To determine if C. elegans TIR-1/SARM1 multimerizes to activate the p38 PMK-1 innate immune pathway, we used CRISPR/Cas9 to insert the fluorescent protein wrmScarlet at the C-terminus of the endogenous C. elegans tir-1 locus, which labeled all tir-1 isoforms. In uninfected animals, TIR-1::wrmScarlet is barely detectable in intestinal epithelial cells (Figure 1A). However, P. aeruginosa infection caused TIR-1::wrmScarlet to multimerize into visible puncta within intestinal epithelial cells (Figure 1A and B). We distinguished TIR-1::wrmScarlet puncta from autofluorescent gut granules by comparing images in the red and green fluorescence channels. TIR-1::wrmScarlet puncta are those that are seen in the red, but not the green fluorescence channel (arrowheads in Figure 1A), as opposed to gut granules, which can be seen in both channels (asterisks in Figure 1A).

C. elegans TIR-1 protein has three characterized domains: a Heat/Armadillo repeat domain, a sterile alpha motif (SAM) domain, and a Toll-interleukin receptor (TIR) domain (Chuang and Bargmann, 2005; Figure 1C). C. elegans TIR-1 oligomerizes in vitro through interactions of its SAM domains (Horsefield et al., 2019). We used CRISPR-Cas9 to delete both SAM domains in tir-1 (tir-1^ΔSAM) and to generate point mutants in two residues within the C. elegans TIR domain that are important for the self-association and activity of mammalian SARM1 (C. elegans tir-1^G747P and tir-1^H833A) (Horsefield et al., 2019; Figure 1D). The C. elegans tir-1^ΔSAM, tir-1^G747P and tir-1^H833A mutants prevented activation of the p38 PMK-1-dependent immune reporter T24B8.5p::gfp in animals infected with P. aeruginosa (Figure 1D). Consistent with these data, the tir-1^ΔSAM, tir-1^G747P and tir-1^H833A mutants have reduced levels of active, phosphorylated p38 PMK-1, equivalent to the tir-1(qd4) null allele (Shivers et al., 2009; Figure 1E and F). Additionally, these mutants are each markedly hypersusceptible to P. aeruginosa infection (Figure 1G).

The TIR domain of C. elegans, TIR-1, and its mammalian homolog, SARM1, possess intrinsic NADase activity (Essuman et al., 2017; Horsefield et al., 2019; Summers et al., 2016). Importantly, oligomerization of mammalian SARM1 and C. elegans TIR-1 is required for maximal NADase activity in vitro (Horsefield et al., 2019). The NADase activity in the TIR domain of mammalian SARM1 requires a putative catalytic glutamate residue (Essuman et al., 2017). We used CRISPR-Cas9 to mutate the homologous glutamate in C. elegans tir-1 (tir-1^E788A) and found that it was required for the immunostimulatory activity of tir-1 – tir-1^E788A mutants do not induce T24B8.5p::gfp following P. aeruginosa infection (Figure 1D), had less active, phosphorylated p38 PMK-1 (Figure 1E and F), and were more susceptible to P. aeruginosa infection (Figure 1G). We confirmed that tir-1^E788A, tir-1^ΔSAM and tir-1^G747P mutants are translated and not degraded by introducing a 3xFLAG tag at the C-terminus of each mutant using CRISPR-Cas9 (Figure 1—figure supplement 1A and B) and probing for epitope-tagged mutant protein in western blots with an anti-FLAG antibody (Figure 1—figure supplement 1A). In addition, the 3xFLAG-tagged wild-type TIR-1 expressed T24B8.5p::gfp, but the tagged mutant TIR-1 proteins did not (Figure 1—figure supplement 1B). Collectively, these data demonstrate that multimerization of TIR-1 and its intrinsic NADase activity are required to activate the p38 PMK-1 innate immune pathway in the intestine during pathogen infection.

To further characterize the mechanism of TIR-1/SARM1 activation, we recombinantly expressed and purified the TIR domain of the TIR-1 protein (called TIR) from E. coli and evaluated its NADase activity in vitro using an etheno-NAD⁺ (ε-NAD) activity assay, in which hydrolysis of the nicotinamide moiety of ε-NAD leads to an increase in fluorescence. Interestingly, purified TIR only shows very modest NADase activity even at high protein concentrations ( > 15 μM) (Figure 2A). Notably, the NADase activity of TIR increased parabolically with increasing TIR concentrations rather than linearly, suggesting that multimerization of TIR-1 potentiates its NADase activity (Figure 2A).

Given that high concentrations of TIR are required to observe NADase activity, we hypothesized that molecular crowding might activate the enzyme. Therefore, we assessed the effect of several macro- and microviscogens on TIR activity. Macroviscogens reduce the free volume available for protein movement and thus promote aggregation of protein complexes that are capable of self-association (Gadda and Sobrado, 2018; Blacklow et al., 1988). Importantly, macroviscogens have minimal impact on the rate of diffusion of small molecules. By contrast, microviscogens, which are much smaller than most enzymes, affect the diffusion of substrates in solution and, thus, the frequency at which enzymes encounter their substrate (Blacklow et al., 1988). Interestingly, macroviscogens (polyethylene glycol [PEG] 3350 and PEG 8000), but not microviscogens (sucrose or glycerol), dramatically increased the NADase activity of TIR (Figure 2B). These effects were most pronounced with higher molecular weight PEGs, as treatment with smaller molecular weight PEGs (e.g. PEG 1500 and PEG 400) did not increase the enzymatic activity of TIR (Figure 2C). Specifically, PEGs 3350 and 8000 increase TIR activity in a concentration-dependent manner (Figure 2C). Crowding agents also increase the activity of the TIR domain of human SARM1, as well as plant TIR domains (Horsefield et al., 2019; Loring et al., 2021), suggesting that the mechanism of TIR regulation is strongly conserved.

Treatment with 25% PEG 3350 led to a roughly linear association between TIR concentration and NADase activity and markedly enhanced TIR activity at each enzyme concentration (Figure 2—figure supplement 1A). Consequently, lower amounts of enzyme (2.5 μM TIR) can be used to obtain robust kinetic data. Using these conditions, we determined the steady-state kinetic parameters (k_cat, K_M, and k_cat/K_M) with 2.5 μM TIR in the presence of increasing concentrations of PEG 3350 (Figure 2D). The K_m of TIR increases and then decreases to level off at ~500 μM of ε-NAD with increasing concentration of PEG 3350 (Figure 2E). On the other hand, k_cat increases nearly linearly with increasing concentrations of PEG 3350 (Figure 2F). In addition, the catalytic efficiency (k_cat/K_m) followed the same trend as k_cat, indicating that increased TIR activity is due to increased substrate turnover by the enzyme and not tighter substrate binding (Figure 2G).

We found that the NAD hydrolase activity of TIR is also activated by the precipitant sodium citrate, providing an orthologous method to characterize the enzymatic activity of the TIR domain (Figure 2—figure supplement 1A and B). Notably, the response to increasing citrate concentration was switch-like, where a concentration of at least 250 mM sodium citrate was needed to observe enzyme activity (Figure 2—figure supplement 1B and C). By contrast, activation with PEG 3350 was more dose-dependent (Figure 2C and D). Nevertheless, the kinetic parameters obtained in the presence of citrate displayed similar trends to those found with PEG 3350: K_m decreased to level off at ~400 µM of ε-NAD (Figure 2—figure supplement 1D), k_cat increases with increasing sodium citrate concentration (Figure 2—figure supplement 1E), and k_cat/K_m follows the k_cat trends (Figure 2—figure supplement 1F).

In the cytoplasm, high concentrations of macromolecules (proteins, nucleic acids, lipids, carbohydrates) cause molecular crowding and induce phase transitions of signaling proteins (Rivas and Minton, 2016). To determine if TIR undergoes a phase transition and whether its NADase activity correlates with the transition, we incubated purified TIR with different concentrations of PEG3350 or citrate, centrifuged the sample, and evaluated TIR NADase activity in the soluble and insoluble fractions (Figure 2H and Figure 2—figure supplement 1G). At low concentrations of both PEG and citrate, TIR protein was present in the supernatant. However, at high concentrations of both PEG and citrate, TIR was principally located in the pelleted (insoluble) fraction, as visualized by Coomassie staining on an SDS-PAGE gel (Figure 2H and Figure 2—figure supplement 1G). Importantly, robust NADase activity was observed in the pelleted fraction and not in the supernatant of samples treated with high concentrations of PEG or citrate (Figure 2H and Figure 2—figure supplement 1G). Taken together, these data demonstrate that precipitation/aggregation of TIR is required to activate the intrinsic NADase activity of TIR-1/SARM1.

