The Intracellular Pathogen Response (IPR) is an innate immune program in Caenorhabditis elegans involving the upregulation of a suite of genes in response to natural intracellular pathogens of the intestine, including Orsay virus and microsporidia species like Nematocida parisii (Sarkies et al., 2013; Bakowski et al., 2014; Reddy et al., 2019). In addition to infection, the IPR is also genetically regulated through several parallel pathways (Reddy et al., 2017; Reddy et al., 2019; Sowa et al., 2020; Lazetic et al., 2023). Two such genetic regulators are enzymes of the highly conserved purine salvage pathway, Adenosine Deaminase ( ADAH-1 , ADA in humans) and Purine Nucleoside Phosphorylase ( PNP-1 , PNP in humans), which recycle purine nucleosides and bases ( Figure 1A ) (Tecle et al., 2021; Wernet et al., 2025). Knockout or knockdown of adah-1 or pnp-1 induces expression of the pals-5 p:: GFP IPR transcriptional reporter ( Figure 1B ) (Bakowski et al., 2014), as well as many other IPR genes, and promotes resistance to intracellular infections. Thus, ADAH-1 and PNP-1 act as negative regulators of the IPR (Tecle et al., 2021; Wernet et al., 2025).

While the IPR is well-defined in C. elegans , the extent to which this immune program is conserved or divergent among related nematode species is unclear. Infection of Caenorhabditis briggsae with Santeuil virus or Nematocida species of microsporidia induces the upregulation of several gene families in common with the C. elegans IPR, such as pals and DUF713 genes, suggesting broadly similar transcriptional responses to intracellular infection (Chen et al., 2017; Wan et al., 2022). However, whether known genetic regulators of the IPR serve similar functions outside of C. elegans has not been investigated.

Here, to determine whether orthologs of adah-1 and pnp-1 have conserved immune regulatory roles, we used C. briggsae and Caenorhabditis remanei strains transgenically modified to express C. elegans SID-2 , which enables uptake of dsRNA to induce systemic RNA interference (RNAi) by feeding (Winston et al., 2007; Nuez & Felix, 2012). We cloned cDNA fragments of Cbr-adah-1 and Cbr-pnp-1 into the L4440 RNAi plasmid vector, transformed HT115 bacteria, and performed RNAi by feeding C. briggsae mfIs42 [Cel-sid-2 + Cel-myo-2::DsRed] animals dsRNA targeting each gene. RT-qPCR showed knockdown of Cbr-adah-1 and Cbr-pnp-1 transcript levels and the upregulation of three Cbr-pals genes ( CBG06463, CBG06596, and CBG30948 ) when compared to control RNAi-treated animals ( Figure 1C ). CBG06596 and CBG30948 were induced to comparable levels as previously demonstrated for pals-5 following adah-1 and pnp-1 RNAi (Wernet et al., 2025). Notably, all three Cbr-pals genes tested were also previously shown to be upregulated during infection by Santeuil virus, N. parisii , or both pathogens, suggesting these genes are both genetically and infection-regulated, like pals genes of the C. elegans IPR (Chen et al., 2017; Wan et al., 2022).

Next, we cloned cDNA fragments for Cre-adah-1 and Cre-pnp-1 into L4440 and performed RNAi by feeding C. remanei mfEx34 [Cel-sid-2 + Cel-myo-2::DsRed] animals dsRNA targeting each gene. As with C. briggsae , we were able to knock down the expression of Cre-adah-1 and Cre-pnp-1 and observed upregulation of three Cre-pals genes ( CRE18779, CRE09158, and CRE27419 ) compared with control RNAi-treated animals ( Figure 1D ). Cre-pals genes were not induced to the same degree as Cbr-pals genes (~3 to 500-fold in C. briggsae versus ~3-14-fold in C. remanei ) ( Figure 1C, D). This reduced induction could be an intrinsic property of pals gene family regulation in this species or the inducibility of the specific genes tested. Alternatively, we note that knockdown of Cre-adah-1 /pnp-1 was not as effective as Cbr- adah-1 /pnp-1 (~0.083 and 0.085-fold in C. briggsae versus ~0.19 and 0.28-fold in C. remanei ) ( Figure 1C, D). This suggests that RNAi may be less efficient in C. remanei mfEx34 , leading to higher background activity of the targeted enzyme and muted upregulation of the Cre-pals genes. We do not attribute this effect to the extrachromosomal inheritance pattern of the mfEx34 transgene, as this strain has previously been reported as a spontaneous integrant; we similarly observe 100% inheritance of this transgene (Nuez & Felix, 2012). Taken together, our results demonstrate that Cbr/ Cre-adah-1 and Cbr/ Cre-pnp-1 negatively regulate pals gene expression, indicating conserved immune regulatory roles for these purine salvage enzymes, like adah-1 and pnp-1 for the IPR.

