triangles) and irf3−/− (black squares) MEFs was measured over time. The data are representative of three independent experiments.(D) MEFs were transfected with vector alone (pcDNA3), pcDNA3:TBK1, or pcDNA3:TBK1-KD where indicated in combination with pcDNA3:GFP as a marker for transfection. Percent infected cells that contained >10 Salmonella, out of 150 infected cells counted per experiment at 8 h p.i., was determined by fluorescence microscopy using DAPI to identify bacteria (n ≥ 3). Quantitation was limited to GFP-positive (transfected) MEFs. * and ** denote statistical significance of p ≤ 0.05 and 0.001, respectively, according to a one-tailed Student t-test comparing each tbk1−/− sample to the tbk1+/+ control.
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ppat-0030029-g001: TBK1 Kinase Activity Suppresses Salmonella Intracellular Growth(A) MEFs were infected with Salmonella-GFP (green), fixed at 8 h p.i., counterstained with TRITC-phalloidin to visualize actin (red), and then analyzed by confocal immunofluorescence microscopy. Images were acquired using either a 100× or 40× objective.(B) Salmonella intracellular growth was measured over time in tbk1+/+ (gray triangles) and tbk1−/− (black squares) MEFs in the absence (solid lines) or presence (dashed lines) of Type I interferon. Where indicated, MEFs were treated with 100 U/ml rIFN-β prior to and during infection. The data are representative of five independent experiments.(C) Salmonella intracellular growth in irf3+/+ (gray triangles) and irf3−/− (black squares) MEFs was measured over time. The data are representative of three independent experiments.(D) MEFs were transfected with vector alone (pcDNA3), pcDNA3:TBK1, or pcDNA3:TBK1-KD where indicated in combination with pcDNA3:GFP as a marker for transfection. Percent infected cells that contained >10 Salmonella, out of 150 infected cells counted per experiment at 8 h p.i., was determined by fluorescence microscopy using DAPI to identify bacteria (n ≥ 3). Quantitation was limited to GFP-positive (transfected) MEFs. * and ** denote statistical significance of p ≤ 0.05 and 0.001, respectively, according to a one-tailed Student t-test comparing each tbk1−/− sample to the tbk1+/+ control.

Mentions: To investigate the role of TBK1 in the cellular response to bacterial infection, we infected tbk1+/+ and tbk1−/− MEFs with Salmonella expressing GFP. Salmonella invaded the wild-type and mutant MEFs similarly; however, at 8 h p.i., the monolayer of tbk1−/− MEFs contained approximately 10-fold more bacteria than the wild-type MEFs (Figure 1A and 1B). Robust bacterial proliferation was observed in 35%–40% of infected tbk1−/− MEFs, which was consistent with immunofluorescence analysis of individual cells showing a more pronounced phenotype than that observed by measuring net increase in bacterial numbers. By immunofluorescence analysis, the remainder of the tbk1−/− infected MEFs appeared similar to tbk1+/+ infected MEFs (Figure 1A; 40× magnification). Infected tbk1−/− MEFs that did not exhibit greater bacterial proliferation might have undergone an unproductive infection, since at 1 h p.i., 20%–30% of the bacteria in either wild-type or mutant MEFs were found in autophagosomes, and an additional 25%–30% in lysosomes, which are likely nonreplicative compartments (Figure S1A and S1B). The increased bacterial growth observed in tbk1−/− MEFs was not suppressed by addition of exogenous Type 1 interferon, nor was a similar phenotype observed when Salmonella infection was compared in irf3+/+ and irf3−/− MEFs (Figures 1B, 1C, and S2A). Inhibition of de novo transcription and translation also had no effect on the phenotype (Figure S2B and S2C; unpublished data). However, the robust bacterial replication in tbk1−/− MEFs was substantially decreased by transient transfection with a plasmid expressing wild-type TBK1, but not a kinase dead mutant protein (TBK1 KD) [6] (Figure 1D). Together, these data indicate that TBK1 kinase activity limits intracellular infection of Salmonella independently of the IRF3-Type I interferon axis.


TBK1 protects vacuolar integrity during intracellular bacterial infection.

Radtke AL, Delbridge LM, Balachandran S, Barber GN, O'Riordan MX - PLoS Pathog. (2007)