To determine whether TIR-1 multimerization is also required for a phase transition and the NADase activity of TIR, we recombinantly expressed and purified the TIR domain of TIR-1, which contained mutations in residues required for oligomerization (TIR^G747P and TIR^H833A) and NAD⁺ hydrolysis (TIR^E788A and TIR^E788Q). Notably, TIR^G747P and TIR^E788Q mutants showed no apparent activity, and TIR^E788A and TIR^H833A showed a > 1 × 10⁸­fold decrease in catalytic efficiency (k_cat/K_m) compared to TIR^WT (Figure 2I). Similarly, kinetic analysis of TIR oligomerization and catalytic mutants in 500 mM citrate revealed that all the mutants were catalytically dead, each showing no apparent activity (Figure 2—figure supplement 1H).

To further characterize the TIR phase transition, we evaluated the precipitation capacity of TIR oligomerization and catalytic mutants in the presence of PEG 3350 and citrate. Consistent with our genetic data, TIR^G747P, which contains a mutation that prevents oligomerization of TIR, was unable to precipitate as readily as TIR^WT (Figure 2J). Compared to TIR^WT, the TIR^G747P mutant in the presence of PEG 3350 demonstrated a 25% decrease in the amount of precipitated protein (Figure 2J). We observed similar results in the presence of citrate – compared to TIR^WT, the TIR^G747P mutant demonstrated a 22% decrease in precipitated protein compared to TIR^WT (Figure 2—figure supplement 1I). Importantly, TIR proteins with two different mutations in the glutamate required for NAD⁺ catalysis, TIR^E788A and TIR^E788Q, precipitated to a similar extent as TIR^WT (Figure 2J and K, and Figure 2—figure supplement 1I and J) but had minimal NADase activity (Figure 2I and Figure 2—figure supplement 1H). Collectively, these data demonstrate that a phase transition of TIR-1 is required for its NADase activity.

During phase separations, macromolecules partition into distinct biochemical compartments characterized by a higher concentration dense phase and a lower concentration dilute phase (Boeynaems et al., 2018; Alberti and Dormann, 2019; Verdile et al., 2019). The dense compartment can have either liquid-like properties in liquid-to-liquid phase separations or solid-like properties in liquid-to-solid phase transitions (Alberti and Dormann, 2019; Patel et al., 2015; Kroschwald et al., 2015; Peskett et al., 2018). To determine whether TIR undergoes a liquid-to-liquid or a liquid-to-solid phase transition, we assayed the enzyme activity of TIR in the presence of 1,6-hexanediol, an aliphatic alcohol that interferes with hydrophobic interactions prominent in liquid-to-liquid separations. Thus, 1,6-hexanediol disrupts liquid-like compartments, but not solid-like compartments (Boeynaems et al., 2018; Verdile et al., 2019; Kroschwald et al., 2015). In the presence of either PEG 3350 or citrate, we observed that 1,6-hexanediol decreases the NADase activity of TIR by <2 fold (Figure 2L). While significant, this modest decrease (the activity of 1,6-hexanediol-treated TIR remains two orders of magnitude higher than the activity of TIR without PEG 3350 or citrate addition) suggests that the NADase activity of TIR is predominantly associated with a solid, rather than a liquid, state or lies on a continuum between a liquid-like and solid-like state (Figure 2L). Notably, in the absence of PEG 3350 or citrate, the low-level TIR NADase activity can be inhibited by 1,6-hexanediol (Figure 2L), suggesting that this activity is driven by hydrophobic interactions. Importantly, 1,6-hexanediol does not interfere with TIR precipitation in the presence of PEG or citrate, regardless of whether it is added before or after precipitate formation (Figure 2—figure supplement 2A and B).

Next, we determined if this liquid-to-solid phase transition of TIR is reversible. The insoluble fraction following treatment of TIR with PEG 3350 or citrate was resuspended in either buffer alone or buffer plus the respective additive (PEG 3350 or citrate). If the phase transition is reversible, resuspension of precipitated TIR in buffer alone should disrupt enzymatic activity. However, if the phase transition is irreversible, activity should be detected when the pellet is resuspended in buffer alone. Notably, we observed some TIR enzymatic activity when the pellet was resuspended in buffer (Figure 2M). With PEG 3350, the activity was lower than that observed in the precentrifugation control or when the pellet was resuspended in buffer with PEG 3350. By contrast, with citrate, the activity was similar to both control conditions (precentrifugation or when the pellet was resuspended in buffer and citrate). These data indicate that the phase transition of TIR is partially reversible in PEG 3350 and irreversible in sodium citrate. To confirm these findings, we centrifuged the resuspended samples to examine the fractions visually by SDS-PAGE (Figure 2—figure supplement 2C). In the sample initially prepared with PEG 3350 and resuspended in buffer alone, a faint band was present in the supernatant fraction. However, this band was absent in the sample initially prepared with sodium citrate (Figure 2—figure supplement 2C). These data confirm that the TIR phase transition is partially reversible in PEG 3350 and irreversible in sodium citrate, at least under these conditions.

Next, we evaluated the effect of pH on TIR precipitation and NADase activity. There was virtually no increase in TIR precipitation in the absence of PEG. However, in the presence of 25% PEG, TIR precipitation increased with increasing pH (Figure 2—figure supplement 2D). Under these same conditions, no activity was apparent below pH 6, K_m remained constant (Figure 2—figure supplement 2E), and k_cat increased from pH 6.5 to pH 8.5 (Figure 2—figure supplement 2F). A corresponding increase in k_cat/K_m was responsible for the increase in catalytic efficiency above pH 7 (Figure 2—figure supplement 2G). Notably, the increase in k_cat/K_m correlated with precipitation (Figure 2—figure supplement 2D and G), again indicating that a phase transition increases TIR activity.

We performed negative stain electron microscopy to directly visualize TIR aggregation in vitro (Figure 2N). Protein visualization is not possible with PEG because macroviscogens themselves are stained, confounding image analysis. Therefore, we performed this experiment with citrate. In the absence of citrate, we observed borderline fibrillar structures and protein aggregates, but overall, there were no consistent structures (Figure 2N). However, in the presence of citrate, circular particles emerged (Figure 2N). These data corroborate our discovery that TIR-1::wrmScarlet aggregates in vivo into visible puncta within intestinal epithelial cells (Figure 1A).

C. elegans is a sterol auxotroph and requires dietary sterols for development, lifespan, fecundity, and resistance to pathogen infection (Hieb and Rothstein, 1968; Chitwood, 1999; Shim et al., 2002; Merris et al., 2003; Yochem et al., 1999; Otarigho and Aballay, 2020; Cheong et al., 2011). As such, 5 μg/mL of cholesterol is a standard additive in C. elegans laboratory growth medium (Brenner, 1974). We found that C. elegans grown in the absence of cholesterol supplementation activated GFP-based transcriptional reporters for two putative immune effector genes, T24B8.5p::gfp and irg-5p::gfp (Figure 3A and B). T24B8.5 and irg-5 are expressed in the intestine, induced during infection with multiple pathogens, including P. aeruginosa, and controlled by the p38 PMK-1 innate immune pathway (Troemel et al., 2006; Shivers et al., 2009; Bolz et al., 2010). qRT-PCR studies confirmed that C. elegans in a low cholesterol environment upregulate T24B8.5 and irg-5, as well as other innate immune effector genes (irg-4 and K08D8.4) (Figure 3C). These data suggest that host defense pathways are activated in the absence of pathogen infection when environmental sterols are scarce.

