The epidermis relies on collagen-rich extracellular matrices (ECMs) to maintain barrier integrity against pathogens. Lysosomes regulate cuticle collagen turnover, yet how neuronal signaling modulates epidermal lysosomal function and collagen organization during infection remains unclear. Using Pseudomonas aeruginosa PA14-Caenorhabditis elegans infection model, we demonstrate that pathogen-induced neuronal signaling disrupts epidermal lysosomal activity and collagen remodeling. PA14 infection triggers neurons to secrete NSIF-1 (Neuronal Secreted Immune Factor 1), which translocates to the epidermis and impairs lysosomal acidification, maturation, and degradation by suppressing the transcription factor ELT-3. This disruption leads to disorganized collagen structure, compromising cuticle integrity and host resistance. Genetic mutation of nsif-1 restores lysosomal function, enhances collagen density, and improves survival, while neuron-specific nsif-1 knockdown confirms its neuronal origin. Moreover, NSIF-1 inhibits ELT-3 nuclear localization, blocking its role in lysosomal-dependent ECM repair. Our study reveals a neuro-epidermal axis wherein pathogens exploit neuronal signals to disrupt lysosomal function and collagen homeostasis, identifying NSIF-1 and ELT-3 as potential targets to counteract infection-driven ECM dysregulation.

PA14 compromises nematode cuticle collagen and triggers epidermal immunity

The nematode cuticle functions similarly to human skin as a physical barrier and first line of immune defense. This protective structure, composed primarily of collagen proteins secreted by epidermal cells, forms a complex ECM [27,28]. We investigated whether PA14 directly damages cuticle collagen and thereby triggers an immune response in the epidermis.

We first validated the infection protocol by initiating infections at the L4 stage (48 h post-L1), thereby bypassing the earlier larval molts (L1-L3). L4-stage synchronization was achieved using vulval substaging criteria [29] (S1A Fig). Following 24 h of exposure to PA14, we evaluated multiple developmental parameters, including body length and egg morphology. No significant differences were observed between PA14-exposed and control animals for either parameter (S1B, S1C Fig), confirming that the 24-h PA14 exposure protocol does not cause detectable developmental delay.

DPY-7 collagen, secreted by epidermal cells and deposited into the cuticle's annular furrows [30], serves as a reliable marker for cuticle morphology when tagged with GFP (DPY-7::sfGFP) [18]. To determine whether PA14 affects the intact cuticle or specifically targets molting phases, we performed a time-course experiment. Cuticle morphology was visualized using DPY-7::sfGFP, and molting status was assessed at five time points: L4 + 0 h, L4 + 3 h, L4 + 6 h, L4 + 12 h, and L4 + 24 h (S2A Fig). Molting was identified by the presence of a mouth plug—a distinctive cap-like structure formed by the loosening of the old cuticle, which temporarily halts feeding [31]. At early intervals (L4 + 3 h), both OP50-fed and PA14-infected animals exhibited mouth plugs, indicating active molting. During these periods, cuticle damage characterized by fragmentation and disorganized DPY-7 patterning was observed in both conditions, reflecting normal cuticle remodeling during molting. At 12 h post-PA14 exposure, cuticle abnormalities began to appear, while animals were not in molting phases (no mouth plugs observed). By 24 h infection, severe cuticle damage (fragmentation and loss of DPY-7 patterning) was evident, again occurring outside molting windows (Figs 1A and S2B–S2E). These observations indicate that PA14 directly compromises the structural integrity of DPY-7 in the cuticle. The phenotype emerging at 24 h post-L4 suggests an active pathogenic mechanism that disrupts cuticle homeostasis independent of developmental remodeling.

