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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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In the Absence of TBK1, Bacteria Enter the Host Cytosol(A) Tbk1+/+ and tbk1−/− MEFs were infected with Salmonella-GFP (green). Infected cells were fixed at 2 h p.i., stained with anti-LAMP-1 antibody followed by a TRITC-labeled secondary antibody (red), and analyzed by confocal immunofluorescence microscopy. Percent colocalization was determined by counting the number of bacteria out of 150 bacteria per experiment that colocalized with LAMP-1. The experiment shown is representative of three independent experiments.(B) MEFs were infected for 1 h with the Salmonella-GFP (green), fixed at 4 h p.i., and stained with an anti-ubiquitin monoclonal antibody followed by a TRITC-labeled secondary antibody (red). The percent of ubiquitin-associated bacteria was determined by counting the number of bacteria out of 150 bacteria per experiment that colocalized with ubiquitin as observed by confocal microscopy. The experiment shown is representative of three independent experiments.(C) Transmission electron microscopy was performed on MEFs infected for 1 h with Salmonella, and images acquired at 64,000× magnification. White arrowheads point to bacterial membranes, black arrowheads point to host vacuolar membranes, and arrows point to bacteria no longer completely surrounded by vacuolar membranes.(D) MEFs were incubated with 125I-EGF and the rate of endocytic uptake (cell associated), degradation (TCA soluble), and accumulation of undegraded product (TCA insoluble) was determined at 0.5, 1, 2, and 3 h after endocytosis. The data represent the mean of four independent experiments.
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ppat-0030029-g002: In the Absence of TBK1, Bacteria Enter the Host Cytosol(A) Tbk1+/+ and tbk1−/− MEFs were infected with Salmonella-GFP (green). Infected cells were fixed at 2 h p.i., stained with anti-LAMP-1 antibody followed by a TRITC-labeled secondary antibody (red), and analyzed by confocal immunofluorescence microscopy. Percent colocalization was determined by counting the number of bacteria out of 150 bacteria per experiment that colocalized with LAMP-1. The experiment shown is representative of three independent experiments.(B) MEFs were infected for 1 h with the Salmonella-GFP (green), fixed at 4 h p.i., and stained with an anti-ubiquitin monoclonal antibody followed by a TRITC-labeled secondary antibody (red). The percent of ubiquitin-associated bacteria was determined by counting the number of bacteria out of 150 bacteria per experiment that colocalized with ubiquitin as observed by confocal microscopy. The experiment shown is representative of three independent experiments.(C) Transmission electron microscopy was performed on MEFs infected for 1 h with Salmonella, and images acquired at 64,000× magnification. White arrowheads point to bacterial membranes, black arrowheads point to host vacuolar membranes, and arrows point to bacteria no longer completely surrounded by vacuolar membranes.(D) MEFs were incubated with 125I-EGF and the rate of endocytic uptake (cell associated), degradation (TCA soluble), and accumulation of undegraded product (TCA insoluble) was determined at 0.5, 1, 2, and 3 h after endocytosis. The data represent the mean of four independent experiments.

Mentions: Previous studies have shown that shortly after invasion, SCV colocalize with the late endosomal marker, LAMP-1 [24]. To determine the nature of the Salmonella-containing compartment in TBK1-deficient cells, we infected MEFs with Salmonella-GFP and analyzed the samples by confocal immunofluorescence microscopy using an anti-LAMP1 antibody (Figures 2A and S3). SCV in tbk1+/+ cells were colocalized with LAMP-1 throughout the entire course of infection, with 94.5% colocalization at 2 h p.i. In contrast, as early as 90 min p.i. in tbk1−/− cells, individual Salmonella lost association with LAMP-1, and at 2 h p.i., only 63.0% of bacteria exhibited colocalization. Loss of LAMP-1 colocalization by individual bacteria was commonly observed early in infection, suggesting that replication per se was not required for this abnormal phenotype. Thus, in the absence of TBK1, many SCV lose the late endosomal marker, LAMP-1, and deviate from the characterized Salmonella endocytic trafficking pathway.


TBK1 protects vacuolar integrity during intracellular bacterial infection.

