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Intestinal epithelial cells are subject to attack by a diverse array of microbes, including intracellular as well as extracellular pathogens. While defense in epithelial cells can be triggered by pattern recognition receptor-mediated detection of microbe-associated molecular patterns, there is much to be learned about how they sense infection via perturbations of host physiology, which often occur during infection. A recently described host defense response in the nematode C. elegans called the Intracellular Pathogen Response (IPR) can be triggered by infection with diverse natural intracellular pathogens, as well as by perturbations to protein homeostasis. From a forward genetic screen, we identified the C. elegans ortholog of purine nucleoside phosphorylase pnp-1 as a negative regulator of IPR gene expression, as well as a negative regulator of genes induced by extracellular pathogens. Accordingly, pnp-1 mutants have resistance to both intracellular and extracellular pathogens. Metabolomics analysis indicates that C. elegans pnp-1 likely has enzymatic activity similar to its human ortholog, serving to convert purine nucleosides into free bases. Classic genetic studies have shown how mutations in human purine nucleoside phosphorylase cause immunodeficiency due to T-cell dysfunction. Here we show that C. elegans pnp-1 acts in intestinal epithelial cells to regulate defense. Altogether, these results indicate that perturbations in purine metabolism are likely monitored as a cue to promote defense against epithelial infection in the nematode C. elegans.Author summaryAll life requires purine nucleotides. However, obligate intracellular pathogens are incapable of generating their own purine nucleotides and thus have evolved strategies to steal these nucleotides from host cells in order to support their growth and replication. Using the small roundworm C. elegans, we show that infection with natural obligate intracellular pathogens is impaired by loss of pnp-1, the C. elegans ortholog of the vertebrate purine nucleoside phosphorylase (PNP), which is an enzyme involved in salvaging purines. Loss of pnp-1 leads to altered levels of purine nucleotide precursors and increased expression of Intracellular Pathogen Response genes, which are induced by viral and fungal intracellular pathogens of C. elegans. In addition, we find that loss of pnp-1 increases resistance to extracellular pathogen infection and increases expression of genes involved in extracellular pathogen defense. Interestingly, studies from 1975 found that mutations in human PNP impair T-cell immunity, whereas our findings here indicate C. elegans pnp-1 regulates intestinal epithelial immunity. Overall, our work indicates that host purine homeostasis regulates resistance to both intracellular and extracellular pathogen infection.

Intestinal epithelial cells are subject to attack by a diverse array of microbes, including intracellular as well as extracellular pathogens. While defense in epithelial cells can be triggered by pattern recognition receptor-mediated detection of microbe-associated molecular patterns, there is much to be learned about how they sense infection via perturbations of host physiology, which often occur during infection. A recently described host defense response in the nematode C. elegans called the Intracellular Pathogen Response (IPR) can be triggered by infection with diverse natural intracellular pathogens, as well as by perturbations to protein homeostasis. From a forward genetic screen, we identified the C. elegans ortholog of purine nucleoside phosphorylase pnp-1 as a negative regulator of IPR gene expression, as well as a negative regulator of genes induced by extracellular pathogens. Accordingly, pnp-1 mutants have resistance to both intracellular and extracellular pathogens. Metabolomics analysis indicates that C. elegans pnp-1 likely has enzymatic activity similar to its human ortholog, serving to convert purine nucleosides into free bases. Classic genetic studies have shown how mutations in human purine nucleoside phosphorylase cause immunodeficiency due to T-cell dysfunction. Here we show that C. elegans pnp-1 acts in intestinal epithelial cells to regulate defense. Altogether, these results indicate that perturbations in purine metabolism are likely monitored as a cue to promote defense against epithelial infection in the nematode C. elegans., Author summary All life requires purine nucleotides. However, obligate intracellular pathogens are incapable of generating their own purine nucleotides and thus have evolved strategies to steal these nucleotides from host cells in order to support their growth and replication. Using the small roundworm C. elegans, we show that infection with natural obligate intracellular pathogens is impaired by loss of pnp-1, the C. elegans ortholog of the vertebrate purine nucleoside phosphorylase (PNP), which is an enzyme involved in salvaging purines. Loss of pnp-1 leads to altered levels of purine nucleotide precursors and increased expression of Intracellular Pathogen Response genes, which are induced by viral and fungal intracellular pathogens of C. elegans. In addition, we find that loss of pnp-1 increases resistance to extracellular pathogen infection and increases expression of genes involved in extracellular pathogen defense. Interestingly, studies from 1975 found that mutations in human PNP impair T-cell immunity, whereas our findings here indicate C. elegans pnp-1 regulates intestinal epithelial immunity. Overall, our work indicates that host purine homeostasis regulates resistance to both intracellular and extracellular pathogen infection.

