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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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MyD88 and Nod signaling alone do not account for the unique response to virulent L. pneumophila, which can be recapitulated by ER stress inducers that also inhibit translation.In all panels, the indicated transcripts were measured by quantitative RT-PCR. (A) Macrophages were infected with ΔflaA L. pneumophila for 6 h. (B) Macrophages were infected with L. pneumophila or were treated with Pam3CSK4 (10 ng/mL) and/or transfected with MDP (10 µg/mL) for 6 h. (C) B6 macrophages were infected with L. pneumophila, wildtype L. monocytogenes or the avirulent L. monocytogenes Δhly mutant for 4 h. (D) B6 macrophages were infected with the indicated strains of L. pneumophila for 6 h. **, p<0.01 compared to wildtype (WT). (E) Uninfected B6 macrophages were treated with thapsigargin (500 nM) or tunicamycin (5 µg/mL) for 6 h alone or in conjunction with Pam3CSK4 (1 ng/mL). All results shown are representative of at least three experiments (mean ± sd). Lm, L. monocytogenes. *, p<0.05; **, p<0.01; ***, p<0.005.
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ppat-1001289-g002: MyD88 and Nod signaling alone do not account for the unique response to virulent L. pneumophila, which can be recapitulated by ER stress inducers that also inhibit translation.In all panels, the indicated transcripts were measured by quantitative RT-PCR. (A) Macrophages were infected with ΔflaA L. pneumophila for 6 h. (B) Macrophages were infected with L. pneumophila or were treated with Pam3CSK4 (10 ng/mL) and/or transfected with MDP (10 µg/mL) for 6 h. (C) B6 macrophages were infected with L. pneumophila, wildtype L. monocytogenes or the avirulent L. monocytogenes Δhly mutant for 4 h. (D) B6 macrophages were infected with the indicated strains of L. pneumophila for 6 h. **, p<0.01 compared to wildtype (WT). (E) Uninfected B6 macrophages were treated with thapsigargin (500 nM) or tunicamycin (5 µg/mL) for 6 h alone or in conjunction with Pam3CSK4 (1 ng/mL). All results shown are representative of at least three experiments (mean ± sd). Lm, L. monocytogenes. *, p<0.05; **, p<0.01; ***, p<0.005.

Mentions: In order to identify the host pathway(s) responsible for induction of the ETR, we first examined innate immune pathways known to recognize L. pneumophila. Induction of the representative genes Il23a, Csf2, and Gem did not require the previously described Naip5/Nlrc4 flagellin-sensing pathway [20], as infection with a flagellin-deficient mutant (ΔflaA) also induced robust expression of these genes (Figure 1A, B and Table S2). Moreover, Il23a, Csf2 and Gem were strongly (>1000-fold) induced in the absence of the Mavs/Irf3/Irf7 signaling axis shown previously to respond to L. pneumophila [11], [13], [18] (Figure 1D, and data not shown). As suggested by previous transcriptional profiling experiments [17], we confirmed that Myd88−/−and Rip2−/−macrophages, which are defective in TLR and Nod1/Nod2 signaling, respectively, strongly upregulated Il23a and Gem following infection with wildtype L. pneumophila (Figure 2A). Induction of Il23a was abrogated in Myd88−/−Rip2−/− and Myd88−/−Nod1−/−Nod2−/− macrophages; however, these macrophages still robustly induced Gem (Figure 2A, and data not shown). These data indicate that TLR/Nod signaling is necessary for induction of some, but not all, genes in the ETR. Furthermore, the intact induction of Gem in Myd88−/−Nod1−/−Nod2−/− macrophages implies the existence of an additional pathway.


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)

MyD88 and Nod signaling alone do not account for the unique response to virulent L. pneumophila, which can be recapitulated by ER stress inducers that also inhibit translation.In all panels, the indicated transcripts were measured by quantitative RT-PCR. (A) Macrophages were infected with ΔflaA L. pneumophila for 6 h. (B) Macrophages were infected with L. pneumophila or were treated with Pam3CSK4 (10 ng/mL) and/or transfected with MDP (10 µg/mL) for 6 h. (C) B6 macrophages were infected with L. pneumophila, wildtype L. monocytogenes or the avirulent L. monocytogenes Δhly mutant for 4 h. (D) B6 macrophages were infected with the indicated strains of L. pneumophila for 6 h. **, p<0.01 compared to wildtype (WT). (E) Uninfected B6 macrophages were treated with thapsigargin (500 nM) or tunicamycin (5 µg/mL) for 6 h alone or in conjunction with Pam3CSK4 (1 ng/mL). All results shown are representative of at least three experiments (mean ± sd). Lm, L. monocytogenes. *, p<0.05; **, p<0.01; ***, p<0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040669&req=5

ppat-1001289-g002: MyD88 and Nod signaling alone do not account for the unique response to virulent L. pneumophila, which can be recapitulated by ER stress inducers that also inhibit translation.In all panels, the indicated transcripts were measured by quantitative RT-PCR. (A) Macrophages were infected with ΔflaA L. pneumophila for 6 h. (B) Macrophages were infected with L. pneumophila or were treated with Pam3CSK4 (10 ng/mL) and/or transfected with MDP (10 µg/mL) for 6 h. (C) B6 macrophages were infected with L. pneumophila, wildtype L. monocytogenes or the avirulent L. monocytogenes Δhly mutant for 4 h. (D) B6 macrophages were infected with the indicated strains of L. pneumophila for 6 h. **, p<0.01 compared to wildtype (WT). (E) Uninfected B6 macrophages were treated with thapsigargin (500 nM) or tunicamycin (5 µg/mL) for 6 h alone or in conjunction with Pam3CSK4 (1 ng/mL). All results shown are representative of at least three experiments (mean ± sd). Lm, L. monocytogenes. *, p<0.05; **, p<0.01; ***, p<0.005.
Mentions: In order to identify the host pathway(s) responsible for induction of the ETR, we first examined innate immune pathways known to recognize L. pneumophila. Induction of the representative genes Il23a, Csf2, and Gem did not require the previously described Naip5/Nlrc4 flagellin-sensing pathway [20], as infection with a flagellin-deficient mutant (ΔflaA) also induced robust expression of these genes (Figure 1A, B and Table S2). Moreover, Il23a, Csf2 and Gem were strongly (>1000-fold) induced in the absence of the Mavs/Irf3/Irf7 signaling axis shown previously to respond to L. pneumophila [11], [13], [18] (Figure 1D, and data not shown). As suggested by previous transcriptional profiling experiments [17], we confirmed that Myd88−/−and Rip2−/−macrophages, which are defective in TLR and Nod1/Nod2 signaling, respectively, strongly upregulated Il23a and Gem following infection with wildtype L. pneumophila (Figure 2A). Induction of Il23a was abrogated in Myd88−/−Rip2−/− and Myd88−/−Nod1−/−Nod2−/− macrophages; however, these macrophages still robustly induced Gem (Figure 2A, and data not shown). These data indicate that TLR/Nod signaling is necessary for induction of some, but not all, genes in the ETR. Furthermore, the intact induction of Gem in Myd88−/−Nod1−/−Nod2−/− macrophages implies the existence of an additional pathway.

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