LIPL-1 and LIPL-2 are TCER-1-regulated lysosomal lipases with distinct roles in immunity and fertility

Abstract

Reproduction and immunity are energy intensive processes that often compete for resources, leading to trade-offs across species. Lipid metabolism integrates these processes, particularly during stressful conditions such as pathogenic infections, yet the underlying molecular mechanisms remain poorly understood. TCER-1, the C. elegans homolog of mammalian TCERG1, suppresses immunity and promotes fertility, especially upon maternal infection. Here, we show that TCER-1 coordinates this balance by regulating two conserved lysosomal lipases, lipl-1 and lipl-2. Using transcriptomic, lipidomic, and molecular-genetic analyses, we demonstrate that both lipases mediate infection-induced lipid remodeling but with distinct outcomes: lipl-1 promotes immunity, whereas, lipl-2 does not. LIPL-1 catalyzes the accumulation of specific ceramide species, including Cer 17:1;O2/24:0, whose supplementation rescues the immunity phenotypes of tcer-1;lipl-1 mutants and enhances post-infection survival of wild-type animals. Both lipases influence fertility with lipl-2 playing a key role in maintaining embryonic-eggshell integrity during maternal infection and aging. Remarkably, expression of human lysosomal acid lipase (hLAL/LIPA), the ortholog of 'lipl' genes, restores immunity defects triggered by lipl-1 loss and enhances immune resilience but does not significantly ameliorate the fertility defects. Together, these findings reveal distinct roles for lipl-1 and lipl-2 in modulating lipid species that link immune defense, reproductive fitness and healthspan through a potentially conserved mechanism.

Fig 1. TCER-1 regulates lipid metabolism upon PA14 infection.

A) RNAseq experimental paradigm. Age-matched wild type (WT) and tcer-1 L4s raised on OP50 exposed to PA14 for 8 hours. B) Overlaps of groups of differentially expressed genes (DEGs) identified. C-F) Overlap of genes upregulated (C, D) and downregulated (E, F) upon PA14 exposure with previously-identified PA14-responsive genes by Troemel et al. 2006 [27]. RF: Representation Factor. Statistical significance of overlap between gene sets calculated using hypergeometric probability formula with normal approximation (see Methods). Comparisons with additional studies in S2 Fig and S2 Table. G) Gene ontology (GO) term analysis of PA14- and TCER-1- driven DEGs using WormCat. Metabolism (Category 1), particularly lipid metabolism, (Category 2) (blue boxes) identified as differentially impacted processes amongst genes upregulated (UP, left) and downregulated (DOWN, right) in infected WT and tcer-1 mutants. Key shown in middle (black box). H-L) lipl-1 and lipl-2 are transcriptionally upregulated in tcer-1 mutants on PA14 infection and lipl-3, lipl-4 and lipl-5 are downregulated. mRNA levels of lipl-1 (H), lipl-2 (I) lipl-3 (J), lipl-4 (K) and lipl-5 (L) measured by qPCR in WT and tcer-1 mutant (blue) adults maintained on OP50 (solid bars) or exposed as L4s to PA14 for 8 h (hashed bars). Data from at least 3 independent trials/biological replicates. Asterisks represent statistical significance of differences observed in unpaired, two-tailed t-tests with P values * ≤ 0.05, *** p ≤ 0.001 and ****p ≤ 0.0001.

Fig 2. tcer-1 inactivation and pathogen exposure induce lipl-1 and lipl-2 transcriptional upregulation. A-F: lipl-1 transcriptional changes visualized in vivo using lipl-1p::mCherry.

Animals raised on bacteria expressing control empty vector (Ctrl, A, C) or tcer-1 dsRNA (RNAi, B, D) until pre-adult, L4 larval stage and transferred to plates seeded with P. aeruginosa PA14 (PA14, C, D) or E. coli OP50 (OP50, A, B) and incubated for 8h at 25 °C. Expression visible in intestine and head; in a WT adult population, varied from low levels to high or very high fluorescence intensities in intestine and head, respectively (categorization and quantification detailed in S3 Fig). Fraction of population with high or very high expression quantified in intestine (E) or head (F). E: Control (Ctrl) RNAi, OP50 (n = 81, 3.7%), tcer-1 RNAi, OP50 (n = 83, 21.69%), Ctrl RNAi, PA14 (n = 69, 23.19%), tcer-1 RNAi, PA14 (n = 68, 58.82%). F: Ctrl RNAi, OP50 (n = 81, 10.52%), tcer-1 RNAi, OP50 (n = 83, 7.895%), Control RNAi, PA14 (n = 69, 7.895%), tcer-1 RNAi, PA14 (n = 67, 23.68%). G-K: lipl-2 transcriptional changes visualized in vivo using lipl-2p::mCherry. lipl-2 expression levels observed using the transcriptional reporter lipl-2p::mCherry. Animals were raised as described above. Expression visible predominantly in the intestine and varied from low to high fluorescence intensities (detailed in S5 Fig). K: Fraction of populations showing high intestinal expression. Ctrl RNAi, OP50 (n = 86, 1.16%), tcer-1 RNAi, OP50 (n = 83, 7.22%), Ctrl RNAi, PA14 (n = 81, 1.23%), tcer-1 RNAi, PA14 (n = 72, 12.5%). A-D and G-J show representative images pseudocolored with ImageJ LUT Fire. Comparisons performed using two-tailed Fisher’s exact test on contingency tables of data from 3 pooled biological replicates in both cases. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001.