The nuclear hormone receptor, NHR-8, a homolog of mammalian liver X receptor (LXR) and pregnane X receptor (PXR), is required for the transport, distribution, and metabolism of cholesterol in C. elegans (Magner et al., 2013; Lindblom et al., 2001). Thus, nhr-8 loss-of-function mutant strains can be used as genetic tools to study conditions of low sterol content. Two previously characterized nhr-8 null alleles are nhr-8(hd117), which lacks the first exon (Magner et al., 2013) and nhr-8(ok186), which is missing most of the ligand-binding domain (Lindblom et al., 2001). Notably, the transcription profile of nhr-8(hd117) and nhr-8(ok186) animals mimics that of wild-type C. elegans infected with the bacterial pathogen P. aeruginosa (Figure 3D, Figure 3—figure supplement 1A, Supplementary files 1 and 2). The correlation between the transcriptional signatures of either the nhr-8(hd117) or the nhr-8(ok186) mutant with the genes that are changed in wild-type animals during pathogen infection was significant across all genes (r = 0.311 and r = 0.370, respectively). Of note, the correlation between these datasets is tighter when comparing only the differentially expressed genes (r = 0.763 and r = 0.849, respectively) and only genes that are also involved in innate immunity (r = 0.677 and r = 0.703, respectively) (Figure 3D and Figure 3—figure supplement 1A). Among the immune effectors that are upregulated in both the nhr-8(hd117) and nhr-8(ok186) mutants, and in wild type animals infected with P. aeruginosa, are T24B8.5, irg-4, irg-5, and K08D8.4; the same genes whose transcription are also induced by cholesterol deprivation (Figure 3C and D, and Figure 3—figure supplement 1A).

To validate these findings, we compared gene expression changes in wild-type C. elegans grown in media lacking supplemented cholesterol versus animals grown under standard culture conditions with 5 μg/mL of cholesterol. Consistent with our transcriptome profiling experiments of nhr-8 loss-of-function mutants, we discovered that innate immune effectors were strongly enriched among genes that were transcriptionally activated during cholesterol deprivation (Figure 3E). Specifically, the expression of innate immune effectors was significantly correlated in wild-type animals infected with P. aeruginosa and in wild-type nematodes starved for cholesterol (r = 0.610) (Figure 3E). Immune effectors that were induced during cholesterol starvation in qRT-PCR studies (irg-4, irg-5, K08D8.4, and T24B8.5) were again found among these differentially regulated genes, confirming the integrity of our RNA-seq analysis (Figure 3C). These observations were confirmed in a separate RNA-seq experiment (Otarigho and Aballay, 2021). Importantly, a comparison of the transcriptional changes in nhr-8(hd117) mutants with the genes regulated by low cholesterol revealed a remarkable correlation across all genes (r = 0.578) and differentially expressed genes (r = 0.836) (Figure 3—figure supplement 1B). Together with previous work characterizing the role of nhr-8 in C. elegans cholesterol metabolism (Magner et al., 2013), these data support the use of nhr-8 mutants to study conditions of low sterol content. In summary, cholesterol-starved, wild-type C. elegans and two different nhr-8 loss-of-function mutants induce the transcription of innate immune defenses.

Two different supplementation experiments using the T24B8.5p::gfp immune reporter demonstrated that immune effector activation in nhr-8(hd117) mutants is due to sterol deficiency in these animals. First, supplementation of exogenous cholesterol at an increased concentration (80 μg/mL) fully suppressed T24B8.5p::gfp activation in the nhr-8(hd117) mutant (Figure 3F). Second, supplementation with the non-ionic detergents Tergitol or Triton X-100, which solubilize hydrophobic, amphipathic compounds, including sterols, suppressed T24B8.5p::gfp activation in nhr-8(hd117) animals in a manner that was dependent on the presence of added cholesterol in the growth media (Figure 3G and H, and Figure 3—figure supplement 1C). Nematode growth media solidified with agarose, rather than agar, contains markedly fewer contaminating sterols (Magner et al., 2013; Chitwood, 1999; Gerisch et al., 2001). Importantly, on agarose, the addition of Tergitol to the growth media also suppressed T24B8.5p::gfp activation in the nhr-8(hd117) mutant, but only in the presence of 5 μg/mL of cholesterol (Figure 3H). Notably, this effect was dose-dependent (Figure 3—figure supplement 1C). These data establish that solubilization of cholesterol by Tergitol in standard nematode growth media suppresses immune activation in the nhr-8(hd117) mutant background. We used qRT-PCR to confirm these findings – Tergitol suppressed activation of T24B8.5, as well as the immune effectors irg-4, irg-5, and K08D8.4 in the nhr-8(hd117) mutant (Figure 3I–L). Of note, activation of immune defenses in nhr-8(hd117) animals is specific to cholesterol deprivation in this genetic background, as supplementation with individual saturated, mono- and polyunsaturated fatty acids, which are also misregulated in the nhr-8(hd117) mutant background (Magner et al., 2013), failed to suppress activation of T24B8.5p::gfp (Figure 3—figure supplement 1D). Together, these data confirm that cholesterol deprivation activates immune effector transcription independent of bacterial infection.

It has been previously shown that wild-type C. elegans raised on media without supplemented cholesterol are hypersusceptible to killing by P. aeruginosa, as are nhr-8(hd117) mutants on media with standard cholesterol supplementation (5 μg/mL) (Otarigho and Aballay, 2020). Importantly, we found that high doses of cholesterol (80 μg/mL) rescued the enhanced susceptibility of nhr-8(hd117) mutants to P. aeruginosa infection (Figure 3M). We confirmed this finding using an orthologous approach. Two detergents (Tergitol or Triton X-100), which solubilize cholesterol in nematode growth media, also restored wild-type pathogen resistance to the nhr-8(hd117) mutant (Figure 3N), consistent with their ability to modulate the expression of innate immune effectors (Figure 3G–L). Importantly, we found that the nhr-8(hd117) mutant accumulated less P. aeruginosa in its intestine during infection than wild-type animals (Figure 3O). Taken together, these data suggest that low cholesterol in the nhr-8(hd117) mutants reduces tolerance to pathogen infection, and that the robust transcriptional induction of immune effectors in this mutant background leads to reduced accumulation of P. aeruginosa during infection.

Additional analyses of the transcriptome profiling data revealed that targets of the p38 PMK-1 innate immune pathway were strongly enriched among the genes induced in wild-type animals during cholesterol deprivation (Figure 4A). Accordingly, the levels of active, phosphorylated PMK-1 were higher in wild-type animals grown on media without supplemented cholesterol than in animals grown under standard culture conditions (Figure 4B and C). Consistent with these data, the p38 PMK-1 pathway is also activated in nhr-8 loss-of-function mutants. A gene set enrichment analysis of the nhr-8(hd117) and nhr-8(ok186) transcriptome profiling experiments revealed strong enrichment of p38 PMK-1 pathway targets among the genes upregulated in each nhr-8 mutant (Figure 4D and Figure 4—figure supplement 1A). In addition, the nhr-8(hd117) and nhr-8(ok186) mutants had an increased ratio of phosphorylated PMK-1 relative to total PMK-1 compared to wild-type controls (Figure 4E and F). RNAi-mediated knockdown of tir-1, the most upstream component of the p38 signaling cassette (Liberati et al., 2004; Couillault et al., 2004), fully suppressed hyperactivation of T24B8.5p::gfp in nhr-8(hd117) animals (Figure 4G). The tir-1(qd4) loss-of-function mutation completely suppressed the induction of T24B8.5, irg-4, irg-5, and K08D8.4 in the nhr-8(hd117) background (Figure 4H–K). The transcription factors ATF-7 and SKN-1 link PMK-1 activity to its transcriptional outputs (Shivers et al., 2010; Inoue et al., 2005). In nhr-8(hd117) animals, knockdown of atf-7, but not skn-1, abrogated T24B8.5p::gfp activation (Figure 4G and Figure 4—figure supplement 1B and C). Finally, to further support our observation that cholesterol scarcity induces immune defenses upstream of p38 PMK-1, we used the MAPK phosphatase vhp-1, a negative regulator of PMK-1 (Kim et al., 2004). Solubilization of cholesterol with Tergitol was unable to suppress activation of T24B8.5p::gfp induced by knockdown of vhp-1 (Figure 4L).

The c-JUN N-terminal kinase MAPK homolog kgb-1, the insulin signaling pathway forkhead box O family (FOXO) transcription factor daf-16, and the G-protein-coupled receptor (GPCR) fshr-1, each function in parallel to the p38 PMK-1 pathway to regulate immune and stress responses in C. elegans (Troemel et al., 2006; Kim et al., 2004; Powell et al., 2009; Garsin et al., 2003). However, knockdown of each of these genes in nhr-8(hd117) animals failed to suppress T24B8.5p::gfp activation (Figure 4—figure supplement 1B), and RNAi-mediated knockdown of nhr-8 did not induce nuclear localization of DAF-16::GFP (Figure 4—figure supplement 1D). Thus, cholesterol scarcity induces C. elegans innate immune responses through specific activation of p38 PMK-1 immune pathway signaling.

