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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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RPW-24 induces the transcription of putative immune effectors in C. elegans.(A) A scatter plot compares gene expression levels of all 22,250 sequences on the Affymetrix GeneChip in wild-type C. elegans exposed to RPW-24 versus DMSO. Black dots highlight the 269 genes that were induced and the 62 that were repressed at least three-fold (P<0.025). Gray dots are genes whose expression levels did not significantly change in this study. The location on the scatter plot of the ten putative immune effectors whose expression was studied further by qRT-PCR are indicated and highlighted with red dots. Genes that fall on the black line are expressed at equal levels in both conditions. AU = arbitrary units. (B) Venn diagram gives the overlap of the C. elegans genes induced three-fold or greater by RPW-24 (P<0.025) in the microarray analysis (this study) with the genes upregulated during P. aeruginosa infection (greater than 2-fold, P<0.01) [8]. P<2.7×10−16 for the degree of overlap between these datasets versus the amount expected by chance alone. (C) Shown are qRT-PCR data of ten putative C. elegans immune effectors in wild-type animals infected with P. aeruginosa for eight hours and exposed to either 70 µM RPW-24 or DMSO, each plotted versus expression of the indicated genes in C. elegans exposed to E. coli OP50 and DMSO. The data are the average of two biological replicates each normalized to a control gene with error bars representing SEM. *P<0.05, **P = 0.06 and ***P = 0.08 for the comparison of fold change of the indicated gene in P. aeruginosa-infected animals exposed to RPW-24 versus DMSO.
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pgen-1002733-g002: RPW-24 induces the transcription of putative immune effectors in C. elegans.(A) A scatter plot compares gene expression levels of all 22,250 sequences on the Affymetrix GeneChip in wild-type C. elegans exposed to RPW-24 versus DMSO. Black dots highlight the 269 genes that were induced and the 62 that were repressed at least three-fold (P<0.025). Gray dots are genes whose expression levels did not significantly change in this study. The location on the scatter plot of the ten putative immune effectors whose expression was studied further by qRT-PCR are indicated and highlighted with red dots. Genes that fall on the black line are expressed at equal levels in both conditions. AU = arbitrary units. (B) Venn diagram gives the overlap of the C. elegans genes induced three-fold or greater by RPW-24 (P<0.025) in the microarray analysis (this study) with the genes upregulated during P. aeruginosa infection (greater than 2-fold, P<0.01) [8]. P<2.7×10−16 for the degree of overlap between these datasets versus the amount expected by chance alone. (C) Shown are qRT-PCR data of ten putative C. elegans immune effectors in wild-type animals infected with P. aeruginosa for eight hours and exposed to either 70 µM RPW-24 or DMSO, each plotted versus expression of the indicated genes in C. elegans exposed to E. coli OP50 and DMSO. The data are the average of two biological replicates each normalized to a control gene with error bars representing SEM. *P<0.05, **P = 0.06 and ***P = 0.08 for the comparison of fold change of the indicated gene in P. aeruginosa-infected animals exposed to RPW-24 versus DMSO.

Mentions: To further study the effects of RPW-24 on C. elegans, we used Affymetrix whole genome GeneChips to generate transcriptome profiles of wild-type nematodes following exposure to either 70 µM RPW-24 or the solvent control DMSO in liquid culture media in the absence of bacterial pathogens for 16 hours at 15°C (Figure 2A). We found that RPW-24 induced a remarkably robust transcriptional response that involved only a small fraction (∼1.3%) of the genes of the C. elegans genome (Figure 2A). 269 genes were upregulated three-fold or greater (P<0.025), 125 of which were induced more than 50-fold during RPW-24 exposure (Table S1). The most highly upregulated gene was expressed more than 3500-fold higher in compound-exposed worms. For confirmation, we used qRT-PCR to analyze 10 genes that exhibited varying degrees of induction and expression levels in the microarray analysis (Figure 2A). We found that the transcriptional changes observed in the transcriptome profiling analysis directly correlated with the values obtained by qRT-PCR for all 10 genes tested (Figure S3). We also observed no difference in C. elegans gene induction whether the RPW-24 exposure occurred in liquid or on solid media (Figure S3). Taken together, these data suggest that RPW-24 strongly induces gene transcription in the absence of pathogen exposure.


