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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 Protects Vacuolar Integrity in Response to Bacterial Infection(A) Intracellular growth of wild-type or mutant Salmonella strains in tbk1+/+ (triangles) and tbk1−/− (squares) MEFs was measured over time. An antibiotic resistant SPI-1-deficient strain (short dashed lines) was coinfected with an antibiotic-sensitive wild-type Salmonella strain to facilitate invasion by the mutant. Only antibiotic resistant colonies are shown for the SPI-1− growth curve.(B) MEFs were infected with Salmonella-GFP and fixed at 2, 3, and 4 h p.i. In the case of coinfection (St SPI-1− + Salmonella), the SPI-1-deficient strain was marked with GFP. The samples were stained as described in Figure 2B. Percent ubiquitin-associated bacteria in tbk1+/+ MEFs (gray) or tbk1−/− MEFs (black) was calculated based on 150 bacteria counted that colocalized with ubiquitin from two to three independent experiments; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a two-tailed Student t-test.(C) MEFs were infected for 1 h with the indicated bacteria and then fixed at 4 h p.i. Samples were subjected to immunofluorescence staining as described in Figure 2A. Percent LAMP-1-associated represents the number of bacteria out of 150 counted per experiment that colocalized with LAMP-1 from three independent experiments; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a two-tailed Student t-test.(D) Transmission electron microscopy was performed on MEFs infected for 1 h with Streptococcus, and images were acquired at 64,000× magnification. White arrowheads point to bacterial membranes, black arrowheads point to host vacuolar membranes, and black arrows point to bacteria that are no longer completely surrounded by vacuolar membranes.
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ppat-0030029-g003: TBK1 Protects Vacuolar Integrity in Response to Bacterial Infection(A) Intracellular growth of wild-type or mutant Salmonella strains in tbk1+/+ (triangles) and tbk1−/− (squares) MEFs was measured over time. An antibiotic resistant SPI-1-deficient strain (short dashed lines) was coinfected with an antibiotic-sensitive wild-type Salmonella strain to facilitate invasion by the mutant. Only antibiotic resistant colonies are shown for the SPI-1− growth curve.(B) MEFs were infected with Salmonella-GFP and fixed at 2, 3, and 4 h p.i. In the case of coinfection (St SPI-1− + Salmonella), the SPI-1-deficient strain was marked with GFP. The samples were stained as described in Figure 2B. Percent ubiquitin-associated bacteria in tbk1+/+ MEFs (gray) or tbk1−/− MEFs (black) was calculated based on 150 bacteria counted that colocalized with ubiquitin from two to three independent experiments; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a two-tailed Student t-test.(C) MEFs were infected for 1 h with the indicated bacteria and then fixed at 4 h p.i. Samples were subjected to immunofluorescence staining as described in Figure 2A. Percent LAMP-1-associated represents the number of bacteria out of 150 counted per experiment that colocalized with LAMP-1 from three independent experiments; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a two-tailed Student t-test.(D) Transmission electron microscopy was performed on MEFs infected for 1 h with Streptococcus, and images were acquired at 64,000× magnification. White arrowheads point to bacterial membranes, black arrowheads point to host vacuolar membranes, and black arrows point to bacteria that are no longer completely surrounded by vacuolar membranes.

Mentions: Since known mediators of TBK1-dependent signaling were not required to suppress intracellular bacterial growth, and endocytic function was not generally compromised, we hypothesized that Salmonella might be triggering a cellular process that requires TBK1. Salmonella contains two Type III secretion systems encoded on SPI-1 (termed SPI-1 T3SS) and SPI-2 (SPI-2 T3SS) that enable the bacterium to secrete proteins directly into the host cell cytosol [22,31]. The SPI-1 T3SS is required for entry into non-phagocytic cells and modulation of endosomal trafficking; later in infection, SPI-2 T3SS-dependent effectors act to regulate membrane dynamics. We tested the possibility that Type III secretion might contribute to triggering the phenotype observed in TBK1-deficient MEFs. Salmonella strains deficient in either the SPI-1 (St SPI-1−) or SPI-2 (St SPI-2−) encoded T3SS were assessed for their ability to replicate within MEFs and access the host cytosol (Figure 3A and 3B). The SPI-2 deficient bacteria proliferated similarly to wild-type Salmonella in both tbk1+/+ and tbk1−/− MEFs. We also analyzed a Salmonella mutant lacking the SPI-2-dependent effector SifA, which exhibits defects in SCV integrity [32–34]. If SifA and TBK1 were acting in concert, we would expect StΔsifA to proliferate equally in tbk1+/+ and tbk1−/−cells; however, we still observed increased cytosolic localization and replication by the mutant bacteria in tbk1−/− MEFs (Figure S4A and S4B). In contrast, the SPI-1-deficient bacteria (tetR), induced to enter independent vacuoles through bystander infection with wild-type Salmonella (tetS), as measured by tetR colony-forming units or immunofluorescence, were unable to replicate in host cells of either genotype and were never released in the cytosol (Figures 3A, 3B, and S5) [35]. A double SPI-1− SPI-2− mutant behaved similarly to the SPI-1− single mutant (unpublished data). These data suggest that a function associated with the Salmonella SPI-1 T3SS stimulates TBK1-dependent modulation of the pathogen-containing vacuole.


TBK1 protects vacuolar integrity during intracellular bacterial infection.