Interestingly, tir-1(qd4);nhr-8(hd117) double mutants were more susceptible to P. aeruginosa infection than nhr-8(hd117) mutants (Figure 4M). These data suggest that the induction of the p38 PMK-1 pathway in the nhr-8(hd117) mutant background promotes resistance to P. aeruginosa infection. In addition, tir-1(qd4);nhr-8(hd117) double mutants were slightly, but significantly and reproducibly, more susceptible than the tir-1(qd4) mutant to killing by P. aeruginosa. We therefore hypothesize that the inherent susceptibility to pathogen-mediated killing in animals that lack sufficient cholesterol (e.g. the nhr-8(hd117) mutant) leads to additive pathogen susceptibility in animals that also lack a functioning p38 PMK-1 host defense pathway (e.g. the tir-1(qd4) mutant).

To determine if sterol scarcity induces oligomerization and the NAD⁺ glycohydrolase activity of TIR-1 to activate the p38 PMK-1 pathway, we examined TIR-1::wrmScarlet puncta formation in the nhr-8(hd117) mutant background (Figure 5A). Consistent with our studies during P. aeruginosa infection (Figure 1A), nhr-8(hd117) mutant animals induced TIR-1::wrmScarlet puncta formation in intestinal epithelial cells (Figure 5A and B). Importantly, TIR-1::wrmScarlet puncta formation in nhr-8(hd117) animals was entirely suppressed by solubilizing cholesterol in the growth media with Tergitol (Figure 5A and B). Additionally, treatment of nhr-8(hd117) animals expressing TIR-1::wrmScarlet with tir-1(RNAi) inhibited fluorescence and puncta formation (Figure 5A and B).

Importantly, the C. elegans tir-1^ΔSAM, tir-1^G747P and tir-1^H833A mutants, which contain specific mutations that block oligomerization of TIR-1, and the tir-1^E788A strain that carries a mutation in the catalytic glutamine required for the NADase activity, each prevented activation of the p38 PMK-1-dependent immune reporter T24B8.5p::gfp in nhr-8(RNAi) animals (Figure 5C) and during cholesterol deprivation (Figure 5D). Using qRT-PCR, we confirmed that these mutations in tir-1 abrogated the induction of the T24B8.5 (Figure 5E) and irg-4 (Figure 5F) immune effectors in nhr-8(RNAi) animals. We also found that these tir-1 mutants phenocopied the effects of the p38 PMK-1 pathway mutants (tir-1(qd4), nys-1(ag3), and pmk-1(km25)) on the basal expression of these immune effector genes (Figure 5E and F).

It is possible that the organization of TIR-1::wrmScarlet into visible puncta in nhr-8 mutants is secondary to non-specific protein aggregation; however, the in vitro and in vivo data presented in this manuscript, when considered together, suggest that this is not the case. Organized multimerization of TIR-1 is a prerequisite for the NADase activity of the protein complex in vitro (Figure 2, Figure 2—figure supplement 1). Accordingly, the mutations that specifically abrogate TIR-1 multimerization in vitro (Figure 2J, Figure 2—figure supplement 1I), also block p38 PMK-1 pathway activation (Figure 1D–F) and prevented immune effector induction in response to cholesterol deprivation in vivo (Figure 5C–F). Furthermore, Tergitol, which solubilizes cholesterol and fully suppresses p38 PMK-1 immune activation in nhr-8 mutants (Figure 3G-N), also suppresses TIR-1::wrmScarlet puncta formation (Figure 5). Finally and perhaps most importantly, P. aeruginosa infection, a separate physiological stress, also induces TIR-1::wrmScarlet puncta formation to activate the p38 PMK-1 pathway (Figure 1).

In summary, the above data demonstrate for the first time that physiological stresses, both pathogen and non-pathogen, induce TIR-1 multimerization into puncta within intestinal epithelial cells, which then activates the p38 PMK-1 innate immune pathway through the intrinsic NADase activity of the TIR-1 protein complex.

Since C. elegans requires cholesterol to survive bacterial infection and must obtain this essential metabolite from its diet, we hypothesized that, when environmental sterols are limited, activation of the p38 PMK-1 pathway represents an evolutionary adaptation that primes immune effector expression to anticipate challenges from bacterial pathogens. To test this hypothesis, we examined the expression of innate immune effector genes during bacterial infection in the presence and absence of cholesterol supplementation. Interestingly, the induction of irg-4p::gfp (Figure 6A), irg-5p::gfp (Figure 6B), and T24B8.5p::gfp (Figure 6C) during P. aeruginosa infection was enhanced when nematodes were infected on media that did not contain supplemented cholesterol. Consistent with these data, P. aeruginosa infection also led to increased activation of irg-4p::gfp (Figure 6D) and irg-5p::gfp (Figure 6E) when nhr-8 was depleted by RNAi.

To provide further support for this hypothesis, we analyzed the expression pattern of p38 PMK-1-dependent transcripts in nhr-8(hd117) mutant animals during P. aeruginosa infection. Of the 472 genes that were induced in wild-type animals during P. aeruginosa infection, 184 were also differentially regulated (either induced or repressed) in nhr-8(hd117) animals that were infected with P. aeruginosa. To perform this analysis in an unbiased manner, we scaled the expression level in each condition for each of these 184 genes by calculating a row z-score and performed hierarchical clustering (Figure 6F). We observed that these 184 genes group into two clusters: Cluster I had 139 genes and Cluster II contained 45 genes (Supplementary file 5). Cluster I was comprised mostly of genes whose expression in the absence of infection was higher in nhr-8(hd117) mutants than wild-type animals (129 of 139 genes). In addition, the majority of Cluster I genes were more strongly induced during P. aeruginosa infection in nhr-8(hd117) animals than wild-type animals. Cluster II, on the other hand, contained genes whose induction on P. aeruginosa were dependent on nhr-8. Importantly, p38 PMK-1-dependent genes were strongly enriched among Cluster I, but not Cluster II, genes (Figure 6G). Thirty-three of the 139 genes in Cluster I are known targets of the p38 PMK-1 immune pathway (24.17-fold enriched, hypergeometric P-value = 7.68 x 10^–37), a group that includes the immune effectors T24B8.5 (Figure 6H), irg-4 (Figure 6I), irg-5 (Figure 6J), and K08D8.4 (Figure 6K). By contrast, only three genes in Cluster II are p38 PMK-1-dependent transcripts (6.95-fold enriched, hypergeometric p-value = 0.01) (Figure 6F and G).

These data demonstrate that sterol scarcity primes the induction of innate immune effectors by activating the p38 PMK-1 immune pathway. Subsequent challenge by P. aeruginosa further drives immune activation in a manner that leads to reduced pathogen accumulation in the intestine (Figure 3O).

The previously published C. elegans strains used in this study were: N2 Bristol (Brenner, 1974), KU25 pmk-1(km25) (Mizuno et al., 2004), AU3 nsy-1(ag3) (Kim et al., 2002), ZD101 tir-1(qd4) (Shivers et al., 2009), AA968 nhr-8(hd117) (Magner et al., 2013), AE501 nhr-8(ok186) (Lindblom et al., 2001), AU78 agIs219 [T24B8.5p::gfp::unc-54–3’UTR; ttx-3p::gfp::unc-54–3’UTR] (Shivers et al., 2009), AU306 agIs44 [irg-4p::gfp::unc-54–3’UTR; myo-2p::mCherry] (Pukkila-Worley et al., 2014), AY101 acIs101 [pDB09.1(irg-5p::gfp); pRF4(rol-6(su1006))] (Bolz et al., 2010), TJ356 zIs356 [daf-16p::daf-16a/b::gfp + pRF4(rol-6(su1006))] (Henderson and Johnson, 2001). The strains developed in this study were: RPW278 nhr-8(hd117);agIs219, RPW317 tir-1(qd4);nhr-8(hd117), RPW339 tir-1(ums47[E788A]);agIs219, RPW369 tir-1(ums54[ΔSAM]);agIs219, RPW374 tir-1(ums55[G747P]);agIs219, RPW381 tir-1(ums56[H833A]);agIs219, RPW386 tir-1(ums57[tir-1::3xFLAG]);agIs219, RPW387 tir-1(ums58[tir-1[E788A]::3xFLAG]);agIs219, RPW388 tir-1 (ums59[tir-1[ΔSAM]::3xFLAG]);agIs219, RPW 389 tir-1(ums60[tir-1[G747P]::3xFLAG]);agIs219, RPW403 tir-1(ums63[tir-1::wrmScarlet]), RPW404 nhr-8(hd117);tir-1(ums63[tir-1::wrmScarlet]). Bacteria used in this study are Escherichia coli OP50, E. coli DH5α, E. coli HT115(DE3), and Pseudomonas aeruginosa strain PA14 (Rahme et al., 1995).