To rule out the possibility that the observed effects reflect a time‑dependent impact on cuticle biosynthesis rather than direct damage, we performed an additional experiment using young adults (L4 + 24 h, well beyond the L4‑to‑adult molt) expressing DPY‑7::sfGFP. These animals were transferred to either OP50 (control) or PA14 lawns and imaged at 0, 3, 6, 12, and 24 h post‑transfer (S3A Fig). PA14-exposed adults exhibited a significant increase in animals with disorganized DPY-7 pattern as early as 3 h post-transfer, and this proportion increased progressively at 6, 12, and 24 h (S3B–S3E Fig). Since the animals were already post‑molt young adults at the time of transfer, these results rule out any artifacts related to molting or cuticle assembly and clearly demonstrate that PA14 directly disrupts cuticle homeostasis.

The cuticle of C. elegans is a complex extracellular matrix composed of distinct layers, each characterized by specific structural proteins that collectively maintain barrier function and mechanical stability [32]. To determine whether P. aeruginosa PA14-induced cuticle damage is specific to DPY-7 or broadly affects cuticular architecture, we examined markers representing multiple layers. Using translational fusions for CUT-2 (cortical layer) [33], BLI-1 (struts) [34], and EPIC-1 (epicuticle) [35], we assessed cuticle integrity following 24-h PA14 infection from L4 stage. In control animals fed E. coli, all markers displayed organized patterns consistent with their established localizations. As a positive control, RNAi knockdown of dpy-7 and bus-8—genes essential for maintaining the cuticle barrier [36]—resulted in abnormal patterning and mislocalization of all markers examined (Fig 2B). After 24 h of PA14 infection, we observed disrupted patterning and mislocalization of all markers tested (Fig 2B). These results indicate that the pathogenic effect extends beyond DPY‑7 and broadly impairs multiple cuticular layers.

To directly assess cuticle barrier integrity, we performed a quantitative cuticle permeability assay using Hoechst 33342 dye penetration [37]. Consistent with published data [36], RNAi knockdown of bus-8 and dpy-7 resulted in a significant increase in Hoechst 33342 fluorescence intensity in the head and tail regions of the animals (S4A Fig). Our findings show that 24-h PA14 infection significantly increases Hoechst 33342 fluorescence intensity in the head and tail regions of wild-type animals compared with uninfected controls (S4A Fig). This enhanced dye penetration provides direct physiological evidence that PA14 infection compromises the structural integrity of the cuticle barrier.

Since damage to the nematode epidermis is known to induce an immune response—specifically through the upregulation of the antimicrobial peptide NLP-29 [38,39], we hypothesized that PA14-induced cuticle damage might similarly trigger epidermal immunity. To test this, we monitored the expression of the epidermal immunity reporter P_nlp-29::GFP and found a significant increase in fluorescence following PA14 infection (Fig 1C). This finding was further corroborated by elevated expression levels of several antimicrobial peptide (AMP) genes in the epidermis, including nlp-27, nlp-28, nlp-29, and cnc-4 (Fig 1D).

To further explore the relationship between cuticle integrity and epidermal immunity, we disrupted cuticle synthesis and structural organization using RNA interference (RNAi) against several key genes dpy-7 [36], sqt-3 [40], col-142 [41], dyn-1, and syx-5 [18,42]. The results demonstrated that compromising cuticle integrity via gene knockdown led to an upregulation of epidermal immunity (S4B Fig). Notably, knockdown of dpy-7—a major furrow collagen [36], and dyn-1—a gene essential for the endocytosis [43]—resulted in the most significant immune activation (S4B Fig). Furthermore, we found that directly knocking down genes related to cuticle synthesis and assembly also reduced the survival rate of nematodes when exposed to PA14 (Fig 1E). This highlights the critical role of cuticle integrity in protecting nematodes against PA14 infection.

In summary, our findings extend the established paradigm of cuticle-immune crosstalk in nematodes [44–46] by demonstrating that pathogens like P. aeruginosa PA14 infection damages the structural integrity of the nematode cuticle, which in turn triggers an upregulation of epidermal immunity (Fig 1F).

