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Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila.

Fontana MF, Banga S, Barry KC, Shen X, Tan Y, Luo ZQ, Vance RE - PLoS Pathog. (2011)

Bottom Line: Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor.L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR.Our results add to this model by providing a striking illustration of how the host immune response to a virulent pathogen can also be shaped by pathogen-encoded activities, such as inhibition of host protein synthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, Berkeley, California, USA.

ABSTRACT
The intracellular bacterial pathogen Legionella pneumophila causes an inflammatory pneumonia called Legionnaires' Disease. For virulence, L. pneumophila requires a Dot/Icm type IV secretion system that translocates bacterial effectors to the host cytosol. L. pneumophila lacking the Dot/Icm system is recognized by Toll-like receptors (TLRs), leading to a canonical NF-κB-dependent transcriptional response. In addition, L. pneumophila expressing a functional Dot/Icm system potently induces unique transcriptional targets, including proinflammatory genes such as Il23a and Csf2. Here we demonstrate that this Dot/Icm-dependent response, which we term the effector-triggered response (ETR), requires five translocated bacterial effectors that inhibit host protein synthesis. Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor. Thus, macrophages infected with wildtype L. pneumophila exhibited prolonged activation of NF-κB, which was associated with transcription of ETR target genes such as Il23a and Csf2. L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR. L. pneumophila mutants expressing enzymatically inactive effectors were also unable to fully induce the ETR, whereas multiple compounds or bacterial toxins that inhibit host protein synthesis via distinct mechanisms recapitulated the ETR when administered with TLR ligands. Previous studies have demonstrated that the host response to bacterial infection is induced primarily by specific microbial molecules that activate TLRs or cytosolic pattern recognition receptors. Our results add to this model by providing a striking illustration of how the host immune response to a virulent pathogen can also be shaped by pathogen-encoded activities, such as inhibition of host protein synthesis.

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Inhibition of host translation by multiple bacterial toxins provokes an inflammatory cytokine response in vitro and in vivo.(A) B6 macrophages were infected for 24 h with the indicated strains of L. pneumophila and/or treated with cycloheximide (5 µg/mL). Protein levels in the supernatant were assayed by ELISA. (B) B6 macrophages were treated for 5 h with Diphtheria Toxin (1 ng/mL; left panel) or with Exotoxin A (500 ng/mL; right panel), alone or in conjunction with Pam3CSK4. Il23a transcript levels were assayed by quantitative RT-PCR. n.d., not detected. (C) B6 mice were treated intranasally with Pam3CSK4 (10 µg/mouse) or ExoA (2 µg/mouse) or both in 25 µL PBS. Bronchoalveolar lavage was performed 24 h post infection. GM-CSF levels in lavage were measured by ELISA. Data are representative of two (A, C) or three (B) experiments (mean ± sd in A, B). CHX, cycloheximide. DT, Diphtheria Toxin. ExoA, Exotoxin A. *, p<0.05. ***, p<0.005.
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ppat-1001289-g007: Inhibition of host translation by multiple bacterial toxins provokes an inflammatory cytokine response in vitro and in vivo.(A) B6 macrophages were infected for 24 h with the indicated strains of L. pneumophila and/or treated with cycloheximide (5 µg/mL). Protein levels in the supernatant were assayed by ELISA. (B) B6 macrophages were treated for 5 h with Diphtheria Toxin (1 ng/mL; left panel) or with Exotoxin A (500 ng/mL; right panel), alone or in conjunction with Pam3CSK4. Il23a transcript levels were assayed by quantitative RT-PCR. n.d., not detected. (C) B6 mice were treated intranasally with Pam3CSK4 (10 µg/mouse) or ExoA (2 µg/mouse) or both in 25 µL PBS. Bronchoalveolar lavage was performed 24 h post infection. GM-CSF levels in lavage were measured by ELISA. Data are representative of two (A, C) or three (B) experiments (mean ± sd in A, B). CHX, cycloheximide. DT, Diphtheria Toxin. ExoA, Exotoxin A. *, p<0.05. ***, p<0.005.

Mentions: Although inhibition of protein synthesis potently induces transcription of certain target genes, a central question is whether this transcriptional response is sufficient to overcome the translational block, and result in increased protein production. Accordingly, we measured the protein levels of GM-CSF (encoded by the Csf2 gene) in the supernatant of infected macrophages. GM-CSF protein was preferentially produced by cells infected with wildtype L. pneumophila as compared to cells infected with Δ5 (Figure 7A). The defect in cytokine production by Δ5-infected macrophages was not due to poor bacterial growth (Figure 3B), increased cytotoxicity (Figure S3A), or defective secretion (Figure S3B), and could be rescued by addition of cycloheximide (Figure 7A). Thus translation inhibition can paradoxically lead to increased production of certain proteins, perhaps because transcriptional superinduction of specific transcripts is sufficient to overcome the partial translational block mediated by L. pneumophila.


Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila.