During animal development, ligand binding releases the intracellular domain of LIN-12/Notch by proteolytic cleavage to translocate to the nucleus, where it associates with the DNA-binding protein LAG-1/CSL to activate target gene transcription. We investigated the spatiotemporal regulation of LAG-1/CSL expression in Caenorhabditis elegans and observed that an increase in endogenous LAG-1 levels correlates with LIN-12/Notch activation in different cell contexts during reproductive system development. We show that this increase is via transcriptional upregulation by creating a synthetic endogenous operon, and identified an enhancer region that contains multiple LAG-1 binding sites (LBSs) embedded in a more extensively conserved high occupancy target (HOT) region. We show that these LBSs are necessary for upregulation in response to LIN-12/Notch activity, indicating that lag-1 engages in direct positive autoregulation. Deletion of the HOT region from endogenous lag-1 reduced LAG-1 levels and abrogated positive autoregulation, but did not cause hallmark cell fate transformations associated with loss of lin-12/Notch or lag-1 activity. Instead, later somatic reproductive system defects suggest that proper transcriptional regulation of lag-1 confers robustness to somatic reproductive system development.

During animal development, ligand binding releases the intracellular domain of LIN-12/Notch by proteolytic cleavage to translocate to the nucleus, where it associates with the DNA-binding protein LAG-1/CSL to activate target gene transcription. We investigated the spatiotemporal regulation of LAG-1/CSL expression in C. elegans and observed that an increase in endogenous LAG-1 levels correlates with LIN-12/Notch activation in different cell contexts during reproductive system development. We show that this increase is via transcriptional upregulation by creating a synthetic endogenous operon, and identified an enhancer region that contains multiple LAG-1 binding sites (LBSs) embedded in a more extensively conserved high occupancy target (HOT) region. We show that these LBSs are necessary for upregulation in response to LIN-12/Notch activity, indicating that lag-1 engages in direct, positive autoregulation. Deletion of the HOT region from endogenous lag-1 reduced LAG-1 levels and abrogated positive autoregulation, but did not cause hallmark cell fate transformations associated with loss of lin-12/Notch or lag-1 activity. Instead, later somatic reproductive system defects suggest that proper transcriptional regulation of lag-1 confers robustness to somatic reproductive system development.

OrthoList, a compendium of Caenorhabditis elegans genes with human orthologs compiled in 2011 by a meta-analysis of four orthology-prediction methods, has been a popular tool for identifying conserved genes for research into biological and disease mechanisms. However, the efficacy of orthology prediction depends on the accuracy of gene-model predictions, an ongoing process, and orthology-prediction algorithms have also been updated over time. Here we present OrthoList 2 (OL2), a new comparative genomic analysis between C. elegans and humans, and the first assessment of how changes over time affect the landscape of predicted orthologs between two species. Although we find that updates to the orthology-prediction methods significantly changed the landscape of C. elegans–human orthologs predicted by individual programs and—unexpectedly—reduced agreement among them, we also show that our meta-analysis approach “buffered” against changes in gene content. We show that adding results from more programs did not lead to many additions to the list and discuss reasons to avoid assigning “scores” based on support by individual orthology-prediction programs; the treatment of “legacy” genes no longer predicted by these programs; and the practical difficulties of updating due to encountering deprecated, changed, or retired gene identifiers. In addition, we consider what other criteria may support claims of orthology and alternative approaches to find potential orthologs that elude identification by these programs. Finally, we created a new web-based tool that allows for rapid searches of OL2 by gene identifiers, protein domains [InterPro and SMART (Simple Modular Architecture Research Tool], or human disease associations ([OMIM (Online Mendelian Inheritence in Man], and also includes available RNA-interference resources to facilitate potential translational cross-species studies.

