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Stimulation of host immune defenses by a small molecule protects C. elegans from bacterial infection.

Pukkila-Worley R, Feinbaum R, Kirienko NV, Larkins-Ford J, Conery AL, Ausubel FM - PLoS Genet. (2012)

Bottom Line: We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested.Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we show that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (∼1.3% of all genes) in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7.These data show that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans-based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The nematode Caenorhabditis elegans offers currently untapped potential for carrying out high-throughput, live-animal screens of low molecular weight compound libraries to identify molecules that target a variety of cellular processes. We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested. Here we show that one of these small molecules, RPW-24, protects C. elegans from bacterial infection by stimulating the host immune response of the nematode. Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we show that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (∼1.3% of all genes) in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7. These data show that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans-based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens.

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The C. elegans transcription factor ATF-7 regulates immune gene induction by RPW-24.(A) Fluorescence microscopy images of C. elegans acIs101 animals, which express a F35E12.5::GFP transgene, exposed to the normal nematode food source E. coli OP50 or P. aeruginosa in the presence of DMSO or 70 µM RPW-24 for 16 hours at 25°C. C. elegans acIs101 animals were raised on L4440 RNAi control bacteria or an RNAi feeder strain designed to knockdown the expression of atf-7. Green is GFP expression. (B and C) Six putative C. elegans immune effectors were analyzed by qRT-PCR in wild-type, atf-7(qd137) and atf-7(qd22 qd130) animals feeding on E. coli and exposed to either 70 µM RPW-24 or DMSO for 16 hours. Data are presented as the average of two biological replicates each normalized to a control gene with error bars representing SEM. In (A), the data are relative to the average expression of the indicated gene in wild-type C. elegans exposed to DMSO. In (B), the fold change of the indicated gene above its expression in C. elegans exposed to DMSO is compared in wild-type, atf-7(qd137) and atf-7(qd22 qd130) animals. An N2-derived strain carrying the acIs219 transgene was used as the control strain because this transgene is also present in the atf-7(lof) strains [12]. *P<0.05, **P = 0.08, ***P = 0.09 for the comparison in (A) of the relative expression levels of both atf-7(lof) strains versus control animals, each exposed to RPW-24, and in (B) for the fold change in wild-type versus both atf-7(lof) mutants.
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pgen-1002733-g005: The C. elegans transcription factor ATF-7 regulates immune gene induction by RPW-24.(A) Fluorescence microscopy images of C. elegans acIs101 animals, which express a F35E12.5::GFP transgene, exposed to the normal nematode food source E. coli OP50 or P. aeruginosa in the presence of DMSO or 70 µM RPW-24 for 16 hours at 25°C. C. elegans acIs101 animals were raised on L4440 RNAi control bacteria or an RNAi feeder strain designed to knockdown the expression of atf-7. Green is GFP expression. (B and C) Six putative C. elegans immune effectors were analyzed by qRT-PCR in wild-type, atf-7(qd137) and atf-7(qd22 qd130) animals feeding on E. coli and exposed to either 70 µM RPW-24 or DMSO for 16 hours. Data are presented as the average of two biological replicates each normalized to a control gene with error bars representing SEM. In (A), the data are relative to the average expression of the indicated gene in wild-type C. elegans exposed to DMSO. In (B), the fold change of the indicated gene above its expression in C. elegans exposed to DMSO is compared in wild-type, atf-7(qd137) and atf-7(qd22 qd130) animals. An N2-derived strain carrying the acIs219 transgene was used as the control strain because this transgene is also present in the atf-7(lof) strains [12]. *P<0.05, **P = 0.08, ***P = 0.09 for the comparison in (A) of the relative expression levels of both atf-7(lof) strains versus control animals, each exposed to RPW-24, and in (B) for the fold change in wild-type versus both atf-7(lof) mutants.

Mentions: The basal regulation of F35E12.5 requires PMK-1, but its induction by RPW-24 occurs in a PMK-1-independent manner (Figure 4A and 4B). We therefore anticipated that a reverse genetic RNAi screen aimed at identifying transcription factors required for the RPW-24-mediated induction of F35E12.5::GFP would identify genetic regulators that act either downstream of or in parallel to the PMK-1 pathway. We used a feeding RNAi library containing bacterial clones that produce double stranded RNA (dsRNA) designed to individually knockdown the expression of 393 transcription factors in C. elegans, corresponding to 30–50% of the transcription factors in the C. elegans genome [18] and screened for RNAi clones that abrogated the RPW-24-mediated induction of F35E12.5::GFP. Among 393 screened, we found that a single clone, corresponding to the transcription factor ATF-7, caused a striking reduction of F35E12.5::GFP expression when nematodes were either growing on their normal laboratory food source (E. coli OP50) or infected with P. aeruginosa (Figure 5A). ATF-7 was previously shown to function downstream of PMK-1 in the regulation of immune response genes during P. aeruginosa infection [12].