Constitutive expression of the C. elegans IPR promotes resistance to intracellular infection in the intestine (Reddy et al., 2019; Tecle et al., 2021; Lazetic et al., 2023; Wernet et al., 2025). While pals gene expression is a useful proxy for IPR induction, only one induced C. elegans pals gene has been shown to confer resistance (Raman et al., 2024). Therefore, analyzing Cbr/Cre-pals expression after knocking down purine salvage enzymes may not fully capture whether a broader IPR-like immune program and its associated phenotypes are induced. To assess intracellular pathogen susceptibility, we performed Cbr- adah-1 /pnp-1 RNAi and infected C. briggsae mfIs42 animals with N. parisii . We focused our analysis on C. briggsae for two reasons: 1) more potent RNAi and induction of pals genes ( Figure 1C ), and 2) both C. briggsae and C. elegans are infected by N. parisii, which allowed for the inclusion of C. elegans strains as controls in our experiment; N. parisii cannot infect C. remanei (Zhang et al., 2016; Wadi et al., 2023). At 30 hours post-infection, we FISH-stained C. briggsae mfIs42 RNAi-treated animals with a probe targeting N. parisii rRNA and quantified FISH fluorescence (Troemel et al., 2008). Knockdown of Cbr-pnp-1 and Cbr-adah-1 resulted in reduced average FISH fluorescence when compared to control RNAi-treated animals, consistent with increased microsporidia resistance ( Figure 1E, F). The degree of resistance observed in C. briggsae was similar to that of C. elegans pnp-1( jy121 ) mutants when compared to wild-type animals ( Figure 1E, F) (Tecle et al., 2021). These findings further support a conserved role for purine salvage enzymes as negative regulators of an innate immune program against intracellular pathogens in a distinct Caenorhabditis species.

In this study, we sought to determine if purine salvage enzymes that act as negative regulators of the C. elegans IPR, ADAH-1 and PNP-1 ( Figure 1A, B), serve similar immune-regulatory roles in related nematode species. We cloned cDNA fragments of Cbr/ Cre-adah-1 and Cbr/ Cre-pnp-1 into L4440 and utilized RNAi-competent C. briggsae and C. remanei strains to knock down these enzymes. In both species, knockdown induced pals gene expression indicative of IPR-like immune activation ( Figure 1C, D). Additionally, knockdown of Cbr-adah-1 and Cbr-pnp-1 promoted N. parisii resistance ( Figure 1E, F). Purine salvage metabolism represents just one of several parallel genetic pathways that regulate the C. elegans IPR. Other regulators include members of the pals gene family that act in antagonistic modules (Reddy et al., 2019; Lazetic et al., 2023), drh-1 as a sensor of dsRNA during viral infection (Sowa et al., 2020; Batachari et al., 2024), and zip-1 as a transcription factor that acts downstream of these factors and controls a subset of IPR genes (Lazetic et al., 2022). Future work can evaluate whether these genetic regulators have broadly conserved immune regulatory roles, as assessed here for Cbr/ Cre-adah-1 and Cbr/ Cre-pnp-1 , whether these genes have divergent functions, or whether novel regulators have evolved in the diverse nematode species that are susceptible to intracellular pathogens.

Worm strain maintenance

C. elegans , C. briggsae , and C. remanei strains (Table 1) were maintained at 21 °C on Nematode Growth Media (NGM) plates seeded with Escherichia coli OP50-1 . RNAi experiments were performed at 22 °C on NGM plates supplemented with 1 mM Carbenicillin and 5 mM IPTG seeded with E. coli HT115 (DE3) carrying the desired dsRNA expression plasmids (Table 2). Seeded RNAi plates were grown in the dark at room temperature (RT) for at least 3 days before adding worms.