Six initially equivalent, multipotential Vulval Precursor Cells (VPCs) in Caenorhabditis elegans adopt distinct cell fates in a precise spatial pattern, with each fate associated with transcription of different target genes. The pattern is centered on a cell that adopts the “1°” fate through Epidermal Growth Factor Receptor (EGFR) activity, and produces a lateral signal composed of ligands that activate LIN-12/Notch in the two flanking VPCs to cause them to adopt “2°” fate. Here, we investigate orthologs of a transcription complex that acts in mammalian EGFR signaling—lin-1/Elk1, sur-2/Med23, and the Cdk8 Kinase module (CKM)—previously implicated in aspects of 1° fate in C. elegans and show they act in different combinations for different processes for 2° fate. When EGFR is inactive, the CKM, but not SUR-2, helps to set a threshold for LIN-12/Notch activity in all VPCs. When EGFR is active, all three factors act to resist LIN-12/Notch, as revealed by the reduced ability of ectopically-activated LIN-12/Notch to activate target gene reporters. We show that overcoming this resistance in the 1° VPC leads to repression of lateral signal gene reporters, suggesting that resistance to LIN-12/Notch helps ensure that P6.p becomes a robust source of the lateral signal. In addition, we show that sur-2/Med23 and lin-1/Elk1, and not the CKM, are required to promote endocytic downregulation of LIN-12-GFP in the 1° VPC. Finally, our analysis using cell fate reporters reveals that both EGFR and LIN-12/Notch signal transduction pathways are active in all VPCs in lin-1/Elk1 mutants, and that lin-1/Elk1 is important for integrating EGFR and lin-12/Notch signaling inputs in the VPCs so that the proper gene complement is transcribed.

Immune genes are under intense, pathogen-induced pressure, which causes these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described a C. elegans-specific gene called pals-22 to be a repressor of “Intracellular Pathogen Response” or IPR genes. Here we describe pals-25, which, like pals-22, is a species-specific gene of unknown biochemical function. We identified pals-25 in a screen for suppression of pals-22 mutant phenotypes and found that mutations in pals-25 suppress all known phenotypes caused by mutations in pals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens, including Nematocida parisii microsporidia and the Orsay virus. Mutations in pals-25 also reverse the reduced lifespan and slowed growth of pals-22 mutants. Transcriptome analysis indicates that pals-22 and pals-25 control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identified pals-22 as a repressor and pals-25 as an activator of epidermal defense gene expression. In summary, the species-specific pals-22 and pals-25 genes act as a switch to regulate a program of gene expression, growth, and defense against diverse natural pathogens in C. elegans., Author summary Infection by microbial pathogens imposes selective pressure on animal and plant hosts. For this reason, host immune genes tend to vary in DNA sequence over evolutionary time and become ‘species-specific’. In this work we describe a pair of species-specific genes called pals-22/pals-25 that promote resistance against natural pathogens of the small roundworm Caenorhabditis elegans. This gene pair acts as an on/off switch for genes that are activated after infection and rewires the physiology of worms to either be in an immune state or a growth state. In particular, the pals-22 gene promotes development and increases lifespan, while the pals-25 gene promotes resistance against heat shock and natural pathogens including virus and fungal-related microbes. While the DNA sequence of pals-22 and pals-25 appears specific to worms, the way that these two genes affect physiology may provide insight into how organisms balance growth and immunity.