Stimulation of host immune defenses by a small molecule protects C. elegans from bacterial infection.

Pukkila-Worley R, Feinbaum R, Kirienko NV, Larkins-Ford J, Conery AL, Ausubel FM - PLoS Genet. (2012)

The C. elegans transcription factor ATF-7 regulates immune gene induction by RPW-24.(A) Fluorescence microscopy images of C. elegans acIs101 animals, which express a F35E12.5::GFP transgene, exposed to the normal nematode food source E. coli OP50 or P. aeruginosa in the presence of DMSO or 70 µM RPW-24 for 16 hours at 25°C. C. elegans acIs101 animals were raised on L4440 RNAi control bacteria or an RNAi feeder strain designed to knockdown the expression of atf-7. Green is GFP expression. (B and C) Six putative C. elegans immune effectors were analyzed by qRT-PCR in wild-type, atf-7(qd137) and atf-7(qd22 qd130) animals feeding on E. coli and exposed to either 70 µM RPW-24 or DMSO for 16 hours. Data are presented as the average of two biological replicates each normalized to a control gene with error bars representing SEM. In (A), the data are relative to the average expression of the indicated gene in wild-type C. elegans exposed to DMSO. In (B), the fold change of the indicated gene above its expression in C. elegans exposed to DMSO is compared in wild-type, atf-7(qd137) and atf-7(qd22 qd130) animals. An N2-derived strain carrying the acIs219 transgene was used as the control strain because this transgene is also present in the atf-7(lof) strains [12]. *P<0.05, **P = 0.08, ***P = 0.09 for the comparison in (A) of the relative expression levels of both atf-7(lof) strains versus control animals, each exposed to RPW-24, and in (B) for the fold change in wild-type versus both atf-7(lof) mutants.
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pgen-1002733-g005: The C. elegans transcription factor ATF-7 regulates immune gene induction by RPW-24.(A) Fluorescence microscopy images of C. elegans acIs101 animals, which express a F35E12.5::GFP transgene, exposed to the normal nematode food source E. coli OP50 or P. aeruginosa in the presence of DMSO or 70 µM RPW-24 for 16 hours at 25°C. C. elegans acIs101 animals were raised on L4440 RNAi control bacteria or an RNAi feeder strain designed to knockdown the expression of atf-7. Green is GFP expression. (B and C) Six putative C. elegans immune effectors were analyzed by qRT-PCR in wild-type, atf-7(qd137) and atf-7(qd22 qd130) animals feeding on E. coli and exposed to either 70 µM RPW-24 or DMSO for 16 hours. Data are presented as the average of two biological replicates each normalized to a control gene with error bars representing SEM. In (A), the data are relative to the average expression of the indicated gene in wild-type C. elegans exposed to DMSO. In (B), the fold change of the indicated gene above its expression in C. elegans exposed to DMSO is compared in wild-type, atf-7(qd137) and atf-7(qd22 qd130) animals. An N2-derived strain carrying the acIs219 transgene was used as the control strain because this transgene is also present in the atf-7(lof) strains [12]. *P<0.05, **P = 0.08, ***P = 0.09 for the comparison in (A) of the relative expression levels of both atf-7(lof) strains versus control animals, each exposed to RPW-24, and in (B) for the fold change in wild-type versus both atf-7(lof) mutants.
Mentions: The basal regulation of F35E12.5 requires PMK-1, but its induction by RPW-24 occurs in a PMK-1-independent manner (Figure 4A and 4B). We therefore anticipated that a reverse genetic RNAi screen aimed at identifying transcription factors required for the RPW-24-mediated induction of F35E12.5::GFP would identify genetic regulators that act either downstream of or in parallel to the PMK-1 pathway. We used a feeding RNAi library containing bacterial clones that produce double stranded RNA (dsRNA) designed to individually knockdown the expression of 393 transcription factors in C. elegans, corresponding to 30–50% of the transcription factors in the C. elegans genome [18] and screened for RNAi clones that abrogated the RPW-24-mediated induction of F35E12.5::GFP. Among 393 screened, we found that a single clone, corresponding to the transcription factor ATF-7, caused a striking reduction of F35E12.5::GFP expression when nematodes were either growing on their normal laboratory food source (E. coli OP50) or infected with P. aeruginosa (Figure 5A). ATF-7 was previously shown to function downstream of PMK-1 in the regulation of immune response genes during P. aeruginosa infection [12].

Bottom Line: We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested.Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we show that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (∼1.3% of all genes) in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7.These data show that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans-based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The nematode Caenorhabditis elegans offers currently untapped potential for carrying out high-throughput, live-animal screens of low molecular weight compound libraries to identify molecules that target a variety of cellular processes. We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested. Here we show that one of these small molecules, RPW-24, protects C. elegans from bacterial infection by stimulating the host immune response of the nematode. Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we show that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (∼1.3% of all genes) in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7. These data show that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans-based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens.

Show MeSH
Related in: MedlinePlus