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

TBK1 Protects Vacuolar Integrity in Response to Bacterial Infection(A) Intracellular growth of wild-type or mutant Salmonella strains in tbk1+/+ (triangles) and tbk1−/− (squares) MEFs was measured over time. An antibiotic resistant SPI-1-deficient strain (short dashed lines) was coinfected with an antibiotic-sensitive wild-type Salmonella strain to facilitate invasion by the mutant. Only antibiotic resistant colonies are shown for the SPI-1− growth curve.(B) MEFs were infected with Salmonella-GFP and fixed at 2, 3, and 4 h p.i. In the case of coinfection (St SPI-1− + Salmonella), the SPI-1-deficient strain was marked with GFP. The samples were stained as described in Figure 2B. Percent ubiquitin-associated bacteria in tbk1+/+ MEFs (gray) or tbk1−/− MEFs (black) was calculated based on 150 bacteria counted that colocalized with ubiquitin from two to three independent experiments; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a two-tailed Student t-test.(C) MEFs were infected for 1 h with the indicated bacteria and then fixed at 4 h p.i. Samples were subjected to immunofluorescence staining as described in Figure 2A. Percent LAMP-1-associated represents the number of bacteria out of 150 counted per experiment that colocalized with LAMP-1 from three independent experiments; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a two-tailed Student t-test.(D) Transmission electron microscopy was performed on MEFs infected for 1 h with Streptococcus, and images were acquired at 64,000× magnification. White arrowheads point to bacterial membranes, black arrowheads point to host vacuolar membranes, and black arrows point to bacteria that are no longer completely surrounded by vacuolar membranes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1808071&req=5

ppat-0030029-g003: TBK1 Protects Vacuolar Integrity in Response to Bacterial Infection(A) Intracellular growth of wild-type or mutant Salmonella strains in tbk1+/+ (triangles) and tbk1−/− (squares) MEFs was measured over time. An antibiotic resistant SPI-1-deficient strain (short dashed lines) was coinfected with an antibiotic-sensitive wild-type Salmonella strain to facilitate invasion by the mutant. Only antibiotic resistant colonies are shown for the SPI-1− growth curve.(B) MEFs were infected with Salmonella-GFP and fixed at 2, 3, and 4 h p.i. In the case of coinfection (St SPI-1− + Salmonella), the SPI-1-deficient strain was marked with GFP. The samples were stained as described in Figure 2B. Percent ubiquitin-associated bacteria in tbk1+/+ MEFs (gray) or tbk1−/− MEFs (black) was calculated based on 150 bacteria counted that colocalized with ubiquitin from two to three independent experiments; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a two-tailed Student t-test.(C) MEFs were infected for 1 h with the indicated bacteria and then fixed at 4 h p.i. Samples were subjected to immunofluorescence staining as described in Figure 2A. Percent LAMP-1-associated represents the number of bacteria out of 150 counted per experiment that colocalized with LAMP-1 from three independent experiments; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a two-tailed Student t-test.(D) Transmission electron microscopy was performed on MEFs infected for 1 h with Streptococcus, and images were acquired at 64,000× magnification. White arrowheads point to bacterial membranes, black arrowheads point to host vacuolar membranes, and black arrows point to bacteria that are no longer completely surrounded by vacuolar membranes.
Mentions: Since known mediators of TBK1-dependent signaling were not required to suppress intracellular bacterial growth, and endocytic function was not generally compromised, we hypothesized that Salmonella might be triggering a cellular process that requires TBK1. Salmonella contains two Type III secretion systems encoded on SPI-1 (termed SPI-1 T3SS) and SPI-2 (SPI-2 T3SS) that enable the bacterium to secrete proteins directly into the host cell cytosol [22,31]. The SPI-1 T3SS is required for entry into non-phagocytic cells and modulation of endosomal trafficking; later in infection, SPI-2 T3SS-dependent effectors act to regulate membrane dynamics. We tested the possibility that Type III secretion might contribute to triggering the phenotype observed in TBK1-deficient MEFs. Salmonella strains deficient in either the SPI-1 (St SPI-1−) or SPI-2 (St SPI-2−) encoded T3SS were assessed for their ability to replicate within MEFs and access the host cytosol (Figure 3A and 3B). The SPI-2 deficient bacteria proliferated similarly to wild-type Salmonella in both tbk1+/+ and tbk1−/− MEFs. We also analyzed a Salmonella mutant lacking the SPI-2-dependent effector SifA, which exhibits defects in SCV integrity [32–34]. If SifA and TBK1 were acting in concert, we would expect StΔsifA to proliferate equally in tbk1+/+ and tbk1−/−cells; however, we still observed increased cytosolic localization and replication by the mutant bacteria in tbk1−/− MEFs (Figure S4A and S4B). In contrast, the SPI-1-deficient bacteria (tetR), induced to enter independent vacuoles through bystander infection with wild-type Salmonella (tetS), as measured by tetR colony-forming units or immunofluorescence, were unable to replicate in host cells of either genotype and were never released in the cytosol (Figures 3A, 3B, and S5) [35]. A double SPI-1− SPI-2− mutant behaved similarly to the SPI-1− single mutant (unpublished data). These data suggest that a function associated with the Salmonella SPI-1 T3SS stimulates TBK1-dependent modulation of the pathogen-containing vacuole.

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