PA14 infection disrupts cuticle structure by impairing epidermal lysosomal function

A previous study revealed that enhanced epidermal lysosomal activity is crucial for cuticle collagen turnover during the molting process in C. elegans [18]. Certain pathogens evade host defenses by secreting proteins that inhibit V-ATPase, thereby disrupting lysosomal acidification, fusion, and overall function [47–50]. Given that PA14 infection causes aberrant DPY-7 localization (Fig 1A), we hypothesized that PA14 impairs lysosomal function, which in turn damages the cuticle. To test this hypothesis, we examined several lysosomal properties—morphology, acidification, and degradation activity—in animals infected with PA14.

Firstly, we evaluated lysosomal morphology using an established reporter-qxIs383(P_hyp-7::NUC-1::CHERRY [18,51], after 24 h of PA14 infection. We observed a significant increase in tubular lysosomal structures under PA14 treatment (Fig 2A, 2B). We also observed a decrease in the number of vesicular lysosomes (Fig 2C), an increase in the length of tubular lysosomes (Fig 2D), and an enlargement in lysosomal diameter (Fig 2E) under PA14 infection.

Secondly, to assess lysosomal acidification, we employed an established transgenic reporter, NUC-1::pHTomato [51,52], in which the pH-sensitive fluorescent protein pHTomato (with a pKa near 7.8) is fused to NUC-1 under the control of a heat-shock promoter. pHTomato exhibits increased fluorescence at higher pH. We found that animals infected with PA14 displayed significantly higher NUC-1::pHTomato fluorescence compared to those treated with OP50 (Fig 2F, 2G), indicating that PA14 infection leads to an increase in lysosomal pH, thereby disrupting acidification—a critical condition for the activity of lysosomal hydrolases.

Thirdly, we monitored lysosomal maturation using the transgenic reporter- qxIs612(P_hsNUC-1::sfGFP::CHERRY) [18,51]. In this system, a heat shock at 34 °C for 30 min induces the expression of the NUC-1::sfGFP::CHERRY fusion protein, which is then trafficked to lysosomes via the endocytic pathway. GFP fluorescence was quenched in the acidic lysosomal environment, while CHERRY fluorescence remained stable, allowing us to assess lysosomal acidification and maturation by the loss of GFP signal (Fig 2H). In PA14-infected animals, GFP fluorescence was not quenched and colocalized strongly with CHERRY (Fig 2I, 2J), indicating impaired lysosomal maturation and acidification.

Finally, we evaluated lysosomal degradation activity using the transgenic reporter-qxIs383(P_hyp-7NUC-1::CHERRY) [18], which expresses the NUC-1::CHERRY fusion protein in the epidermis. Upon delivery to lysosomes, CHERRY is cleaved by cathepsins, and the cleaved product can be quantified by western blot (Fig 2K). We observed a reduction in cleaved CHERRY levels following PA14 infection (Fig 2L, 2M), demonstrating a decline in lysosomal degradation activity.

To investigate the role of specific bacterial virulence factors in modulating lysosomal morphology and cuticle collagen organization, we compared the effects of wild-type P. aeruginosa-PA14 with those of attenuated mutant strains lacking key virulence regulators (ΔgacA, ΔlasR, ΔmvfR, ΔpqsF) [53]. In contrast to wild-type PA14, these mutants significantly attenuated the PA14-induced lysosomal alterations and cuticle disruption (S5A, S5B Fig), indicating that functional virulence expression is required for PA14-dependent dysregulation of both lysosomal architecture and cuticle integrity.

In summary, our findings reveal that PA14 infection drives lysosomes into an inactive state characterized by altered morphology, reduced acidification, impaired maturation, and diminished degradation activity. This impairment of lysosomal function likely contributes to the disruption of cuticle collagen, thereby compromising the nematode's physical barrier and triggering epidermal immunity.

NSIF-1, expressed in neuron, is a signaling factor that responds to PA14 infection and modulates lysosomal and cuticle integrity

C. elegans uses various sensory neurons to detect pathogenic bacteria, triggering both avoidance behaviors and immune responses [54]. Neuronal signaling also influences peripheral mitochondrial homeostasis, affecting aging, lipid metabolism, and immunity [55–58]. However, whether neuronal signals can regulate lysosomal functions in peripheral tissues remains unclear.