Fig 3. tcer-1 mutants’ enhanced immunity is dependent upon lipl-1 but not lipl-2. A, B: Impact of lipl-1 deletion on survival upon pathogen infection and lifespan.

Survival of wild type (WT, black), tcer-1 (blue), tcer-1;lipl-1 (dark green) and lipl-1 (light green) raised on OP50 till L4 stage and exposed to PA14 (A) or retained on OP50 (B). A: WT (m = 51.13 ± 1.32, n = 86/113), tcer-1 (m = 70.61 ± 2.09, n = 87/112), lipl-1 (m = 52.17 ± 1.1, n = 102/134), tcer-1;lipl-1 (m = 56.04 ± 1.23, n = 85/120). B: WT (m = 14.96 ± 0.34, n = 94/121), tcer-1 (m = 14.23 ± 0.48, n = 92/120), lipl-1 (m = 15.79 ± 0.57, n = 80/106), tcer-1;lipl-1 (m = 15.36 ± 0.51, n = 77/120). C, D: Impact of lipl-2 deletion on survival upon pathogen infection and lifespan. Survival of wild type (WT, black), tcer-1 (blue), tcer-1;lipl-2 (grape) and lipl-2 (magenta) raised on OP50 till L4 stage and exposed to PA14 (C) or retained on OP50 (D). C: WT (m = 51.13 ± 1.32, n = 86/113), tcer-1 (m = 70.61 ± 2.09, n = 87/112), lipl-2 (m = 70.64 ± 1.5, n = 61/123), tcer-1; lipl-2 (m = 78.23 ± 2.66, n = 66/124). D) Lifespan on OP50. WT (m = 14.96 ± 0.34, n = 94/121), tcer-1 (m = 14.23 ± 0.48, n = 92/120), lipl-2 (m = 15.93 ± 0.56, n = 89/113), tcer-1;lipl-2 (m = 16.72 ± 0.58, n = 81/103). Data from additional PA14 survival and lifespan trials in S3 and S4 Tables, respectively. Statistical significance was determined using log-rank Mantel Cox method and shown in each panel next to a given strain/condition. Asterisks are color coded to indicate the strain/condition being used for the comparison. p ≤ 0.05(*), p < 0.01 (**), < 0.001 (***), < 0.0001 (****).

Fig 4. lipl-1 and lipl-2 lof result in decreased brood size, sterility, and embryonic inviability, particularly in combination with tcer-1 lof.