C. elegans strains were maintained on standard nematode growth medium (NGM) plates (0.25% bacto peptone, 0.3% sodium chloride, 1.7% agar [Fisher], 5 μg/mL cholesterol [Sigma-Aldrich, BioReagent grade], 25 mM potassium phosphate pH 6.0, 1 mM magnesium sulfate, 1 mM calcium chloride)with E. coli OP50 as a food source, as described (Brenner, 1974). For low-cholesterol medium (0 μg/mL cholesterol), NGM was prepared without cholesterol supplementation, while 0.1% ethanol was added to maintain an equivalent ethanol concentration. For high-cholesterol medium, cholesterol was dissolved in ethanol at 20 mg/mL and added to NGM at a final concentration of 80 μg/mL immediately prior to pouring plates. For all assays with high-cholesterol medium, NGM containing 0.4% ethanol and 5 μg/mL cholesterol were used as control plates. Cholesterol solubilization assays were performed by supplementing NGM containing 5 μg/mL cholesterol with either 0.1% Tergitol (Sigma-Aldrich) or 0.1% Triton X-100 (Sigma-Aldrich). For assays using media solidified with agarose, NGM plates were prepared with 1.7% Ultrapure agarose (ThermoFisher Scientific) in place of agar. All fatty acids were purchased from Nu-Check-Prep Inc, and supplementation performed as previously described with modification (Anderson et al., 2019; Nandakumar and Tan, 2008). Fatty acids were dissolved in 50% ethanol and added at a final concentration of 1 mM to NGM agarose containing 0 μg/mL cholesterol and 0.1% Tergitol immediately prior to plate pouring. Prior to all assays, plates supplemented with lipids and control plates were seeded with E. coli OP50 and grown for 24 hr at room temperature. Assays were performed by picking 10–20 gravid adult animals to either lipid-supplemented or matched control plates. Animals were maintained on the plates for 14 hr at 20 °C, after which they were removed. Eggs laid on the plate were allowed to hatch and develop to the L4 stage at 20 °C. For low-cholesterol assays, animals were grown for two generations on NGM containing 0 μg/mL cholesterol.

CRISPR/Cas9 was used to generate tir-1 mutants in both wild-type and TIR-1::3xFLAG backgrounds, as described (Dokshin et al., 2018; Ghanta and Mello, 2020). All CRISPR/Cas9 reagents were purchased from Integrated DNA Technologies. Target guide sequences were selected using the CHOPCHOP web tool (Labun et al., 2019). ssODN and dsDNA repair templates contained indicated edits, deletions or insertions with 35 bp flanking homology arms. crRNA guide and ssODN sequences are listed in Supplementary file 6. For wrmScarlet dsDNA repair template, wrmScarlet was PCR amplified with 35 bp flanking homology arms. PCR was gel purified, diluted to 100 ng/μL, melted and reannealed using a thermal cycler (95 °C – 2 min; 85 °C – 10 s, 75 °C – 10 s, 65 °C – 10 s, 55 °C – 10 s, 45 °C – 10 s, 35 °C – 10 s, 25 °C – 10 s, 4 °C – hold. Ramp down 1 °C per s), and used immediately for injection. A mixture of 0.25 μg/μL Cas9, 0.1 μg/μL tracrRNA and 0.056 μg/μL crRNA were incubated for 15 min at 37 °C. 0.11 μg/μL ssODN or 25 ng/μL dsDNA and 40 ng/μL pRF4(rol-6(su1006)) plasmid were added to the mixture, centrifuged, and microinjected into young adult animals carrying the agIs219 transgene, tir-1(ums57[tir-1::3xFLAG]);agIs219, or N2. The F1 progeny were screened for Rol phenotypes 3–4 days after injection and then for indicated edits using PCR and Sanger sequencing. Primer sequences used for genotyping are listed in Supplementary file 6.

Knockdown of target genes was performed by feeding C. elegans E. coli HT115 expressing dsRNA targeting the gene of interest, as previously described with modification (Fire et al., 1998; Timmons et al., 2001; Conte et al., 2015). In brief, HT115 bacteria expressing dsRNA targeting genes of interest were grown in Lysogeny broth (LB) Lennox medium containing 50 μg/mL ampicillin and 15 μg/mL tetracycline overnight with shaking (250 rpm) at 37 °C. Overnight cultures were seeded onto NGM containing 5 mM IPTG and 50 μg/mL carbenicillin and incubated at 37 °C for 16 hr, after which synchronized L1 animals were transferred to bacterial lawns and allowed to grow until the L4 stage.

“Slow killing” P. aeruginosa infection experiments were performed as previously described (Tan et al., 1999; Foster et al., 2020b). Wild-type is either N2 or agIs219. In brief, a single colony of P. aeruginosa PA14 was inoculated into 3 mL of LB medium and grown with shaking (250 rpm) at 37 °C for 14 hr. Ten μL of overnight culture was spread onto 35 mm petri dishes containing 4 mL slow killing agar (0.35% peptone, 0.3% sodium chloride, 1.7% agar, 5 μg/mL cholesterol, 25 mM potassium phosphate pH 6.0, 1 mM magnesium sulfate, 1 mM calcium chloride). Plates were incubated for 24 hr at 37 °C and for approximately 24 hr at 25 °C. Immediately prior to starting the assay, 0.1 mg/mL 5-fluorodeoxyuridine (FUDR) was added on top of the agar to prevent progeny from hatching. Animals used in all assays were grown at 20 °C with specified growth conditions. For assays involving high cholesterol or nonionic detergents, slow-killing agar plates were prepared with either 80 μg/mL cholesterol (Sigma-Aldrich), 0.1% Tergitol (Sigma-Aldrich), or 0.1% Triton X-100 (Sigma-Aldrich). For experiments using plates containing 80 μg/mL cholesterol, matched control plates containing the equivalent ethanol concentration (0.4%) and 5 μg/mL cholesterol were prepared. All pathogenesis and lifespan assays are representative of three biological replicates. Sample sizes, mean lifespan, and p values for all trials are shown in Supplementary file 4.

CFU of P. aeruginosa were quantified in the intestine of C. elegans as previously described with modifications (Foster et al., 2020b; Singh and Aballay, 2019). Briefly, C. elegans animals were exposed to lawns of P. aeruginosa, which were prepared as previously described, for 24 hr. Animals were then picked to NGM plates lacking bacteria and incubated for 10 min to remove external P. aeruginosa. Animals were then transferred to a second NGM plate, after which 10–11 animals per replicate were collected, washed with M9 buffer containing 25 mM tetramisole (Sigma-Aldrich) and 0.01% Triton X-100 (Sigma-Aldrich), and ground with 1.0 mm silicon carbide beads (BioSpec Products). P. aeruginosa CFUs were quantified from serial dilutions of the lysate grown on LB agar.