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

In the Absence of TBK1, Bacteria Enter the Host Cytosol(A) Tbk1+/+ and tbk1−/− MEFs were infected with Salmonella-GFP (green). Infected cells were fixed at 2 h p.i., stained with anti-LAMP-1 antibody followed by a TRITC-labeled secondary antibody (red), and analyzed by confocal immunofluorescence microscopy. Percent colocalization was determined by counting the number of bacteria out of 150 bacteria per experiment that colocalized with LAMP-1. The experiment shown is representative of three independent experiments.(B) MEFs were infected for 1 h with the Salmonella-GFP (green), fixed at 4 h p.i., and stained with an anti-ubiquitin monoclonal antibody followed by a TRITC-labeled secondary antibody (red). The percent of ubiquitin-associated bacteria was determined by counting the number of bacteria out of 150 bacteria per experiment that colocalized with ubiquitin as observed by confocal microscopy. The experiment shown is representative of three independent experiments.(C) Transmission electron microscopy was performed on MEFs infected for 1 h with Salmonella, and images acquired at 64,000× magnification. White arrowheads point to bacterial membranes, black arrowheads point to host vacuolar membranes, and arrows point to bacteria no longer completely surrounded by vacuolar membranes.(D) MEFs were incubated with 125I-EGF and the rate of endocytic uptake (cell associated), degradation (TCA soluble), and accumulation of undegraded product (TCA insoluble) was determined at 0.5, 1, 2, and 3 h after endocytosis. The data represent the mean of four independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1808071&req=5

ppat-0030029-g002: In the Absence of TBK1, Bacteria Enter the Host Cytosol(A) Tbk1+/+ and tbk1−/− MEFs were infected with Salmonella-GFP (green). Infected cells were fixed at 2 h p.i., stained with anti-LAMP-1 antibody followed by a TRITC-labeled secondary antibody (red), and analyzed by confocal immunofluorescence microscopy. Percent colocalization was determined by counting the number of bacteria out of 150 bacteria per experiment that colocalized with LAMP-1. The experiment shown is representative of three independent experiments.(B) MEFs were infected for 1 h with the Salmonella-GFP (green), fixed at 4 h p.i., and stained with an anti-ubiquitin monoclonal antibody followed by a TRITC-labeled secondary antibody (red). The percent of ubiquitin-associated bacteria was determined by counting the number of bacteria out of 150 bacteria per experiment that colocalized with ubiquitin as observed by confocal microscopy. The experiment shown is representative of three independent experiments.(C) Transmission electron microscopy was performed on MEFs infected for 1 h with Salmonella, and images acquired at 64,000× magnification. White arrowheads point to bacterial membranes, black arrowheads point to host vacuolar membranes, and arrows point to bacteria no longer completely surrounded by vacuolar membranes.(D) MEFs were incubated with 125I-EGF and the rate of endocytic uptake (cell associated), degradation (TCA soluble), and accumulation of undegraded product (TCA insoluble) was determined at 0.5, 1, 2, and 3 h after endocytosis. The data represent the mean of four independent experiments.
Mentions: Previous studies have shown that shortly after invasion, SCV colocalize with the late endosomal marker, LAMP-1 [24]. To determine the nature of the Salmonella-containing compartment in TBK1-deficient cells, we infected MEFs with Salmonella-GFP and analyzed the samples by confocal immunofluorescence microscopy using an anti-LAMP1 antibody (Figures 2A and S3). SCV in tbk1+/+ cells were colocalized with LAMP-1 throughout the entire course of infection, with 94.5% colocalization at 2 h p.i. In contrast, as early as 90 min p.i. in tbk1−/− cells, individual Salmonella lost association with LAMP-1, and at 2 h p.i., only 63.0% of bacteria exhibited colocalization. Loss of LAMP-1 colocalization by individual bacteria was commonly observed early in infection, suggesting that replication per se was not required for this abnormal phenotype. Thus, in the absence of TBK1, many SCV lose the late endosomal marker, LAMP-1, and deviate from the characterized Salmonella endocytic trafficking pathway.

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