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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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Induction of the ‘effector-triggered response’ can be recapitulated by pharmacological inhibitors of translation.(A) B6 macrophages were infected for 6 h with the indicated strains, alone or with CHX (5 µg/mL). (B, C, D) B6 macrophages were infected or were treated for 4 h with CHX (10 µg/mL; B), puromycin (20 µg/mL; C) or bruceantin (50 nM; D) alone or in conjunction with Pam3CSK4 (10 ng/mL). CHX, cycloheximide. Data shown are representative of two (C, D) or three (A, B) experiments (mean ± sd). *, p<0.05. **, p<0.01.
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ppat-1001289-g004: Induction of the ‘effector-triggered response’ can be recapitulated by pharmacological inhibitors of translation.(A) B6 macrophages were infected for 6 h with the indicated strains, alone or with CHX (5 µg/mL). (B, C, D) B6 macrophages were infected or were treated for 4 h with CHX (10 µg/mL; B), puromycin (20 µg/mL; C) or bruceantin (50 nM; D) alone or in conjunction with Pam3CSK4 (10 ng/mL). CHX, cycloheximide. Data shown are representative of two (C, D) or three (A, B) experiments (mean ± sd). *, p<0.05. **, p<0.01.

Mentions: We then tested more directly whether the ETR was induced by translation inhibition. The defective induction of Il23a, Csf2, and Gem in macrophages infected with ΔdotA or Δ5 was rescued by addition of the translation inhibitor cycloheximide (Figure 4A, and data not shown). These results support the hypothesis that induction of the ETR by L. pneumophila involves inhibition of translation by the five deleted effectors. Importantly, the potent induction of Il23a, Csf2 and Gem by L. pneumophila could be recapitulated in uninfected macrophages by treatment with the translation elongation inhibitors cycloheximide (Figure 4B) or puromycin (Figure 4C), or the initiation inhibitor bruceantin (Figure 4D), in conjunction with the TLR2 ligand Pam3CSK4. These three translation inhibitors possess different targets and modes of action, making it unlikely that the common host response to each of them is due to nonspecific drug effects. Thus, translation inhibition in the context of TLR signaling provokes a specific transcriptional response. Translation inhibitors alone were capable of inducing some, but not all, effector-triggered transcriptional targets (Figure 4B, C, and D), supporting our model that translation inhibition acts in concert with classical PRR signaling to generate the full effector-dependent signature. Microarray analysis indicated that the five effectors accounted for induction of at least 54 (∼30%) of the Dot/Icm-dependent genes (Figure 5A and Table S4).


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)

Induction of the ‘effector-triggered response’ can be recapitulated by pharmacological inhibitors of translation.(A) B6 macrophages were infected for 6 h with the indicated strains, alone or with CHX (5 µg/mL). (B, C, D) B6 macrophages were infected or were treated for 4 h with CHX (10 µg/mL; B), puromycin (20 µg/mL; C) or bruceantin (50 nM; D) alone or in conjunction with Pam3CSK4 (10 ng/mL). CHX, cycloheximide. Data shown are representative of two (C, D) or three (A, B) experiments (mean ± sd). *, p<0.05. **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040669&req=5

ppat-1001289-g004: Induction of the ‘effector-triggered response’ can be recapitulated by pharmacological inhibitors of translation.(A) B6 macrophages were infected for 6 h with the indicated strains, alone or with CHX (5 µg/mL). (B, C, D) B6 macrophages were infected or were treated for 4 h with CHX (10 µg/mL; B), puromycin (20 µg/mL; C) or bruceantin (50 nM; D) alone or in conjunction with Pam3CSK4 (10 ng/mL). CHX, cycloheximide. Data shown are representative of two (C, D) or three (A, B) experiments (mean ± sd). *, p<0.05. **, p<0.01.
Mentions: We then tested more directly whether the ETR was induced by translation inhibition. The defective induction of Il23a, Csf2, and Gem in macrophages infected with ΔdotA or Δ5 was rescued by addition of the translation inhibitor cycloheximide (Figure 4A, and data not shown). These results support the hypothesis that induction of the ETR by L. pneumophila involves inhibition of translation by the five deleted effectors. Importantly, the potent induction of Il23a, Csf2 and Gem by L. pneumophila could be recapitulated in uninfected macrophages by treatment with the translation elongation inhibitors cycloheximide (Figure 4B) or puromycin (Figure 4C), or the initiation inhibitor bruceantin (Figure 4D), in conjunction with the TLR2 ligand Pam3CSK4. These three translation inhibitors possess different targets and modes of action, making it unlikely that the common host response to each of them is due to nonspecific drug effects. Thus, translation inhibition in the context of TLR signaling provokes a specific transcriptional response. Translation inhibitors alone were capable of inducing some, but not all, effector-triggered transcriptional targets (Figure 4B, C, and D), supporting our model that translation inhibition acts in concert with classical PRR signaling to generate the full effector-dependent signature. Microarray analysis indicated that the five effectors accounted for induction of at least 54 (∼30%) of the Dot/Icm-dependent genes (Figure 5A and Table S4).

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