A-C: Comparisons of maternal reproductive success in wild type (WT, black), lipl-1 (light green), lipl-2 (pink), lipl-2 lipl-1 (light brown), tcer-1 (blue), tcer-1;lipl-1 (dark green), tcer-1;lipl-2 (grape) and tcer-1;lipl-2 lipl-1 (dark brown) strains. A: Brood size: Total number of live progeny counted per worm per strain. Each dot represents one worm from an aggregate of 3 independent trials. WT (n = 28, m = 330.9 ± 59.19), lipl-1 (n = 29, m = 275.8 ± 51.97), lipl-2 (n = 23, m = 288.2 ± 39.97), lipl-2 lipl-1 (n = 24, m = 316.3 ± 33.23), tcer-1 (n = 25, m = 236.2 ± 97.14) tcer-1;lipl-1 (n = 23, m = 68.09 ± 68.12), tcer-1;lipl-2 (n = 36, m = 99.31 ± 116.1), tcer-1;lipl-2 lipl-1 (n = 20, m = 32.80 ± 44.87). B: Sterility: Percentage of animals which never produced a live progeny counted for each strain. WT (n = 28, m = 0), lipl-1 (n = 29, m = 0), lipl-2 (n = 23, m = 0), lipl-2 lipl-1 (n = 24, m = 0), tcer-1 (n = 25, m = 0), tcer-1;lipl-1 (n = 23, m = 27.48 ± 8.82), tcer-1;lipl-2 (n = 36, m = 48.87 ± 27.60), tcer-1;lipl-2 lipl-1 (n = 20, m = 30.67 ± 9.81). C: Hatching: calculated from [#progeny/(#progeny+ unhatched eggs)]. WT (m = 99.01 ± 0.29), lipl-1 (m = 97.09 ± 1.24), lipl-2 (m = 98.81 ± 0.65), lipl-2 lipl-1 (m = 99.47 ± 0.15), tcer-1 (m = 97.75 ± 1.36), tcer-1;lipl-1 (m = 59.95 ± 18.07), tcer-1;lipl-2 (m = 81.75 ± 7.78), tcer-1;lipl-2 lipl-1 (m = 74.79 ± 3.24). Data obtained from 3 independent trials in all cases. D-F: Embryonic eggshell defects induced by tcer-1, lipl-1 and lipl-2 inactivation. Representative images of embryos expressing cpg-2p::mCherry::CPG-2 and pie-1p::GFP::PH PLC1delta1, labeling eggshell chondroitin proteoglycan layer and embryonic plasma membrane, respectively, incubated with lipid-labeling dye, BODIPY. D: BODIPY is excluded in healthy embryo with intact lipid-permeability barrier (LBP) and CPG-2 is sequestered away from the embryo, in a characteristic ‘wavy’ pattern in the perivitelline space of the eggshell. E: Embryo with defective LPB exhibits widespread BODIPY staining, and mCherry::CPG-2 which freely diffuses between the outer eggshell and the embryo. F: Quantification of BODIPY-permeable embryos. Average fold-change of the fraction of BODIPY stained embryos laid by Day 4 mothers, normalized to tcer-1 mutants. tcer-1 (n = 1964, m = 0), WT (n = 1930, m = 0), lipl-1 (n = 2811, m = 0.7153 ± 0.7889), tcer-1;lipl-2 (n = 1940, m = 6.222 ± 3.030), tcer-1;lipl-1 (n = 2257, m = 1.563 ± 1.587), lipl-2 (n = 2546, m = 2.244 ± 1.980). Data obtained from 4 independent trials in all cases except lipl-2 for which 3 trials were conducted. In A-C, statistical significance was calculated using one-way ANOVA with Tukey’s correction. In F, student’s t-test was used. p ≤ 0.05(*), p < 0.01 (**), < 0.001 (***), < 0.0001 (****).

Fig 5. lipl-1 and lipl-2 have both shared and distinct impacts on the lipidome of tcer-1 mutants.

A-C: Effects of lipl-1 (A) and lipl-2 (B) loss on broad neutral lipid (NL) and phospholipid (PL) populations in wild type (WT, black) and tcer-1 mutant (blue) animals. Bars indicate the fold-change of each data point normalized to the WT average. Statistical significance calculated using mixed-effects analysis with Tukey’s correction. C: Relative abundance of overall phosphatidylcholine (PC) levels. D: Categorization of NL (top, blue) and PL (bottom, green) species whose abundance is altered in tcer-1 mutants. For each class, number of species identified and those changed in tcer-1 mutants shown in the table. E: Relative abundance of PC 34:2 and PC 34:3 compared to all PCs in different strains. F-I: Distinct impacts of lipl-1 (F-H) and lipl-2 (I) on lipidome of tcer-1 mutants. F-H: Relative abundance of Cer 17:1;O2/22:0 (Cer 22) (F), Cer 17:1;O2/24:0 (Cer 24) (G) and Cer 17:1;O2/26:0 (Cer 26) (H) compared to all ceramides. I: Relative abundance of lysophosphatidyl choline (LPC) 18:2 compared to all LPCs. Data derived from HPLC-MS/MS analysis of Day 1 young adult hermaphrodites isolated in 5–6 independent biological trials. Statistical significance was calculated using two-way ANOVA with Tukey’s correction. J-K: Ceramide supplementation enhances survival upon PA14 infection. Survival of wild type Ctrl (WT, black), lipl-1;tcer-1 ctrl (green), lipl-1;tcer-1 + Cer 24 (Olive), WT + Cer 24 (Red) raised on OP50 till Day1 and exposed to PA14. J: WT ctrl (m = 63.09 ± 1.53, n = 74/90), lipl-1;tcer-1 ctrl (m = 58.1 ± 1.33, n = 82/90), lipl-1;tcer-1 + Cer 24 (m = 73.74 ± 2.0, n = 71/90). K: WT ctrl (m = 63.09 ± 1.53, n = 74/90), WT + Cer 24 (m = 70.57 ± 1.78, n = 58/90). Data from additional PA14 survival trials in S7 Table. Statistical significance was determined using log-rank Mantel Cox method and shown in each panel next to a given strain/condition. Asterisks indicate the statistical significance and their color the strain being used for the comparison. p ≤ 0.05(*), p < 0.01 (**), < 0.001 (***), < 0.0001 (****), n.s: not significant.