Given that PA14 infection disrupts epidermal lysosomal function (Fig 2A) and collagen integrity (Fig 1A), an important question arises: Does the nervous system relay signals to epidermal lysosomes, thereby exacerbating cuticle damage during infection? Since secreted proteins are crucial mediators of intercellular communication—as hormones, cytokines, or ligands binding to receptors [59], we hypothesized that PA14 infection might induce the secretion of specific proteins that impair lysosomal function (Fig 3A).

To identify these factors, we collected supernatants from worms treated with PA14 and various control conditions (M9 buffer + worms; OP50 + worms; OP50 only; PA14 only). Silver staining revealed distinct protein bands in PA14-treated samples (Fig 3B), suggesting that infection stimulates the secretion of certain proteins. Mass spectrometry identified 35 candidate proteins induced by PA14 (S6A Fig, S1 Table). By applying two criteria—(1) the presence of signal peptides (as predicted by SignalP-5.0) [60] and (2) neuronal expression based on the C. elegans Neuronal Gene Expression Map & Network (CeNGEN https://www.cengen.org/) database [61], we narrowed this list to 5 potential neuronal secreted proteins (S6A Fig).

We then investigated whether any of these PA14-induced proteins impair lysosomal function, consequently affecting collagen organization (Fig 3A). If PA14-stimulated neuronal proteins disrupt lysosomal activity, then knocking them down should restore lysosomal function. Using RNAi to reduce the expression of rla-5, dao-2, nlp-77, tag-293, and lec-5, we assessed lysosomal acidification with NUC-1::pHTomato. Remarkably, knockdown of tag-293 significantly enhanced lysosomal acidification under PA14 infection (S6B Fig). Consistently, the tag-293(ok1337) deletion mutant exhibited elevated lysosomal acidification (Fig 3C). Furthermore, analysis of supernatants from worms expressing TAG-293::Flag::GFP showed that PA14 infection indeed promotes the secretion of TAG-293 (S6C Fig). These data imply that PA14-induced TAG-293 secretion may impair lysosomal function. Given that TAG-293 is a neuron-derived secreted factor that responds to pathogen infection and likely plays a role in immune regulation, we have renamed TAG-293 as NSIF-1 (Neuronal Secreted Immune Factor-1).

Next, we assessed whether the enhanced lysosomal activity in nsif-1 mutants impacts cuticle collagen integrity. We generated a second null-like allele, nsif-1(ylf82), using CRISPR/Cas9 to introduce a stop codon in the second exon (S7A Fig). Under normal OP50 feeding conditions, two independent nsif-1 mutant alleles (ok1337, ylf82) displayed a higher density of cuticular structural units compared to wild-type worms (Figs 3D and S7B–S7D). Moreover, during PA14 infection, the disrupted collagen organization observed in wild-type worms was significantly alleviated in nsif-1 mutants (Figs 3E and S7B–S7D).

Survival assays further confirmed that nsif-1(ok1337) mutants exhibit enhanced resistance to PA14 infection relative to wild-type animals (Figs 3F and S8). In contrast, overexpression of NSIF-1 under its native promoter in wild-type animals did not result in increased susceptibility to PA14 (S8 Fig), indicating that only pathogen-induced NSIF-1 expression contributes to infection progression. This supports the idea that PA14-induced NSIF-1 contributes to cuticle damage.

To determine the expression and localization of NSIF-1, we generated transcriptional (P_nsif-1::GFP) and translational (P_nsif-1NSIF-1::GFP) reporters. The transcriptional reporter confirmed that nsif-1 is exclusively expressed in neurons (Fig 3G), whereas the translational reporter revealed that the NSIF-1 protein is distributed across various tissues, including head and body neurons, muscle, and intestine (S9A Fig). Using a CRISPR knock-in strain (syb9508) expressing NSIF-1::mNeonGreen::3xFLAG from the endogenous locus, we further confirmed that NSIF-1 also localizes to the epidermis (S9B Fig). Together, these findings indicate that neuronally expressed NSIF-1 may be secreted and act on peripheral tissues, likely modulating epidermal lysosomal function and collagen organization (Fig 3H).