Fontana MF, Banga S, Barry KC, Shen X, Tan Y, Luo ZQ, Vance RE - PLoS Pathog. (2011)

Inhibition of host translation by multiple bacterial toxins provokes an inflammatory cytokine response in vitro and in vivo.(A) B6 macrophages were infected for 24 h with the indicated strains of L. pneumophila and/or treated with cycloheximide (5 µg/mL). Protein levels in the supernatant were assayed by ELISA. (B) B6 macrophages were treated for 5 h with Diphtheria Toxin (1 ng/mL; left panel) or with Exotoxin A (500 ng/mL; right panel), alone or in conjunction with Pam3CSK4. Il23a transcript levels were assayed by quantitative RT-PCR. n.d., not detected. (C) B6 mice were treated intranasally with Pam3CSK4 (10 µg/mouse) or ExoA (2 µg/mouse) or both in 25 µL PBS. Bronchoalveolar lavage was performed 24 h post infection. GM-CSF levels in lavage were measured by ELISA. Data are representative of two (A, C) or three (B) experiments (mean ± sd in A, B). CHX, cycloheximide. DT, Diphtheria Toxin. ExoA, Exotoxin A. *, p<0.05. ***, p<0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040669&req=5

ppat-1001289-g007: Inhibition of host translation by multiple bacterial toxins provokes an inflammatory cytokine response in vitro and in vivo.(A) B6 macrophages were infected for 24 h with the indicated strains of L. pneumophila and/or treated with cycloheximide (5 µg/mL). Protein levels in the supernatant were assayed by ELISA. (B) B6 macrophages were treated for 5 h with Diphtheria Toxin (1 ng/mL; left panel) or with Exotoxin A (500 ng/mL; right panel), alone or in conjunction with Pam3CSK4. Il23a transcript levels were assayed by quantitative RT-PCR. n.d., not detected. (C) B6 mice were treated intranasally with Pam3CSK4 (10 µg/mouse) or ExoA (2 µg/mouse) or both in 25 µL PBS. Bronchoalveolar lavage was performed 24 h post infection. GM-CSF levels in lavage were measured by ELISA. Data are representative of two (A, C) or three (B) experiments (mean ± sd in A, B). CHX, cycloheximide. DT, Diphtheria Toxin. ExoA, Exotoxin A. *, p<0.05. ***, p<0.005.
Mentions: Although inhibition of protein synthesis potently induces transcription of certain target genes, a central question is whether this transcriptional response is sufficient to overcome the translational block, and result in increased protein production. Accordingly, we measured the protein levels of GM-CSF (encoded by the Csf2 gene) in the supernatant of infected macrophages. GM-CSF protein was preferentially produced by cells infected with wildtype L. pneumophila as compared to cells infected with Δ5 (Figure 7A). The defect in cytokine production by Δ5-infected macrophages was not due to poor bacterial growth (Figure 3B), increased cytotoxicity (Figure S3A), or defective secretion (Figure S3B), and could be rescued by addition of cycloheximide (Figure 7A). Thus translation inhibition can paradoxically lead to increased production of certain proteins, perhaps because transcriptional superinduction of specific transcripts is sufficient to overcome the partial translational block mediated by L. pneumophila.

Bottom Line: Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor.L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR.Our results add to this model by providing a striking illustration of how the host immune response to a virulent pathogen can also be shaped by pathogen-encoded activities, such as inhibition of host protein synthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, Berkeley, California, USA.

ABSTRACT
The intracellular bacterial pathogen Legionella pneumophila causes an inflammatory pneumonia called Legionnaires' Disease. For virulence, L. pneumophila requires a Dot/Icm type IV secretion system that translocates bacterial effectors to the host cytosol. L. pneumophila lacking the Dot/Icm system is recognized by Toll-like receptors (TLRs), leading to a canonical NF-κB-dependent transcriptional response. In addition, L. pneumophila expressing a functional Dot/Icm system potently induces unique transcriptional targets, including proinflammatory genes such as Il23a and Csf2. Here we demonstrate that this Dot/Icm-dependent response, which we term the effector-triggered response (ETR), requires five translocated bacterial effectors that inhibit host protein synthesis. Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor. Thus, macrophages infected with wildtype L. pneumophila exhibited prolonged activation of NF-κB, which was associated with transcription of ETR target genes such as Il23a and Csf2. L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR. L. pneumophila mutants expressing enzymatically inactive effectors were also unable to fully induce the ETR, whereas multiple compounds or bacterial toxins that inhibit host protein synthesis via distinct mechanisms recapitulated the ETR when administered with TLR ligands. Previous studies have demonstrated that the host response to bacterial infection is induced primarily by specific microbial molecules that activate TLRs or cytosolic pattern recognition receptors. Our results add to this model by providing a striking illustration of how the host immune response to a virulent pathogen can also be shaped by pathogen-encoded activities, such as inhibition of host protein synthesis.

Show MeSH
Related in: MedlinePlus