A total of 2000 synchronized L1 stage C. elegans of the indicated genotypes were grown to the L4 stage and harvested by washing with M9. For expression analysis of C. elegans genes during P. aeruginosa infection, animals at the L4 stage animals were transferred by washing to plates containing E. coli OP50 or P. aeruginosa PA14 lawns. Animals were exposed for four hours and subsequently harvested by washing with M9. RNA was isolated using TriReagent (Sigma-Aldrich), column purified (Qiagen), and analyzed by 100 bp paired-end mRNA-sequencing using the BGISEQ-500 platform (BGIAmericasCorp) with > 20 million reads per sample. Raw fastq reads were evaluated by FastQC (version 0.11.5), clean reads were aligned to the C. elegans reference genome (WBcel235) and quantified using Kallisto (version 0.45.0) (Bray et al., 2016). Differentially expressed genes were identified using Sleuth (version 0.30.0) (Pimentel et al., 2017). Pearson correlation statistical analysis was performed using Prism 9.0. Innate immune genes were identified using DAVID Bioinformatics database biological process gene ontology (GO) term innate immune response (Huang et al., 2009). Heatmaps of differentially expressed genes were generated using pheatmap (version 1.0.12). Gene set enrichment analysis of RNA-seq was performed using GSEA (version 4.1.0) (Subramanian et al., 2005) with a custom gene set database (Supplementary file 7) containing p38 PMK-1-dependent genes generated by analyzing previously published RNA-seq of uninfected pmk-1(km25) animals (Fletcher et al., 2019) with the RNA-seq data analysis pipeline described above. Differential gene expression was defined as a fold change (FC) versus wild-type greater than two and q less than 0.01.

For the qRT-PCR studies, RNA was reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc), amplified and detected using Syber Green (Bio-Rad Laboratories, Inc) and a CFX384 machine (Bio-Rad Laboratories, Inc). The sequences of primers that were designed for this study are presented in Supplementary file 6. Other primers were previously published (Troemel et al., 2006; Taubert et al., 2008; Richardson et al., 2010; Estes et al., 2010). All values were normalized against the geometric mean of the control genes snb-1 and act-3. Fold change was calculated using the Pfaffl method (Pfaffl, 2001).

Protein lysates from 2000 C. elegans grown to the L4 larval stage on E. coli OP50 on NGM agar were prepared as previously described with modification (Peterson et al., 2019; Cheesman et al., 2016). Harvested animals were washed twice with M9 buffer and resuspended in RIPA Buffer (Cell Signaling Technology, Inc) containing 1 x Halt Protease and Phosphatase inhibitor (ThermoFisher Scientific). Samples were lysed using a teflon homogenizer, centrifuged, and protein was quantified from the supernatant of each sample using the DC protein assay (Bio-Rad Laboratories, Inc). NuPAGE LDS sample buffer (ThermoFisher Scientific) was added to a concentration of 1 X, and 12.5–30 μg of total protein from each sample was resolved on NuPAGE 4–12% BisTris (Phospho-PMK-1 and Total-PMK-1) or NuPAGE 3–8% TrisAcetate (TIR-1::3xFLAG) gels (ThermoFisher Scientific), transferred to nitrocellulose membranes using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc), blocked with 5% milk powder in TBST and probed with a 1:1,000 dilution of an antibody that recognizes the doubly-phosphorylated TGY motif of PMK-1 (Cell Signaling Technology, #9211), a previously characterized total PMK-1 antibody (Peterson et al., 2019), a monoclonal mouse anti-FLAG antibody (Sigma-Aldrich, M2), or a monoclonal mouse anti-alpha-tubulin antibody (Sigma-Aldrich, Clone B-5-1-2). Horseradish peroxidase (HRP)-conjugated anti-rabbit (Cell Signaling Technology, #7074) and anti-mouse IgG secondary antibodies (Abcam, #ab6789) were diluted 1:10,000 and used to detect the primary antibodies following the addition of ECL reagents (Thermo Fisher Scientific, Inc), which were visualized using a BioRad ChemiDoc MP Imaging System. The band intensities were quantified using Fiji/ImageJ, and the ratio of active phosphorylated PMK-1 to total PMK-1 was calculated.

For TIR-1::wrmScarlet puncta visualization, all TIR-1::wrmScarlet expressing animals were depleted of autofluorescent gut granules by exposure to glo-3(RNAi) from the L1 stage prior to all experiments. P. aeruginosa was prepared as described for bacterial infection and colonization assays. L4 animals expressing TIR-1::wrmScarlet were transferred to either OP50 or P. aeruginosa containing plates for 24 hr. For imaging, animals were transferred to 2% agarose pads, paralyzed with 300 mM sodium azide, and imaged with a 63 x oil immersion lens. For Tergitol experiments, animals were grown on RNAi plates containing 0.1% Tergitol and the respective RNAi strains and imaged at the L4 stage.

Nematodes were mounted onto agar pads, paralyzed with 10 mM levamisole (Sigma) or 300 mM sodium azide and photographed using a Zeiss AXIO Imager Z2 microscope with a Zeiss Axiocam 506mono camera and Zen 2.3 (Zeiss) software.

Image processing and analysis were performed using Fiji image analysis software. In each panel, the exposure time and processing for all images were identical. To quantify T24B8.5p::gfp fluorescence intensity, the DIC channel was used to identify and select the intestine of each animal using the Freehand Tool. The integrated density, which is the product of the area and mean gray value, was then calculated for the green channel of each selected region using the Measurement function. To quantify TIR-1::wrmScarlet puncta, the last posterior pair of intestinal epithelial cells were identified in the DIC channel and selected using the Freehand Tool. We then filtered the red and green channel images using Gaussian Smoothing (sigma radius = 1). The Find Maxima tool was then used to identify puncta in the red channel within the selected area. The Find Maxima settings used for the analysis of P. aeruginosa puncta were prominence: 5, strict: no, exclude edge maxima: no. The settings for the nhr-8(hd117) and Tergitol experiments were prominence: 15, strict: no, exclude edge maxima: no. We then overlayed the location of the red channel puncta in the green channel and determined if there was an autofluorescent puncta in this exact location. TIR-1::wrmScarlet puncta were those that were present in the red, but not the green, channel. Importantly, we only reported puncta that were positively identified by the image analysis software.

The recombinant C. elegans TIR-1 TIR domain (TIR) was expressed in bacteria as previously described (Loring et al., 2020). Briefly, the TIR domain cloned into the pET-30a(+) vector was transformed into chemically competent E. coli BL21(DE3) cells and maintained as a glycerol stock at –80 °C. An inoculation loop was used to transfer the transformed bacteria into 5 mL of LB media with 50 µg/mL (final concentration) of kanamycin, and the culture was grown overnight at 37 °C while rotating. The next day, the cultures were diluted 1:400 in LB media with 50 µg/mL (final concentration) of kanamycin and grown at 37 °C while shaking at 215 rpm until an OD₆₀₀ of 0.7–0.8 was reached. After cooling, 50 µM IPTG (final concentration) was added to the culture to induce protein expression. The incubator temperature was decreased to 16 °C, and cells were incubated for an additional 16–18 hr. Bacterial cells were collected by centrifugation at 3000 x g for 15 min at 4 °C, flash frozen in liquid nitrogen, and stored at –80 °C until purification.

For purification, bacterial pellets were thawed on ice and then resuspended in Lysis Buffer (50 mM Tris•HCl pH 7.0, 300 mM NaCl, 10% (w/v) glycerol, 0.001% Tween 20) with Pierce EDTA-free protease inhibitor mini tablets (ThermoFisher Scientific). The resuspension was incubated with 100 µg/mL lysozyme for 10 min at 4 °C and sonicated with a Fisher Scientific Sonic Dismembrator sonicator (FB-705) in 50 mL batches at an amplitude of 30 for 20 s, pulsing for 1 sec on and 1 s off, followed by a delay period of 20 s for a series of 12 cycles. Crude lysate was clarified at 21,000 x g for 25 min at 4 °C, at which point the supernatant was applied to pre-equilibrated Strep-Tactin XT Superflow high-capacity resin (IBA Lifesciences) and allowed to enter the column by gravity flow; the Strep-Tactin resin had been equilibrated in Strep Wash Buffer (50 mM Tris•HCl pH 7.0, 300 mM NaCl). The column was washed with 30 column volumes of Strep Wash Buffer, and the protein was eluted with 25 column volumes of Strep Elution Buffer (Strep Wash Buffer with 50 mM biotin). The protein eluted from the Strep-Tactin column was then applied to pre-equilibrated TALON Metal Affinity Resin (Takara) and allowed to enter the column by gravity flow; the TALON resin was equilibrated in His Wash 1 (50 mM Tris•HCl pH 7.0, 150 mM NaCl, 5 mM imidazole). A series of 15 column volume washes were applied (His Wash 1; His Wash 2: 50 mM Tris•HCl pH 7.0, 150 NaCl, 10 mM imidazole), and the protein was eluted in 20 column volumes of His Elution Buffer (50 mM Tris•HCl pH 7.0, 150 mM NaCl, 150 mM imidazole). The eluted protein was dialyzed overnight in Dialysis Buffer (50 mM Tris•HCl, pH 7.0, 150 mM NaCl). The next day, the protein was concentrated using a 10,000 NMWL Amicon Ultra-15 Centrifugal Filter Unit at 4 °C, and the protein concentration was determined by the Bradford assay. TIR was flash frozen in liquid nitrogen and stored at –80 °C in 25 µL aliquots.