Under OP50 feeding conditions, this NISF-1 (endogenous reporter: NSIF-1::mNeonGreen::3xFLAG) exhibited low-level fluorescence. However, upon 24-h infection with wild-type P. aeruginosa-PA14, we observed a marked increase in fluorescence intensity specifically within the epidermis (S10A Fig). Furthermore, the NSIF-1::mNeonGreen signal showed clear co-localization with epidermal lysosomal markers (S10B Fig). These effects were significantly attenuated when infection was carried out with attenuated PA14 strains lacking key virulence factors (gacA, lasR, mvfR, and pqsF) (S10B Fig). These results confirm that pathogen-induced upregulation and epidermal lysosomal localization of NSIF-1 are specific responses to active virulence.

In summary, we propose that PA14 infection triggers the neuronal secretion of NSIF-1—a signaling factor that modulates epidermal lysosomal function and cuticle integrity (Fig 3H). This disruption of lysosomal activity leads to impaired collagen organization, compromising the cuticle barrier and increasing susceptibility to PA14 infection.

PA14 infection induces the secretion of NSIF-1 from neurons to the epidermis

To directly demonstrate that NSIF-1 is synthesized in neurons and secreted to epidermis, we employed a GFP-binding nanobody (GBP) capture system in epidermis. In this system, tissue-specific expression of GBP::mKate2 enables detection of trace amounts of secreted GFP-tagged proteins originating from other tissue [62,63]. We generated transgenic C. elegans strains expressing GBP::mKate2 in the epidermis (P_col-12GBP::mKate2) in a background where NSIF-1::GFP is expressed pan-neuronally under the Prgef-1 promoter (P_rgef-1NSIF-1::GFP). If NSIF-1 is secreted from neurons and reaches the epidermis, the epidermal GBP should capture NSIF-1::GFP, resulting in detectable green fluorescence (Fig 4A).

Our results confirmed that GBP::mKate2 expressed in the epidermis successfully captured neuron-derived NSIF-1, producing clear green fluorescence signals in these tissues (Fig 4B). In contrast, when the signal peptide (SP) of NSIF-1 was removed, no green fluorescence was detected in epidermis, despite the presence of GBP::mKate2 (Fig 4B). These results demonstrate that NSIF-1 is indeed secreted from neurons and taken up by the epidermis. Furthermore, upon PA14 infection, we observed a marked increase in NSIF-1 signal specifically in the epidermis (Fig 4C), indicating that infection induces enhanced secretion of NSIF-1 from neurons to epidermis. These effects were suppressed when worms were infected with attenuated PA14 strains-lacking key virulence regulators (gacA, lasR, mvfR, and pqsF) (S11 Fig), indicating that NSIF-1 secretion is specifically dependent on PA14 virulence factors.

To determine whether NSIF-1 secretion is a specific response to PA14 virulence rather than a general response to cuticle damage, we disrupted cuticle integrity using RNAi against collagen and extracellular matrix genes (col-142, dpy-7, dyn-1, sqt-3, and syx-5). None of these RNAi induced NSIF-1 secretion into epidermis (S12 Fig), confirming that NSIF-1 is specifically upregulated in response to PA14 virulence factors rather than general barrier damage.

We further investigated whether NSIF-1 secretion depends on canonical neuropeptide processing pathways by testing in animals with RNAi of unc-31(regulating dense-core vesicle secretion) [64] and egl-3 (proprotein convertase) [65]. NSIF-1 secretion remained unchanged in these RNAi animals (S13 Fig), indicating that its release occurs through a noncanonical pathway independent of traditional neuropeptide processing mechanisms.

To identify which specific neurons express nsif-1 and thus secret it to the epidermis, we first examined its expression profile. Analysis using the CeNGEN database revealed that nsif-1 is predominantly expressed in ASJ neurons (S6A Fig). Subsequent co-localization studies, using transcriptional (P_nsif-1::GFP) and translational (P_nsif-1NSIF-1::GFP) reporters along with multiple neuronal markers, confirmed that nsif-1 is expressed exclusively in ASI and ASJ neurons, rather than in RIS or ADF neurons. (Figs 4D, 4E and S14).

