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Tyrosine-phosphorylated caveolin-1 blocks bacterial uptake by inducing Vav2-RhoA-mediated cytoskeletal rearrangements.

Boettcher JP, Kirchner M, Churin Y, Kaushansky A, Pompaiah M, Thorn H, Brinkmann V, Macbeath G, Meyer TF - PLoS Biol. (2010)

Bottom Line: A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2.Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake.Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.

ABSTRACT
Certain bacterial adhesins appear to promote a pathogen's extracellular lifestyle rather than its entry into host cells. However, little is known about the stimuli elicited upon such pathogen host-cell interactions. Here, we report that type IV pili (Tfp)-producing Neisseria gonorrhoeae (P(+)GC) induces an immediate recruitment of caveolin-1 (Cav1) in the host cell, which subsequently prevents bacterial internalization by triggering cytoskeletal rearrangements via downstream phosphotyrosine signaling. A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake. Our findings, which have been extended to enteropathogenic Escherichia coli, highlight how Tfp-producing bacteria avoid host cell uptake. Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function.

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Expression and recruitment of Cav1 prevents internalization of P+GC by host cells.(A) Recruitment of endogenous Cav1 (white, middle panel) to attached P+GC (green, right panel) in ME-180 cells 2 h post-infection. (B) Excerpts of Movie 1: Cav1-GFP (green) is recruited within seconds to microcolonies and individually attached P+GC (red) in ME-180 cells (lower panels). Recruitment continues as infection proceeds (upper panels). Attached P+GC are indicated by arrows. (C) Knockdown of Cav1 in ME-180 cells and in Cav1 expressing AGS cells (AGS-Cav1) results in P+GC internalization. Cav1 expression was downregulated by transfection of two different siRNAs (Cav1-I or Cav1-II) in ME-180 and AGS-Cav1 cells using lamin A/C as a control siRNA. Gentamicin protection assays were performed 2 h post-infection (upper panel). Experiments were performed in triplicate. Data are mean ± standard deviation. Cav1 knockdown efficiency was confirmed by Western blot analysis (lower panel). (D) shRNA-mediated downregulation of Cav1 in ME-180 cells results in P+GC internalization. Intracellular bacteria (red) are detected in ME-180 shCav1 cells (upper right panels), whereas only extracellular bacteria (yellow-green) are detected in ME-180 shLuciferase control cells (upper left panels). Efficiency of Cav1 knockdown in ME-180 cells after lentiviral transduction of Luciferase (control) or Cav1 shRNA constructs (lower panel). Scale bar: 20 µm. (E) Numbers of viable intracellular P+GC in AGS cells decrease rapidly over time. Infected cells were initially treated with gentamicin for 2 h, then further incubated in gentamicin and serum-free medium and lysed at indicated time points. Experiments were performed in triplicate. Data are mean ± standard deviation.
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pbio-1000457-g001: Expression and recruitment of Cav1 prevents internalization of P+GC by host cells.(A) Recruitment of endogenous Cav1 (white, middle panel) to attached P+GC (green, right panel) in ME-180 cells 2 h post-infection. (B) Excerpts of Movie 1: Cav1-GFP (green) is recruited within seconds to microcolonies and individually attached P+GC (red) in ME-180 cells (lower panels). Recruitment continues as infection proceeds (upper panels). Attached P+GC are indicated by arrows. (C) Knockdown of Cav1 in ME-180 cells and in Cav1 expressing AGS cells (AGS-Cav1) results in P+GC internalization. Cav1 expression was downregulated by transfection of two different siRNAs (Cav1-I or Cav1-II) in ME-180 and AGS-Cav1 cells using lamin A/C as a control siRNA. Gentamicin protection assays were performed 2 h post-infection (upper panel). Experiments were performed in triplicate. Data are mean ± standard deviation. Cav1 knockdown efficiency was confirmed by Western blot analysis (lower panel). (D) shRNA-mediated downregulation of Cav1 in ME-180 cells results in P+GC internalization. Intracellular bacteria (red) are detected in ME-180 shCav1 cells (upper right panels), whereas only extracellular bacteria (yellow-green) are detected in ME-180 shLuciferase control cells (upper left panels). Efficiency of Cav1 knockdown in ME-180 cells after lentiviral transduction of Luciferase (control) or Cav1 shRNA constructs (lower panel). Scale bar: 20 µm. (E) Numbers of viable intracellular P+GC in AGS cells decrease rapidly over time. Infected cells were initially treated with gentamicin for 2 h, then further incubated in gentamicin and serum-free medium and lysed at indicated time points. Experiments were performed in triplicate. Data are mean ± standard deviation.