Immune genes are under intense pressure from pathogens, which cause these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described aC. elegans-specific gene calledpals-22to be a repressor of “Intracellular Pathogen Response” or IPR genes. Here we describepals-25, which, likepals-22, is a species-specific gene of unknown biochemical function. We identifiedpals-25in a screen for suppression ofpals-22mutant phenotypes and found that mutations inpals-25suppress all known phenotypes caused by mutations inpals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens. Mutations inpals-25also reverse the reduced lifespan and slowed growth ofpals-22mutants. Transcriptome analysis indicates thatpals-22andpals-25control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identifiedpals-22as a repressor andpals-25as an activator of epidermal defense gene expression. These phenotypic and evolutionary features ofpals-22andpals-25are strikingly similar to species-specific R gene pairs in plants that control immunity against co-evolved pathogens.

OrthoList, a compendium of Caenorhabditis elegans genes with human orthologs compiled in 2011 by a meta-analysis of four orthology-prediction methods, has been a popular tool for identifying conserved genes for research into biological and disease mechanisms. However, the efficacy of orthology prediction depends on the accuracy of gene-model predictions, an ongoing process, and orthology-prediction algorithms have also been updated over time. Here we present OrthoList 2 (OL2), a new comparative genomic analysis between C. elegans and humans, and the first assessment of how changes over time affect the landscape of predicted orthologs between two species. Although we find that updates to the orthology-prediction methods significantly changed the landscape of C. elegans–human orthologs predicted by individual programs and—unexpectedly—reduced agreement among them, we also show that our meta-analysis approach “buffered” against changes in gene content. We show that adding results from more programs did not lead to many additions to the list and discuss reasons to avoid assigning “scores” based on support by individual orthology-prediction programs; the treatment of “legacy” genes no longer predicted by these programs; and the practical difficulties of updating due to encountering deprecated, changed, or retired gene identifiers. In addition, we consider what other criteria may support claims of orthology and alternative approaches to find potential orthologs that elude identification by these programs. Finally, we created a new web-based tool that allows for rapid searches of OL2 by gene identifiers, protein domains [InterPro and SMART (Simple Modular Architecture Research Tool], or human disease associations ([OMIM (Online Mendelian Inheritence in Man], and also includes available RNA-interference resources to facilitate potential translational cross-species studies.

Six initially equivalent, multipotential Vulval Precursor Cells (VPCs) in Caenorhabditis elegans adopt distinct cell fates in a precise spatial pattern, with each fate associated with transcription of different target genes. The pattern is centered on a cell that adopts the “1°” fate through Epidermal Growth Factor Receptor (EGFR) activity, and produces a lateral signal composed of ligands that activate LIN-12/Notch in the two flanking VPCs to cause them to adopt “2°” fate. Here, we investigate orthologs of a transcription complex that acts in mammalian EGFR signaling—lin-1/Elk1, sur-2/Med23, and the Cdk8 Kinase module (CKM)—previously implicated in aspects of 1° fate in C. elegans and show they act in different combinations for different processes for 2° fate. When EGFR is inactive, the CKM, but not SUR-2, helps to set a threshold for LIN-12/Notch activity in all VPCs. When EGFR is active, all three factors act to resist LIN-12/Notch, as revealed by the reduced ability of ectopically-activated LIN-12/Notch to activate target gene reporters. We show that overcoming this resistance in the 1° VPC leads to repression of lateral signal gene reporters, suggesting that resistance to LIN-12/Notch helps ensure that P6.p becomes a robust source of the lateral signal. In addition, we show that sur-2/Med23 and lin-1/Elk1, and not the CKM, are required to promote endocytic downregulation of LIN-12-GFP in the 1° VPC. Finally, our analysis using cell fate reporters reveals that both EGFR and LIN-12/Notch signal transduction pathways are active in all VPCs in lin-1/Elk1 mutants, and that lin-1/Elk1 is important for integrating EGFR and lin-12/Notch signaling inputs in the VPCs so that the proper gene complement is transcribed.