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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 unique transcriptional response in macrophages infected with virulent L. pneumophila.(A) Caspase-1−/− macrophages were infected for 6 h with the specified strains of L. pneumophila. RNA was amplified and hybridized to MEEBO microarrays. Black and red dots, genes exhibiting greater than 2-fold difference in induction between wildtype (WT) and mutant. Red dots indicate labeled genes. Data shown are the average of two experiments. (B) B6 macrophages were infected for 6 h with the specified strains of L. pneumophila. Levels of the indicated transcripts were measured by quantitative RT-PCR. (C) Mice were infected intranasally with 2×106 L. pneumophila and bronchoalveolar lavage was performed 24 h post infection. Host cells recovered from bronchoalveolar lavage fluid (BALF) were counted with a hemocytometer. A portion of each sample was plated on BCYE plates to enumerate cfu. (D) Macrophages were infected for 6 h with L. pneumophila. Levels of the indicated transcripts were measured by quantitative RT-PCR. N.S., not significant. Data shown are representative of two (a, d) or at least three (B, C) experiments (mean ± sd in b, d). *, p<0.05 versus uninfected. ***, p<0.005 versus uninfected.
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ppat-1001289-g001: A unique transcriptional response in macrophages infected with virulent L. pneumophila.(A) Caspase-1−/− macrophages were infected for 6 h with the specified strains of L. pneumophila. RNA was amplified and hybridized to MEEBO microarrays. Black and red dots, genes exhibiting greater than 2-fold difference in induction between wildtype (WT) and mutant. Red dots indicate labeled genes. Data shown are the average of two experiments. (B) B6 macrophages were infected for 6 h with the specified strains of L. pneumophila. Levels of the indicated transcripts were measured by quantitative RT-PCR. (C) Mice were infected intranasally with 2×106 L. pneumophila and bronchoalveolar lavage was performed 24 h post infection. Host cells recovered from bronchoalveolar lavage fluid (BALF) were counted with a hemocytometer. A portion of each sample was plated on BCYE plates to enumerate cfu. (D) Macrophages were infected for 6 h with L. pneumophila. Levels of the indicated transcripts were measured by quantitative RT-PCR. N.S., not significant. Data shown are representative of two (a, d) or at least three (B, C) experiments (mean ± sd in b, d). *, p<0.05 versus uninfected. ***, p<0.005 versus uninfected.

Mentions: We initially sought to identify host responses that discriminate between pathogenic and non-pathogenic bacteria. Our strategy was to compare the host response to wildtype virulent L. pneumophila with the host response to an avirulent L. pneumophila mutant, ΔdotA. ΔdotA mutants lack a functional Dot/Icm secretion system, and thus fail to translocate effectors into the host cytosol, but they nevertheless express the normal complement of PAMPs that engage Toll-like receptor pathways. We performed transcriptional profiling experiments on macrophages infected with either wildtype L. pneumophila or the avirulent ΔdotA mutant. In the microarray experiments, Caspase-1−/− macrophages were used to eliminate flagellin-dependent macrophage death, which would otherwise differ between wildtype and ΔdotA infections [12], [14], [16], but our results were later validated with wildtype macrophages (see below). RNA was collected from macrophages at a timepoint when there were similar numbers of bacteria in both wildtype-infected and ΔdotA-infected macrophages. Microarray analysis revealed 166 genes that were differentially induced >2-fold in a manner dependent on type IV secretion (Figure 1A and Table S1). The induction of some of the Dot/Icm-dependent genes, e.g. Ifnb, could be explained by cytosolic sensing pathways that have been previously characterized [11], [13], [18]. However, much of the response to Dot/Icm+ bacteria did not appear to be accounted for by host pathways known to recognize L. pneumophila. For reasons discussed below, we refer specifically to this unexplained Dot/Icm-dependent transcriptional signature as the ‘effector-triggered response,’ or 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 unique transcriptional response in macrophages infected with virulent L. pneumophila.(A) Caspase-1−/− macrophages were infected for 6 h with the specified strains of L. pneumophila. RNA was amplified and hybridized to MEEBO microarrays. Black and red dots, genes exhibiting greater than 2-fold difference in induction between wildtype (WT) and mutant. Red dots indicate labeled genes. Data shown are the average of two experiments. (B) B6 macrophages were infected for 6 h with the specified strains of L. pneumophila. Levels of the indicated transcripts were measured by quantitative RT-PCR. (C) Mice were infected intranasally with 2×106 L. pneumophila and bronchoalveolar lavage was performed 24 h post infection. Host cells recovered from bronchoalveolar lavage fluid (BALF) were counted with a hemocytometer. A portion of each sample was plated on BCYE plates to enumerate cfu. (D) Macrophages were infected for 6 h with L. pneumophila. Levels of the indicated transcripts were measured by quantitative RT-PCR. N.S., not significant. Data shown are representative of two (a, d) or at least three (B, C) experiments (mean ± sd in b, d). *, p<0.05 versus uninfected. ***, p<0.005 versus uninfected.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040669&req=5

ppat-1001289-g001: A unique transcriptional response in macrophages infected with virulent L. pneumophila.(A) Caspase-1−/− macrophages were infected for 6 h with the specified strains of L. pneumophila. RNA was amplified and hybridized to MEEBO microarrays. Black and red dots, genes exhibiting greater than 2-fold difference in induction between wildtype (WT) and mutant. Red dots indicate labeled genes. Data shown are the average of two experiments. (B) B6 macrophages were infected for 6 h with the specified strains of L. pneumophila. Levels of the indicated transcripts were measured by quantitative RT-PCR. (C) Mice were infected intranasally with 2×106 L. pneumophila and bronchoalveolar lavage was performed 24 h post infection. Host cells recovered from bronchoalveolar lavage fluid (BALF) were counted with a hemocytometer. A portion of each sample was plated on BCYE plates to enumerate cfu. (D) Macrophages were infected for 6 h with L. pneumophila. Levels of the indicated transcripts were measured by quantitative RT-PCR. N.S., not significant. Data shown are representative of two (a, d) or at least three (B, C) experiments (mean ± sd in b, d). *, p<0.05 versus uninfected. ***, p<0.005 versus uninfected.
Mentions: We initially sought to identify host responses that discriminate between pathogenic and non-pathogenic bacteria. Our strategy was to compare the host response to wildtype virulent L. pneumophila with the host response to an avirulent L. pneumophila mutant, ΔdotA. ΔdotA mutants lack a functional Dot/Icm secretion system, and thus fail to translocate effectors into the host cytosol, but they nevertheless express the normal complement of PAMPs that engage Toll-like receptor pathways. We performed transcriptional profiling experiments on macrophages infected with either wildtype L. pneumophila or the avirulent ΔdotA mutant. In the microarray experiments, Caspase-1−/− macrophages were used to eliminate flagellin-dependent macrophage death, which would otherwise differ between wildtype and ΔdotA infections [12], [14], [16], but our results were later validated with wildtype macrophages (see below). RNA was collected from macrophages at a timepoint when there were similar numbers of bacteria in both wildtype-infected and ΔdotA-infected macrophages. Microarray analysis revealed 166 genes that were differentially induced >2-fold in a manner dependent on type IV secretion (Figure 1A and Table S1). The induction of some of the Dot/Icm-dependent genes, e.g. Ifnb, could be explained by cytosolic sensing pathways that have been previously characterized [11], [13], [18]. However, much of the response to Dot/Icm+ bacteria did not appear to be accounted for by host pathways known to recognize L. pneumophila. For reasons discussed below, we refer specifically to this unexplained Dot/Icm-dependent transcriptional signature as the ‘effector-triggered response,’ or 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