Nicotinamide 1,N⁶-ethenoadenine dinucleotide (ε-NAD, Sigma-Aldrich) is a fluorescent analog of NAD⁺ and was utilized in kinetic assays as a TIR-1 substrate. TIR-1 cleaves the nicotinamide moiety from ε-NAD to release nicotinamide and etheno-ADPR (ε-ADPR), which fluoresces (λ_ex = 330 nm, λ_em = 405 nm). Enzymatic activity was assayed in Assay Buffer (50 mM Tris pH 8.0, 150 mM NaCl; final concentration) using Corning 96–well Half Area Black Flat Bottom Polystyrene NBS Microplates for a final reaction volume of 60 μL, or Corning 384-well Low Volume Black Round Bottom Polystyrene NBS Microplates for a final volume of 20 µL; reactions were initiated by the addition of ε-NAD. ε-ADPR fluorescence intensity readings were taken in real time every 15 s for 15–30 min using Wallac EnVision Manager Software and a PerkinElmer EnVision 2,104 Multilabel Reader. Fluorescence intensity readings (λ_ex = 330 nm, λ_em = 405 nm) were converted to [ε-ADPR] with an ε-ADPR standard curve, which was produced by incubating fixed concentrations (0–400 µM) of ε-NAD with excess ADP-ribosyl cyclase and plotting the peak fluorescence intensity values against [ε-ADPR]. The activity was linear with respect to time under all conditions tested.

The enzymatic activity of the TIR domain was evaluated using the Fluorescent Assay described above. First, the enzyme concentration dependence was determined. For purified protein, the enzyme (0–32.5 µM; final concentration) was added to Assay Buffer in duplicate, briefly incubated at room temperature for 10 min, and the reaction was initiated with 1 mM ε-NAD. Fluorescence intensity was monitored in real time every 15 s for 15 min.

To evaluate the effect of crowding agents and sodium citrate on TIR activity, stock solutions of the additives were made. 50% (w/v) solutions of PEGs 8000, 3500, 1500, and 400, as well as 60% (w/v) solutions of dextran, sucrose, and glycerol were prepared and filtered. Initially, the concentration dependence of TIR in the presence of PEG 3350 was determined. TIR (0–15 µM; final concentration) was added to Assay Buffer with 25% PEG 3350 (final concentration) in duplicate, incubated for 10 min at room temperature, initiated with 1 mM ε-NAD, and monitored every 15 sec for 15 min. The concentration dependence of TIR in 25% PEG 3350 was used to establish that 2.5 µM TIR could be used to enable robust kinetic analyses. Next, the effect of viscogens on TIR activity was evaluated by adding 2.5 μM TIR (final concentration) in duplicate to Assay Buffer with or without 25% w/v of the viscogens (final concentration). Following a brief 10-min incubation period, the reaction was initiated with 1 mM ε-NAD and monitored every 15 s for 20 min. The dose response of 2.5 μM TIR to the viscogens (0, 10, 20, and 30% final concentrations) was evaluated in the same manner and monitored every 15 s for 15 min.

Sodium citrate was prepared as a stock solution of 2 M and filtered. The concentration dependence of TIR in the presence of 500 mM sodium citrate was determined. TIR (0–15 µM; final concentration) was added to Assay Buffer with 500 mM citrate (final concentration) in duplicate, incubated for 10 min at room temperature, and then the reaction was initiated with 1 mM ε-NAD and monitored every 15 s for 15 min. Additionally, the dose dependence of sodium citrate was determined in duplicate. 2.5 µM TIR was added to Assay Buffer with (0–1000 mM) sodium citrate and briefly incubated at room temperature for 10 min. The reaction was initiated with 1 mM ε-NAD, and fluorescence was monitored every 15 s for 15 min.

In all cases, the fluorescence intensity was converted to ε-ADPR using the ε-ADPR standard curve described above. Slopes of the progress curves yielded the velocities of the reactions, which were plotted in GraphPad Prism.

Steady-state kinetic reactions were carried out in Assay buffer with either PEG 3350 (0%–25%; final concentration) or sodium citrate (0–1000 mM; final concentration); a constant concentration of 2.5 µM TIR was used in these assays. Reaction components were mixed in duplicate and incubated at room temperature for 10 min before initiating the reaction with ε-NAD (0–4000 µM, final concentration). Fluorescence intensity was monitored every 15 s for 15 min. Using the ε-ADPR standard curve, the fluorescence was converted to [ε-ADPR]. The velocity of the reactions was calculated from the slope of the progress curve at each ε-NAD concentration and plotted in GraphPad Prism. Kinetic parameters were determined by fitting these velocities to the Michaelis-Menten equation (Eq. 1) at each PEG 3350 or sodium citrate concentration. K_m, k_cat, and k_cat/K_m values were plotted against PEG 3350 or sodium citrate concentration.,(1)v=VmaxSKm+S

where V_max is the maximum velocity, [S] is the substrate concentration, and K_m is the substrate concentration at half the maximum velocity.

Five µM TIR was incubated in Assay Buffer with PEG 3350 (0, 10, 17.5, and 25%; final concentration) or sodium citrate (0, 125, 250, 500, 750, and 1000 mM; final concentration) in duplicate at room temperature for 15 min. The precentrifugation control was removed, and the remainder of the sample was centrifuged at 17,000 x g at 4 °C for 10 min. Following centrifugation, the supernatant was separated from the pellet, and the pellet was resuspended in Assay Buffer with the respective concentration of PEG 3350 or sodium citrate. Samples were diluted 1:2 with gel loading buffer and run on an SDS-PAGE gel. Protein bands were stained by Coomassie and visualized on a BioRad Gel Doc EZ Gel Documentation System with Image Lab Software. Representative images are shown.

Following resuspension of the pellet, all fractions were analyzed in the Fluorescent Assay. After the samples were aliquoted into the assay plate in duplicate, the enzymatic reaction was initiated with 1 mM ε-NAD and monitored every 15 s for 15 min. Fluorescence intensity was converted to ε-ADPR concentration using the ε-ADPR standard curve. Slopes of the progress curves yielded the velocities of the reactions, which were plotted in GraphPad Prism.

To evaluate the effect of 1,6-hexanediol on TIR activity, the Fluorescent Assay was performed in triplicate in the absence and presence of PEG 3350 or sodium citrate. For pure protein, 35 µM TIR (final concentration) was incubated in Assay Buffer at room temperature for 10 min with or without 2% 1,6-hexanediol (final concentration). For PEG 3350 or citrate, 2.5 µM TIR was incubated in Assay Buffer with 25% PEG 3350 or 500 mM sodium citrate (final concentrations) for 10 min at room temperature with or without 2% 1,6-hexanediol. The reactions were initiated with 1 mM ε-NAD and monitored every 15 s for 15 min. Using the ε-ADPR standard curve, fluorescence was converted to [ε-ADPR], and the reaction velocities (i.e. slopes of the progress curves) were obtained. Velocities were normalized for enzyme concentration, and these normalized velocities were plotted in GraphPad Prism.