IPR reporter imaging

P0 L4s of C. elegans jyIs8 were picked to 6-cm RNAi plates seeded with control, adah-1, or pnp-1 RNAi. F1 adults were then picked to the same RNAi treatments. F2 adults were imaged for IPR reporter expression on a Zeiss Axioscope 7.

Cloning

cDNA sequences for Cbr/ Cre-adah-1 or Cbr/ Cre-pnp-1 were synthesized as gene fragments (Twist Biosciences) and inserted into linearized pL4440 between the T7 promoters by NEBuilder HiFi assembly (New England Biolabs). Assembled plasmids were transformed into DH5α chemically competent cells (Thermo Fisher Scientific). Candidate clones were miniprepped (Qiagen) and sequence-confirmed for the desired insertion using Sanger and PlasmidEZ whole-vector sequencing (Azenta Life Sciences). Verified plasmids were subsequently transformed into chemically competent HT115 (DE3) bacteria prepared with the Mix & Go E. coli Transformation Kit (Zymo Research). Genbank files for each plasmid vector generated in this study are included as Extended Data.

RNA extraction and RT-qPCR

For C. briggsae mfIs42 , ~10-12 P0 L4s were picked to 10-cm RNAi plates seeded with control, Cbr-adah-1, or Cbr-pnp-1 RNAi. ~10-12 F1 adults were then picked to 2 x 10-cm RNAi plates of the same treatments. After 3 days, F2s were enriched for L4 and adult life stages by gravity settling in M9 in a 15 mL conical tube for RNA extraction. For C. remanei mfEx34 , a mixed-stage plate was chunked to 10-cm RNAi plates seeded with control, Cre-adah-1 , or Cre-pnp-1 RNAi. After 3 days, F1 mixed-stage worms were chunked again to 2 x 10-cm RNAi plates of the same treatments. After 3 days, F2s were enriched for L4 and adult life stages by gravity settling in M9 in a 15 mL conical tube for RNA extraction. For both species, total RNA was purified as previously described (Gang et al., 2022) and converted to cDNA using iScript (Bio-Rad). RT-qPCR was performed with iQ SYBR Green using a CFX Connect machine running CFX Maestro v1.1 (Bio-Rad). RT-qPCR data was normalized to the Cbr/ Cre-eef-2 gene using the Pfaffl method (Pfaffl, 2001). To assess expression, Cbr/ Cre-adah-1 and Cbr/ Cre-pnp-1 knockdown samples were normalized to control RNAi-treated samples. RT-qPCR primers are listed in Table 3.

Microsporidia infection

mfIs42 P0 and F1 generations were picked to seeded 10-cm RNAi plates as described above. ~125 F2 L4s for each treatment were then picked to 6-cm NGM plates seeded with a thin lawn of OP50-1 covering the entire surface. A mixture of 1 million N. parisii spores, 50 µL of 10X-concentrated OP50-1 , and M9 to a total volume of 300 µL was then top-plated, spread evenly across the surface, and dried at RT for 10 minutes. The infection plates were transferred to 25 °C for 3 h. Infected worms were washed off the plates in M9, pelleted, and transferred to 6-cm RNAi plates seeded with HT115 (DE3) to sustain knockdown of the desired genes, without additional N. parisii spores, and returned to 25 °C for 27 h. At 30 hpi, worms were fixed in 4% paraformaldehyde for 30 min at RT. Worms were FISH-stained with 5 ng/µL N. parisii -specific MicroB CF610 probe (Biosearch Technologies), imaged on a Zeiss Axioscope 7, and infection was quantified using ImageJ as previously described (Gang et al., 2022).

Statistics

All statistical tests were performed in GraphPad Prism version 11. The statistical test used in each experiment, number of replicates performed, and summary of test results are described in the corresponding figure legend panel. All individual data points and the complete results of statistical tests performed are included as Extended Data.

Table 1. Worm Strains

Table 2. Plasmids

Table 3. qPCR Primers