Mentions: To assess the role of Cav1 in the Tfp-mediated binding of P+GC to host cells, we began by monitoring the cellular localization of Cav1 in ME-180 cells, a human epidermoid carcinoma cell line, immediately following infection. We found that endogenous Cav1 localized close to P+GC microcolonies after 2 h of infection (Figure 1A). Using live-cell imaging, we likewise observed a substantial accumulation of Cav1-GFP at sites of bacterial infection, which was induced even by single diplococci and within seconds after P+GC attachment (Figure 1B, lower panel). Cav1-GFP recruitment occurred throughout the early stages of infection, resulting ultimately in a conspicuous accumulation of the protein (Figure 1B, upper panel and Video S1). By contrast, using a non-piliated isogenic GC strain (P− Opa57+GC is a non-piliated isogenic strain of P+GC that produces an Opa57 adhesin specific for CEACAM receptors), we found no recruitment of endogenous Cav1 in ME-180 cells (Figure S1A). Interestingly, despite the observed recruitment of Cav1 after P+GC infection, microarray and Western blot analysis failed to detect any increase in Cav1 expression (unpublished data). This rather points to a cellular Cav1 reorganization leading to Cav1-accumulation than de novo synthesis.


Tyrosine-phosphorylated caveolin-1 blocks bacterial uptake by inducing Vav2-RhoA-mediated cytoskeletal rearrangements.

Boettcher JP, Kirchner M, Churin Y, Kaushansky A, Pompaiah M, Thorn H, Brinkmann V, Macbeath G, Meyer TF - PLoS Biol. (2010)

Expression and recruitment of Cav1 prevents internalization of P+GC by host cells.(A) Recruitment of endogenous Cav1 (white, middle panel) to attached P+GC (green, right panel) in ME-180 cells 2 h post-infection. (B) Excerpts of Movie 1: Cav1-GFP (green) is recruited within seconds to microcolonies and individually attached P+GC (red) in ME-180 cells (lower panels). Recruitment continues as infection proceeds (upper panels). Attached P+GC are indicated by arrows. (C) Knockdown of Cav1 in ME-180 cells and in Cav1 expressing AGS cells (AGS-Cav1) results in P+GC internalization. Cav1 expression was downregulated by transfection of two different siRNAs (Cav1-I or Cav1-II) in ME-180 and AGS-Cav1 cells using lamin A/C as a control siRNA. Gentamicin protection assays were performed 2 h post-infection (upper panel). Experiments were performed in triplicate. Data are mean ± standard deviation. Cav1 knockdown efficiency was confirmed by Western blot analysis (lower panel). (D) shRNA-mediated downregulation of Cav1 in ME-180 cells results in P+GC internalization. Intracellular bacteria (red) are detected in ME-180 shCav1 cells (upper right panels), whereas only extracellular bacteria (yellow-green) are detected in ME-180 shLuciferase control cells (upper left panels). Efficiency of Cav1 knockdown in ME-180 cells after lentiviral transduction of Luciferase (control) or Cav1 shRNA constructs (lower panel). Scale bar: 20 µm. (E) Numbers of viable intracellular P+GC in AGS cells decrease rapidly over time. Infected cells were initially treated with gentamicin for 2 h, then further incubated in gentamicin and serum-free medium and lysed at indicated time points. Experiments were performed in triplicate. Data are mean ± standard deviation.
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Related In: Results  -  Collection