Fig 6. Human Lysosomal Acid Lipase (hLAL/LIPA) rescues immune deficits induced by lipl-1 mutation and enhances survival upon PA14 infection.

A: hLAL/LIPA rescues survival of tcer-1;lipl-1 mutants on PA14 exposure to tcer-1 level. Survival upon PA14 exposure from late-L4 stage onwards compared between wild type WT (black, m = 35.06 ± 1.91, n = 54/90), tcer-1 (blue, m = 54.8 ± 3.25, n = 63/90), tcer-1;lipl-1 (green, m = 36.65 ± 1.79, n = 52/90), tcer-1;lipl-1;hLAL (red, m = 55.77 ± 2.53, n = 73/90). B: hLAL/LIPA expression in C. elegans enhances survival upon PA14 infection. Survival upon PA14 exposure from late-L4 stage onwards compared between WT (black, m = 57.24 ± 2.01, n = 35/110) and two transgenic strains expressing hLAL broadly in somatic tissues. COP2983 (orange, m = 83.18 ± 2.22, n = 46/96) and COP2589 (red, m = 82.84 ± 2.22, n = 63/103). Data from additional trials in S8 Table. C-E: hLAL/LIPA does not rescue the reproductive defects of tcer-1;lipl-1 and tcer-1;lipl-2 mutants. Comparisons of reproductive success in wild type (WT, black), tcer-1;lipl-1 (green), tcer-1;lipl-2 (purple), tcer-1;lipl-1;hLAL (red) and tcer-1;lipl-2;hLAL (maroon) strains. C, D: Brood size: Total number of live progeny counted per worm per strain. Each dot represents one worm from an aggregate for two independent trials. C: WT (n = 15, m = 275.7 ± 16.37), tcer-1 (n = 15, m = 64.20 ± 18.40), tcer-1;lipl-1 (n = 15, m = 122.4 ± 6.80), tcer-1;lipl-1;hLAL (n = 15, m = 91.53 ± 11.99). D: WT (n = 26, m = 281.5 ± 26.43), tcer-1 (n = 25, m = 323.6 ± 21.83), tcer-1;lipl-2 (n = 23, m = 161.9 ± 23.66), tcer-1;lipl-2;hLAL (n = 26, m = 248.3 ± 21.53). E: Quantification of Embryonic egg-shell defects. Average-fold change of the fraction of BODIPY stained embryos laid by Day 4 mothers, normalized to tcer-1 mutants. WT (n = 126, m = 0.3687 ± 0.1515), tcer-1 (n = 30, m = 1.0 ± 0.1389), tcer-1;lipl-2 (n = 45, m = 1.741 ± 0.6080), tcer-1;lipl-2;hLAL (n = 36, m = 1.459 ± 0.2039). In A-B, statistical significance was calculated using the log-rank Mantel Cox method and is shown on each panel next to a given strain/condition with the color of the asterisk indicating strain being used for comparison. In C-D, statistical significance was calculated using one-way ANOVA with Tukey’s correction. In E, Student’s t-test was used. p ≤ 0.05(*), p < 0.01 (**), < 0.001 (***), < 0.0001(****), n.s: not significant.

Fig 7. Schematic depicting the regulation of lysosomal lipases, lipl-1 and lipl-2, by TCER-1 and their distinct roles in immunity and fertility identified in this study.

TCER-1 represses the expression of lipl-1 upon infection, which in turn modulates levels of the specific fatty-acid species, such as the Ceramide 24 (Cer 24) that promote immune resilience. lipl-1 also acts with TCER-1 to promote fertility (left panel). In contrast, the relationship between TCER-1 and lipl-2 appears more complex. Although TCER-1 represses lipl-2 induction upon infection, both genes repress immunity and promote fertility, suggesting that TCER-1 may act through additional pathways to coordinate these processes (right panel). Arrows denote activation, and blunt-ended lines denote repression. Figure created using Biorender.