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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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A mutant L. pneumophila lacking 5 bacterial effectors that inhibit host protein synthesis is defective in induction of the host ‘effector-triggered response’.Growth of the indicated strains of L. pneumophila was measured in amoebae (A) or A/J macrophages (B). (C) Global host protein synthesis was measured by 35S-methionine incorporation in macrophages infected for 2.5 h with the indicated strains. (D) Myd88−/− (bottom right graph) or Caspase-1−/− (all others) macrophages were infected for 6 h with the specified strains. The indicated transcripts were measured by quantitative RT-PCR. (E) Caspase-1−/− macrophages were infected for 6 h with the specified strains. Indicated strains carried plasmids that constitutively expressed either a functional (plgt2, plgt3) or a catalytically inactive (plgt2*, plgt3*) bacterial effector. Data shown are representative of two (b, c) or at least three (A, D, E) experiments (mean ± sd). Δ5, Δlgt1Δlgt2Δlgt3ΔsidIΔsidL. Δ4, Δlgt1Δlgt2Δlgt3ΔsidI. *, p<0.05. ***, p<0.005.
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ppat-1001289-g003: A mutant L. pneumophila lacking 5 bacterial effectors that inhibit host protein synthesis is defective in induction of the host ‘effector-triggered response’.Growth of the indicated strains of L. pneumophila was measured in amoebae (A) or A/J macrophages (B). (C) Global host protein synthesis was measured by 35S-methionine incorporation in macrophages infected for 2.5 h with the indicated strains. (D) Myd88−/− (bottom right graph) or Caspase-1−/− (all others) macrophages were infected for 6 h with the specified strains. The indicated transcripts were measured by quantitative RT-PCR. (E) Caspase-1−/− macrophages were infected for 6 h with the specified strains. Indicated strains carried plasmids that constitutively expressed either a functional (plgt2, plgt3) or a catalytically inactive (plgt2*, plgt3*) bacterial effector. Data shown are representative of two (b, c) or at least three (A, D, E) experiments (mean ± sd). Δ5, Δlgt1Δlgt2Δlgt3ΔsidIΔsidL. Δ4, Δlgt1Δlgt2Δlgt3ΔsidI. *, p<0.05. ***, p<0.005.

Mentions: These 5 effectors appear to be important for survival within the pathogen's natural host, since the Δ5 mutant displayed a ∼10-fold growth defect in Dictyostelium amoebae (Figure 3A). By contrast, the Δ5 mutant showed no growth defect in macrophages (Figure 3B), but was defective, compared to wildtype, in its ability to inhibit host protein synthesis (Figure 3C). Although to a lesser degree than wildtype bacteria, the Δ5 mutant still appears to partially inhibit host protein synthesis, suggesting that L. pneumophila may encode additional inhibitors of host translation. Nevertheless, macrophages infected with Δ5 exhibited striking defects in induction of the ETR, including a ∼50-fold defect in induction of Il23a, Gem, and Csf2 (Figure 3D and Table S4). Importantly, the Dot/Icm-dependent induction of Ifnb, which is induced via a separate pathway [11], [13], [15], remained intact (Figure 3D), implying that the Δ5 mutant was competent for infection and Dot/Icm function. Individual deletion mutants of each of the five effectors showed no defect in Il23a, Csf2, or Gem induction, whereas a mutant lacking four of the five (Δlgt1Δlgt2Δlgt3ΔsidI) had a partial defect (Figure 3D, and data not shown). Complementation of Δ5 with wildtype lgt2 or lgt3 restored induction of Il23a and Gem, but complementation with mutant lgt2 or lgt3 lacking catalytic activity did not (Figure 3E). These results are significant because they show that macrophages make an innate response to a pathogen-encoded activity and that recognition of the effector molecules themselves is not likely to explain the ETR.


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)

A mutant L. pneumophila lacking 5 bacterial effectors that inhibit host protein synthesis is defective in induction of the host ‘effector-triggered response’.Growth of the indicated strains of L. pneumophila was measured in amoebae (A) or A/J macrophages (B). (C) Global host protein synthesis was measured by 35S-methionine incorporation in macrophages infected for 2.5 h with the indicated strains. (D) Myd88−/− (bottom right graph) or Caspase-1−/− (all others) macrophages were infected for 6 h with the specified strains. The indicated transcripts were measured by quantitative RT-PCR. (E) Caspase-1−/− macrophages were infected for 6 h with the specified strains. Indicated strains carried plasmids that constitutively expressed either a functional (plgt2, plgt3) or a catalytically inactive (plgt2*, plgt3*) bacterial effector. Data shown are representative of two (b, c) or at least three (A, D, E) experiments (mean ± sd). Δ5, Δlgt1Δlgt2Δlgt3ΔsidIΔsidL. Δ4, Δlgt1Δlgt2Δlgt3ΔsidI. *, 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-g003: A mutant L. pneumophila lacking 5 bacterial effectors that inhibit host protein synthesis is defective in induction of the host ‘effector-triggered response’.Growth of the indicated strains of L. pneumophila was measured in amoebae (A) or A/J macrophages (B). (C) Global host protein synthesis was measured by 35S-methionine incorporation in macrophages infected for 2.5 h with the indicated strains. (D) Myd88−/− (bottom right graph) or Caspase-1−/− (all others) macrophages were infected for 6 h with the specified strains. The indicated transcripts were measured by quantitative RT-PCR. (E) Caspase-1−/− macrophages were infected for 6 h with the specified strains. Indicated strains carried plasmids that constitutively expressed either a functional (plgt2, plgt3) or a catalytically inactive (plgt2*, plgt3*) bacterial effector. Data shown are representative of two (b, c) or at least three (A, D, E) experiments (mean ± sd). Δ5, Δlgt1Δlgt2Δlgt3ΔsidIΔsidL. Δ4, Δlgt1Δlgt2Δlgt3ΔsidI. *, p<0.05. ***, p<0.005.
Mentions: These 5 effectors appear to be important for survival within the pathogen's natural host, since the Δ5 mutant displayed a ∼10-fold growth defect in Dictyostelium amoebae (Figure 3A). By contrast, the Δ5 mutant showed no growth defect in macrophages (Figure 3B), but was defective, compared to wildtype, in its ability to inhibit host protein synthesis (Figure 3C). Although to a lesser degree than wildtype bacteria, the Δ5 mutant still appears to partially inhibit host protein synthesis, suggesting that L. pneumophila may encode additional inhibitors of host translation. Nevertheless, macrophages infected with Δ5 exhibited striking defects in induction of the ETR, including a ∼50-fold defect in induction of Il23a, Gem, and Csf2 (Figure 3D and Table S4). Importantly, the Dot/Icm-dependent induction of Ifnb, which is induced via a separate pathway [11], [13], [15], remained intact (Figure 3D), implying that the Δ5 mutant was competent for infection and Dot/Icm function. Individual deletion mutants of each of the five effectors showed no defect in Il23a, Csf2, or Gem induction, whereas a mutant lacking four of the five (Δlgt1Δlgt2Δlgt3ΔsidI) had a partial defect (Figure 3D, and data not shown). Complementation of Δ5 with wildtype lgt2 or lgt3 restored induction of Il23a and Gem, but complementation with mutant lgt2 or lgt3 lacking catalytic activity did not (Figure 3E). These results are significant because they show that macrophages make an innate response to a pathogen-encoded activity and that recognition of the effector molecules themselves is not likely to explain the ETR.

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