To determine whether 1,6-hexanediol can alter TIR precipitation, 1,6-hexanediol was added either before or after TIR precipitation in duplicate. To assess whether 1,6-hexanediol disrupts TIR precipitation, TIR precipitates were formed first by incubating 10 μM TIR (final concentration) with 25% PEG3350 or 500 mM sodium citrate (final concentration) at room temperature for 15 min. 1,6-hexanediol (0, 1, or 2%; final concentrations) was added to the mixture and incubated at room temperature for an additional 10 min. The precentrifugation control was removed, and the remaining mixture was centrifuged at 21,000 x g for 10 min at 4 °C. Supernatant fractions were removed, and the pellet was resuspended in Assay Buffer with the respective additive and concentration of 1,6-hexanediol. All fractions were run on an SDS-PAGE and stained with Coomassie Blue, and gels were imaged on a BioRad Gel Doc EZ Gel Documentation System with Image Lab Software. Next, we determined if 1,6-hexanediol could prevent TIR precipitation. 10 µM TIR was incubated in Assay Buffer with 1,6-hexanediol (0, 1, or 2%; final concentration) for 15 min at room temperature. 25% PEG 3350 or 500 mM sodium citrate was added to the TIR-buffer-hexanediol mixture and incubated further for 10 min. Controls were removed and the samples were centrifuged at 21,000 x g for 10 min at 4 °C. As before, supernatant fractions were removed, and pellet fractions were resuspended in Assay Buffer with respective additives and hexanediol concentrations. Fractions were analyzed by SDS-PAGE/Coomassie staining; representative images are shown.

To evaluate the reversibility of the TIR phase transition, 5 µM of TIR was mixed with Assay Buffer and 25% PEG 3350 or 500 mM sodium citrate in duplicate (final concentrations). Following a 15-min incubation period at room temperature, precentrifugation controls were removed and the sample remaining was centrifuged at 17,000 x g for 10 min at 4 °C. Supernatant fractions were separated from the pellet, which was resuspended in either Assay Buffer alone or Assay Buffer with respective additive. All fractions were analyzed for enzymatic activity in the Fluorescent Assay. Briefly, the precentrifugation, supernatant, and pellet fractions were aliquoted into the assay plates in duplicate and the reaction was initiated with 1 mM ε-NAD. Fluorescence was converted to [ε-ADPR] concentration with the ε-ADPR curve to yield the progress curves. The velocity of the reactions was taken as the slope of the line and the velocities were plotted in GraphPad Prism.

To validate the kinetic data, 10 µM TIR (final concentration) was incubated in Assay Buffer with either 25% PEG 3350 or 500 mM sodium citrate (final concentration) for 15 min at room temperature; control samples were incubated in Assay Buffer only. Samples were centrifuged at 21,000 x g for 10 min at 4 °C, after which the supernatant was separated from the pellet. The pellets from samples initially prepared with additives were resuspended in either Assay Buffer alone or Assay Buffer plus the respective additive; this was not necessary for the sample initially prepared without additive since the protein is primarily located in the supernatant in this case. After removing another control sample (Additive lanes on gel), the resuspended samples were centrifuged again at 21,000 x g for 10 min at 4 °C. As before, the supernatant was removed, and the pellet was resuspended in Assay Buffer with the respective additive. All samples were analyzed for protein content on an SDS-PAGE gel and stained with Coomassie Blue.

To determine the effect of pH on TIR precipitation, the experiments described above were carried out in duplicate at pH values from 4.5 to 9.0. Briefly, 5 µM TIR (final concentration) was mixed with Assay Buffer (50 mM buffer; 150 NaCl) with and without 25% PEG 3350 (final concentration); for pH 4.5–5, sodium acetate buffer was used; for pH 5.5–6.5, MES was used; for pH 7–9, Tris was used. The samples were incubated for 15 min at ambient temperature, at which point the samples were centrifuged at 21,000 x g for 10 min at 4 °C. The supernatant was removed, and the pellet was resuspended in either buffer alone or buffer with 25% PEG 3350 (final concentration); the presence or absence of 25% PEG 3350 and the buffer identity of the resuspension solution corresponded to the initial sample preparation. To neutralize the buffer, 10 µL of 1 M Tris (pH 6.8) was added to each sample before running on an SDS-PAGE gel and staining with Coomassie blue. Images of the gels were obtained on the BioRad Gel Doc EZ Gel Documentation System with Image Lab Software. Representative images are shown. ImageJ was used to quantify the bands, which were plotted in GraphPad Prism.

Steady-state kinetic analyses were also performed at each pH in 25% PEG 3350. A constant concentration of 2.5 µM TIR was used in these assays. Reaction components were mixed in quadruplicate and incubated at room temperature for 10 min before initiating the reaction with 0–2000 µM of ε-NAD (final concentration). Fluorescence intensity was monitored every 15 s for 15 min. Using the ε-ADPR standard curve, the fluorescence was converted to [ε-ADPR]. The velocity of the reactions was calculated from the slope of the progress curve at each ε-NAD concentration. Kinetic parameters were determined by fitting these velocities to the Michaelis-Menten equation (Eq. 1) at each pH. The log of K_m, k_cat, and k_cat/K_m values were determined and plotted in GraphPad Prism.

Negative stain EM on TIR (270 µg/mL) was performed in Assay Buffer (50 mM Tris, pH 8; 150 mM NaCl) with or without 500 mM sodium citrate. Samples were applied to glow-discharged, carbon- and formvar-coated copper grids and allowed to sit for 1 min and 30 s before blotting excess liquid away. 1% uranyl acetate was used to fix the samples before imaging. Samples were imaged on an FEI Tecnai Spirit 12 microscope. The diameters of particles in the samples with citrate were analyzed in ImageJ.

TIR domain mutants were made using PCR-based methods. The pET30a + TIR-1 TIR domain construct was used as a template (0.2–2 ng/µL; final concentration). The manufacturer’s protocol for iProof High-Fidelity DNA Polymerase (Bio-Rad Laboratories, Inc) was followed using iProof HF buffer supplemented with 3% DMSO. 50 µL reaction volumes were used in the following protocol: initial denaturation for 3 min at 98 °C, denaturation for 45 s at 98 °C, annealing for 1:30 min at 45°C–72°C, extension for 6 min at 72 °C, final extension for 10 min at 72 °C. Denaturation, annealing, and extension steps were repeated 30 times. The following day, DpnI was added to the PCR reactions and incubated at 37 °C for 2 hr to digest the template DNA. The digest was transformed into chemically competent E. coli XL1-Blue cells. Transformants were grown overnight in LB media with 50 µg/mL kanamycin and mini-prepped (Promega). Mutagenesis was validated by Sanger sequencing (Genewiz). The mutants were expressed and purified as described for WT TIR.

Steady-state kinetic analyses of the mutants were carried out in Assay Buffer with 25% PEG 3350 or 500 mM sodium citrate with 2.5 µM of the mutants (final concentrations). Reaction components were mixed in triplicate and incubated at room temperature for 10 min before initiating the reaction with 0–2000 µM of ε-NAD for PEG 3350 or 0–4000 µM for citrate. Fluorescence intensity was monitored every 15 s for 17.5 min. Using the ε-ADPR standard curve, the fluorescence was converted to [ε-ADPR]. The velocity of the reactions was calculated from the slope of the progress curve at each ε-NAD concentration and plotted in GraphPad Prism. Kinetic parameters were determined by fitting these velocities to the Michaelis-Menten equation (Eq. 1) for each mutant.

To evaluate the precipitation capacity of TIR mutants, 10 µM (WT, G747P, E788Q, and H833A) or 3 µM (WT, E788A) of the enzyme was mixed with Assay Buffer (50 mM Tris, pH 8.0; 150 NaCl) with and without 25% PEG 3350 or 500 mM sodium citrate (final concentrations). Precipitation of the TIR^E788A mutant was evaluated at 3 µM due to low yields of the protein; WT TIR at 3 µM was included as the proper control. The samples were incubated for 15 min at ambient temperature, at which point the samples were centrifuged at 21,000 x g for 10 min at 4 °C. The supernatant was removed, and the pellet was resuspended buffer with the respective additive (final concentration). Samples were diluted 1:1 with water before running on an SDS-PAGE gel. Stain-free images of the gels were obtained on the BioRad Gel Doc EZ Gel Documentation System with Image Lab Software. Representative images are shown. ImageJ was used to quantify the bands, which were plotted in GraphPad Prism.

Differences in the survival of C. elegans in the P. aeruginosa pathogenesis assays were determined with the log-rank test after survival curves were estimated for each group with the Kaplan-Meier method. OASIS two was used for these statistical analyses (Han et al., 2016). qRT-PCR studies, intestinal CFU quantification, western blot band intensity quantification, TIR NADase activity, and TIR protein precipitation are presented as the mean ± standard error of the mean. Statistical hypothesis testing was performed with Prism 9 (GraphPad Software) using methods indicated in the figure legends. Sample sizes, mean lifespan, and p-values for all trials are shown in Supplementary file 4.