TBK1 Kinase Activity Suppresses Salmonella Intracellular Growth(A) MEFs were infected with Salmonella-GFP (green), fixed at 8 h p.i., counterstained with TRITC-phalloidin to visualize actin (red), and then analyzed by confocal immunofluorescence microscopy. Images were acquired using either a 100× or 40× objective.(B) Salmonella intracellular growth was measured over time in tbk1+/+ (gray triangles) and tbk1−/− (black squares) MEFs in the absence (solid lines) or presence (dashed lines) of Type I interferon. Where indicated, MEFs were treated with 100 U/ml rIFN-β prior to and during infection. The data are representative of five independent experiments.(C) Salmonella intracellular growth in irf3+/+ (gray triangles) and irf3−/− (black squares) MEFs was measured over time. The data are representative of three independent experiments.(D) MEFs were transfected with vector alone (pcDNA3), pcDNA3:TBK1, or pcDNA3:TBK1-KD where indicated in combination with pcDNA3:GFP as a marker for transfection. Percent infected cells that contained >10 Salmonella, out of 150 infected cells counted per experiment at 8 h p.i., was determined by fluorescence microscopy using DAPI to identify bacteria (n ≥ 3). Quantitation was limited to GFP-positive (transfected) MEFs. * and ** denote statistical significance of p ≤ 0.05 and 0.001, respectively, according to a one-tailed Student t-test comparing each tbk1−/− sample to the tbk1+/+ control.
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Related In: Results  -  Collection

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ppat-0030029-g001: TBK1 Kinase Activity Suppresses Salmonella Intracellular Growth(A) MEFs were infected with Salmonella-GFP (green), fixed at 8 h p.i., counterstained with TRITC-phalloidin to visualize actin (red), and then analyzed by confocal immunofluorescence microscopy. Images were acquired using either a 100× or 40× objective.(B) Salmonella intracellular growth was measured over time in tbk1+/+ (gray triangles) and tbk1−/− (black squares) MEFs in the absence (solid lines) or presence (dashed lines) of Type I interferon. Where indicated, MEFs were treated with 100 U/ml rIFN-β prior to and during infection. The data are representative of five independent experiments.(C) Salmonella intracellular growth in irf3+/+ (gray triangles) and irf3−/− (black squares) MEFs was measured over time. The data are representative of three independent experiments.(D) MEFs were transfected with vector alone (pcDNA3), pcDNA3:TBK1, or pcDNA3:TBK1-KD where indicated in combination with pcDNA3:GFP as a marker for transfection. Percent infected cells that contained >10 Salmonella, out of 150 infected cells counted per experiment at 8 h p.i., was determined by fluorescence microscopy using DAPI to identify bacteria (n ≥ 3). Quantitation was limited to GFP-positive (transfected) MEFs. * and ** denote statistical significance of p ≤ 0.05 and 0.001, respectively, according to a one-tailed Student t-test comparing each tbk1−/− sample to the tbk1+/+ control.
Mentions: To investigate the role of TBK1 in the cellular response to bacterial infection, we infected tbk1+/+ and tbk1−/− MEFs with Salmonella expressing GFP. Salmonella invaded the wild-type and mutant MEFs similarly; however, at 8 h p.i., the monolayer of tbk1−/− MEFs contained approximately 10-fold more bacteria than the wild-type MEFs (Figure 1A and 1B). Robust bacterial proliferation was observed in 35%–40% of infected tbk1−/− MEFs, which was consistent with immunofluorescence analysis of individual cells showing a more pronounced phenotype than that observed by measuring net increase in bacterial numbers. By immunofluorescence analysis, the remainder of the tbk1−/− infected MEFs appeared similar to tbk1+/+ infected MEFs (Figure 1A; 40× magnification). Infected tbk1−/− MEFs that did not exhibit greater bacterial proliferation might have undergone an unproductive infection, since at 1 h p.i., 20%–30% of the bacteria in either wild-type or mutant MEFs were found in autophagosomes, and an additional 25%–30% in lysosomes, which are likely nonreplicative compartments (Figure S1A and S1B). The increased bacterial growth observed in tbk1−/− MEFs was not suppressed by addition of exogenous Type 1 interferon, nor was a similar phenotype observed when Salmonella infection was compared in irf3+/+ and irf3−/− MEFs (Figures 1B, 1C, and S2A). Inhibition of de novo transcription and translation also had no effect on the phenotype (Figure S2B and S2C; unpublished data). However, the robust bacterial replication in tbk1−/− MEFs was substantially decreased by transient transfection with a plasmid expressing wild-type TBK1, but not a kinase dead mutant protein (TBK1 KD) [6] (Figure 1D). Together, these data indicate that TBK1 kinase activity limits intracellular infection of Salmonella independently of the IRF3-Type I interferon axis.

Bottom Line: TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function.In tbk1(-/-)cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles.Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
TANK-binding kinase-1 (TBK1) is an integral component of Type I interferon induction by microbial infection. The importance of TBK1 and Type I interferon in antiviral immunity is well established, but the function of TBK1 in bacterial infection is unclear. Upon infection of murine embryonic fibroblasts with Salmonella enterica serovar Typhimurium (Salmonella), more extensive bacterial proliferation was observed in tbk1(-/-) than tbk1(+/+) cells. TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function. In tbk1(-/-)cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles. Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types. Taken together, these data demonstrate a requirement for TBK1 in control of bacterial infection distinct from its established role in antiviral immunity.

Show MeSH
Related in: MedlinePlus