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)

RPW-24 induces the transcription of putative immune effectors in C. elegans.(A) A scatter plot compares gene expression levels of all 22,250 sequences on the Affymetrix GeneChip in wild-type C. elegans exposed to RPW-24 versus DMSO. Black dots highlight the 269 genes that were induced and the 62 that were repressed at least three-fold (P<0.025). Gray dots are genes whose expression levels did not significantly change in this study. The location on the scatter plot of the ten putative immune effectors whose expression was studied further by qRT-PCR are indicated and highlighted with red dots. Genes that fall on the black line are expressed at equal levels in both conditions. AU = arbitrary units. (B) Venn diagram gives the overlap of the C. elegans genes induced three-fold or greater by RPW-24 (P<0.025) in the microarray analysis (this study) with the genes upregulated during P. aeruginosa infection (greater than 2-fold, P<0.01) [8]. P<2.7×10−16 for the degree of overlap between these datasets versus the amount expected by chance alone. (C) Shown are qRT-PCR data of ten putative C. elegans immune effectors in wild-type animals infected with P. aeruginosa for eight hours and exposed to either 70 µM RPW-24 or DMSO, each plotted versus expression of the indicated genes in C. elegans exposed to E. coli OP50 and DMSO. The data are the average of two biological replicates each normalized to a control gene with error bars representing SEM. *P<0.05, **P = 0.06 and ***P = 0.08 for the comparison of fold change of the indicated gene in P. aeruginosa-infected animals exposed to RPW-24 versus DMSO.
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Related In: Results  -  Collection

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pgen-1002733-g002: RPW-24 induces the transcription of putative immune effectors in C. elegans.(A) A scatter plot compares gene expression levels of all 22,250 sequences on the Affymetrix GeneChip in wild-type C. elegans exposed to RPW-24 versus DMSO. Black dots highlight the 269 genes that were induced and the 62 that were repressed at least three-fold (P<0.025). Gray dots are genes whose expression levels did not significantly change in this study. The location on the scatter plot of the ten putative immune effectors whose expression was studied further by qRT-PCR are indicated and highlighted with red dots. Genes that fall on the black line are expressed at equal levels in both conditions. AU = arbitrary units. (B) Venn diagram gives the overlap of the C. elegans genes induced three-fold or greater by RPW-24 (P<0.025) in the microarray analysis (this study) with the genes upregulated during P. aeruginosa infection (greater than 2-fold, P<0.01) [8]. P<2.7×10−16 for the degree of overlap between these datasets versus the amount expected by chance alone. (C) Shown are qRT-PCR data of ten putative C. elegans immune effectors in wild-type animals infected with P. aeruginosa for eight hours and exposed to either 70 µM RPW-24 or DMSO, each plotted versus expression of the indicated genes in C. elegans exposed to E. coli OP50 and DMSO. The data are the average of two biological replicates each normalized to a control gene with error bars representing SEM. *P<0.05, **P = 0.06 and ***P = 0.08 for the comparison of fold change of the indicated gene in P. aeruginosa-infected animals exposed to RPW-24 versus DMSO.
Mentions: To further study the effects of RPW-24 on C. elegans, we used Affymetrix whole genome GeneChips to generate transcriptome profiles of wild-type nematodes following exposure to either 70 µM RPW-24 or the solvent control DMSO in liquid culture media in the absence of bacterial pathogens for 16 hours at 15°C (Figure 2A). We found that RPW-24 induced a remarkably robust transcriptional response that involved only a small fraction (∼1.3%) of the genes of the C. elegans genome (Figure 2A). 269 genes were upregulated three-fold or greater (P<0.025), 125 of which were induced more than 50-fold during RPW-24 exposure (Table S1). The most highly upregulated gene was expressed more than 3500-fold higher in compound-exposed worms. For confirmation, we used qRT-PCR to analyze 10 genes that exhibited varying degrees of induction and expression levels in the microarray analysis (Figure 2A). We found that the transcriptional changes observed in the transcriptome profiling analysis directly correlated with the values obtained by qRT-PCR for all 10 genes tested (Figure S3). We also observed no difference in C. elegans gene induction whether the RPW-24 exposure occurred in liquid or on solid media (Figure S3). Taken together, these data suggest that RPW-24 strongly induces gene transcription in the absence of pathogen exposure.

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