To test ASI/ASJ (neuron)-to-hypodermis NSIF-1 transfer, we generated four transgenic lines expressing full-length or ΔSP NSIF-1::GFP under ASI-(str-3) or ASJ-specific (trx-1) promoters, in a hypodermal GBP capture background (P_col-12GBP::mKate2). Full-length yielded robust hypodermal GFP signal, whereas ΔSP yielded little to none (Fig 4F). These results directly demonstrate that NSIF-1 produced specifically in ASI or ASJ neurons is secreted and taken up by the hypodermis. Thus, ASI/ASJ-derived NSIF-1 may influence epidermal lysosomal function.

NSIF-1 enhances susceptibility to PA14 by inhibiting lysosomal function

To investigate how NSIF-1 modulates immune responses and lysosomal activity in C. elegans, we performed transcriptome analysis (RNA-seq) on wild-type N2 worms and nsif-1(ok1337) mutants. KEGG pathway enrichment analysis of the upregulated genes in nsif-1(ok1337) mutants revealed a significant enrichment of lysosomal-related genes—54 in total (S15A, S15B Fig, S2 and S3 Tables). Basic categorization showed that among these genes, 32 encode membrane proteins, 12 encode proteases, and 6 encode nonhydrolytic enzymes (S15B Fig, S3 Table). Subsequent qPCR analysis confirmed the RNA-seq results (S15C Fig), demonstrating that genes related to lysosomal proteases and nonhydrolytic enzymes were significantly upregulated. These data suggest that lysosomal function is enhanced in the nsif-1 mutant.

We next examined lysosomal morphology under normal OP50 feeding and PA14 infection conditions. In wild-type animals, PA14 infection increased the number of tubular lysosomes and decreased the number of vesicular lysosomes compared to OP50-fed controls; however, these morphological changes were restored in the nsif-1 mutant background (Fig 5A – 5C). Moreover, by measuring lysosomal degradation activity using qxIs383(P_hyp-7NUC-1::CHERRY), we found that cleaved CHERRY is increased in nsif-1(ok1337) mutant animals compared with wild-type under E. coli (Fig 5D) or PA14 (Fig 5E) condition, indicating an increased lysosomal degradation activity in nsif-1mutant animals.

Using a NUC-1::sfGFP::CHERRY marker to monitor lysosomal maturation, we observed that in nsif-1(ok1337) mutant, lysosomes matured within 6 h after heat shock, whereas wild-type lysosomes had not yet fully matured, as evidenced by a higher degree of colocalization between GFP and CHERRY (Fig 5F, 5G) under OP50 feeding condition. Moreover, we observed lysosomal maturation in NUC-1::sfGFP::CHERRY, infected them with PA14 for 12 h, subjected them to a heat shock at 34 °C for 30 min, followed by a 20 °C recovery and continued infection for an additional 12 h. The results revealed that GFP fluorescence was quenched in the nsif-1 mutant but not wild-type worms after PA14 infection (Fig 5H). Together, these data indicate that nsif-1 mutation promotes lysosomal maturation, increases acidification and degradation activity.

Loss of the C. elegans lysosomal Ca²⁺ channel CUP-5 (homologous to human TRPML1) is known to disrupt lysosomal biogenesis and transport, leading to impaired degradation of endocytic and autophagic cargo [51]. When we introduced a cup-5 mutation into the nsif-1 deletion background to disrupt lysosomal function, the enhanced resistance to PA14 infection observed in nsif-1 mutants was significantly reduced (Fig 5I). In summary, NSIF-1 acts as a negative regulator of lysosomal function, thereby increasing C. elegans susceptibility to PA14 infection.