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pbio-1000457-g001: Expression and recruitment of Cav1 prevents internalization of P+GC by host cells.(A) Recruitment of endogenous Cav1 (white, middle panel) to attached P+GC (green, right panel) in ME-180 cells 2 h post-infection. (B) Excerpts of Movie 1: Cav1-GFP (green) is recruited within seconds to microcolonies and individually attached P+GC (red) in ME-180 cells (lower panels). Recruitment continues as infection proceeds (upper panels). Attached P+GC are indicated by arrows. (C) Knockdown of Cav1 in ME-180 cells and in Cav1 expressing AGS cells (AGS-Cav1) results in P+GC internalization. Cav1 expression was downregulated by transfection of two different siRNAs (Cav1-I or Cav1-II) in ME-180 and AGS-Cav1 cells using lamin A/C as a control siRNA. Gentamicin protection assays were performed 2 h post-infection (upper panel). Experiments were performed in triplicate. Data are mean ± standard deviation. Cav1 knockdown efficiency was confirmed by Western blot analysis (lower panel). (D) shRNA-mediated downregulation of Cav1 in ME-180 cells results in P+GC internalization. Intracellular bacteria (red) are detected in ME-180 shCav1 cells (upper right panels), whereas only extracellular bacteria (yellow-green) are detected in ME-180 shLuciferase control cells (upper left panels). Efficiency of Cav1 knockdown in ME-180 cells after lentiviral transduction of Luciferase (control) or Cav1 shRNA constructs (lower panel). Scale bar: 20 µm. (E) Numbers of viable intracellular P+GC in AGS cells decrease rapidly over time. Infected cells were initially treated with gentamicin for 2 h, then further incubated in gentamicin and serum-free medium and lysed at indicated time points. Experiments were performed in triplicate. Data are mean ± standard deviation.
Mentions: To assess the role of Cav1 in the Tfp-mediated binding of P+GC to host cells, we began by monitoring the cellular localization of Cav1 in ME-180 cells, a human epidermoid carcinoma cell line, immediately following infection. We found that endogenous Cav1 localized close to P+GC microcolonies after 2 h of infection (Figure 1A). Using live-cell imaging, we likewise observed a substantial accumulation of Cav1-GFP at sites of bacterial infection, which was induced even by single diplococci and within seconds after P+GC attachment (Figure 1B, lower panel). Cav1-GFP recruitment occurred throughout the early stages of infection, resulting ultimately in a conspicuous accumulation of the protein (Figure 1B, upper panel and Video S1). By contrast, using a non-piliated isogenic GC strain (P− Opa57+GC is a non-piliated isogenic strain of P+GC that produces an Opa57 adhesin specific for CEACAM receptors), we found no recruitment of endogenous Cav1 in ME-180 cells (Figure S1A). Interestingly, despite the observed recruitment of Cav1 after P+GC infection, microarray and Western blot analysis failed to detect any increase in Cav1 expression (unpublished data). This rather points to a cellular Cav1 reorganization leading to Cav1-accumulation than de novo synthesis.

Bottom Line: A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2.Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake.Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.

ABSTRACT
Certain bacterial adhesins appear to promote a pathogen's extracellular lifestyle rather than its entry into host cells. However, little is known about the stimuli elicited upon such pathogen host-cell interactions. Here, we report that type IV pili (Tfp)-producing Neisseria gonorrhoeae (P(+)GC) induces an immediate recruitment of caveolin-1 (Cav1) in the host cell, which subsequently prevents bacterial internalization by triggering cytoskeletal rearrangements via downstream phosphotyrosine signaling. A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake. Our findings, which have been extended to enteropathogenic Escherichia coli, highlight how Tfp-producing bacteria avoid host cell uptake. Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function.

Show MeSH
Related in: MedlinePlus