Neuronal secreted NSIF-1 regulates epidermal lysosomes

NSIF-1 is secreted by neurons and taken up by the epidermis under PA14 infection. To test whether neuronally secreted NSIF-1 specifically regulates epidermal epidermal lysosomal and cuticular function, we expressed NSIF-1 under muscle-specific (myo-3) or intestine-specific (vha-6) promoters in nsif-1(ok1337) mutants. Neither construct reversed the protective cuticle (DPY-7::sfGFP) and lysosomal phenotypes observed in the mutant during PA14 infection, ruling out these tissues as primary targets (S16A, S16B Fig). In contrast, hypodermal expression (col-12) of either full-length NSIF-1::GFP or signal peptide-deleted (ΔSP) NSIF-1::GFP abolished these protective benefits and even disrupted lysosomes and cuticle organization under nonpathogenic (OP50) conditions (S16C, S16D Fig). Importantly, ΔSP NSIF-1 was as effective as the full-length protein when expressed locally, confirming that NSIF-1 acts within hypodermal cells. Together, these data indicate that neuronally secreted NSIF-1 functions in the hypodermis to impair lysosomal health and cuticle integrity.

To test whether ASI/ASJ-derived NSIF-1 influences epidermal lysosomal function, we generated ASI/ASJ-specific RNAi strains qxIs383;sid-1(qt9);ylfEx432(P_ASISID-1::3 × FLAG;P_ASINSIF-1::GFP) and qxIs383;sid-1(qt9);ylfEx433(P_ASJSID-1::3 × FLAG;P_ASJNSIF-1::GFP) to knock down nsif-1 exclusively in these two neurons. Comparing animals fed nsif-1 RNAi versus the HT115 empty-vector control, NSIF-1::GFP fluorescence in ASI/ASJ was markedly and significantly reduced upon nsif-1 RNAi, confirming efficient neuron-specific knockdown (Fig 6A, 6B). Under PA14 infection, worms with ASI/ASJ-specific nsif-1 knockdown maintained healthier lysosomal morphology, similar to that observed in nsif-1(ok1337) mutants (Fig 6A, 6B). Direct measurement of NUC-1::CHERRY cleavage efficiency further confirmed that lysosomal proteolytic activity was significantly higher in the ASI/ASJ-specific nsif-1 knockdown animals—both under PA14 infection and normal feeding—relative to wild-type worms (Fig 5D, 5E).

To ensure that our findings reflect the physiological role of NSIF-1, we performed comprehensive rescue experiments in the nsif-1(ok1337) mutant background. We expressed the NSIF-1 coding sequence under its native promoter, as well as under ASJ- (trx-1) or ASI-(str-3) specific promoters. Under PA14 infection, both the native and neuron-specific expression restored wild-type lysosomal morphology and rescued cuticle organization in the mutant (Fig 6C, 6D). These results support our model that neuronal secreted NSIF-1 acts noncell-autonomously to regulate lysosomal function in peripheral tissues. However, we also examined versions lacking the signal peptide (ΔSP). Unexpectedly, ΔSP NSIF‑1 expressed from ASI- or ASJ-specific promoters still rescued lysosomal morphology and cuticle organization in the mutant (S17A, S17B Fig). One plausible explanation is that the elevated level of ΔSP NSIF‑1 in ASI or ASJ may trigger other unknown signaling pathways that restore wild‑type lysosomal morphology and rescue cuticle organization in the mutant.

To further validate the neuronal origin of NSIF-1 in regulation epidermal lysosomes, we genetically ablated ASJ/ASI neurons using the cell death activator EGL-1 [66]. To confirm efficacy of EGL-1-mediated ASI/ASJ ablation, we co-expressed cell-death activator EGL-1 with ASI/ASJ-specific NSIF-1::GFP in the qxIs383 background. Compared with sibling controls lacking EGL-1, animals carrying the EGL-1 transgene showed a marked reduction in NSIF-1::GFP fluorescence in the targeted neurons, consistent with successful cell ablation (S18A Fig). Ablation of ASJ/ASI neurons significantly reduced NSIF-1 secretion (S18B Fig), as measured by GFP-binding nanobody (GBP) capture system, confirming that these neurons are the primary source of NSIF-1.

Moreover, pathogen-induced expansion of tubular lysosomes was significantly suppressed in both nsif-1(ok1337) mutants and animals subjected to ASI/ASJ neuronal ablation (S18A and S19A, S19B Figs). Furthermore, assessment of lysosomal proteolytic activity revealed consistent trends: cleaved CHERRY levels—a direct indicator of lysosomal degradation efficiency—were elevated in nsif-1(ok1337) mutants and neuron-ablated animals relative to wild-type controls under both standard (OP50) and pathogenic (PA14) conditions (Fig 6E, 6F).

In summary, our data indicate that ASI/ASJ neuronal secreted NSIF-1 acts noncell-autonomously to impair lysosomal health and cuticle integrity in the hypodermis.

NSIF-1 impairs lysosomal function via inhibiting ELT-3

We have demonstrated that PA14-induced impairment of lysosomal function requires NSIF-1, particularly its neuronal secretion, which depends on its signal peptide (Fig 4). To directly test whether NSIF-1 is sufficient to impair epidermal lysosomal function, we expressed either full-length NSIF-1 (FL NSIF-1) or a signal peptide-deleted version (ΔSP NSIF-1) under the native Pnsif-1 promoter or the epidermis-specific col-12 promoter in wild-type animals. Overexpression of either construct from the native Pnsif-1 promoter in wild-type animals produced no detectable change in hypodermal lysosomal morphology (S20A Fig) or cuticle organization (S20B Fig) compared to wild-type control under OP50 feeding condition. In contrast, when the same constructs were driven by the hypodermal col-12 promoter, both FL NSIF-1 and ΔSP NSIF-1 markedly disrupted lysosomal morphology (S20A Fig) and cuticle organization (S20B Fig), phenocopying the effects of PA14 infection (Figs 1A and 2A). These results indicate two key points: (i) overexpression of NSIF-1 under noninfection conditions from its native promoter is well tolerated, consistent with our model that it is the PA14-induced secretion of NSIF-1 that drives pathology; and (ii) forced expression of NSIF-1 directly in the hypodermis is sufficient to recapitulate the lysosomal and cuticle defects, supporting the conclusion that the hypodermis is the site of NSIF-1 action.

Using cNLS Mapper (https://nls-mapper.iab.keio.ac.jp/), we identified a putative NLS sequence within NSIF-1 with a score of 3.3 (S21 Fig). According to the cNLS Mapper scoring system established by Kosugi and colleagues [67], scores of 3–5 indicate proteins that localize to both the nucleus and the cytoplasm. Moreover, we found that ΔSP NSIF-1 localizes to the nucleus (S22A Fig). This observation led us to explore its potential interaction with ELT-3, a transcription factor known to enhance lysosomal function [18] and regulate collagen organization [8]. Consistent with this hypothesis, we observed strong nuclear co-localization between ΔSP NSIF-1 and ELT-3 (S22A Fig). Furthermore, transcription factor enrichment analysis through WormExp (https://wormexp.zoologie.uni-kiel.de/wormexp/) [68] revealed approximately 200 upregulated genes in nsif-1 mutants that are putative ELT-3 targets (S22B Fig; S3 Table). This suggests that loss of NSIF-1 may enhance ELT-3 activity, potentially driving lysosomal functional enhancement. To further test this hypothesis, we examined ELT-3 localization in nsif-1 mutant animals. We found that loss of nsif-1 resulted in significantly enhanced nuclear expression of ELT-3 (Fig 7A). Moreover, we performed the qRT-PCR on synchronized L4 + 24 h wild-type and nsif-1(ok1337) animals and find that endogenous elt-3 mRNA is significantly upregulated in nsif-1(ok1337) compared with wild type (S22C Fig). These data indicate that NSIF-1 normally suppresses ELT-3 nuclear expression.

If NSIF-1 functions through ELT-3 inhibition, then the lysosomal enhancement observed in nsif-1 mutants should depend on ELT-3 activity. To validate this hypothesis, we performed a series of genetic epistasis experiments using nsif-1;elt-3 double mutants. Firstly, we observed lysosomal morphology. The reduction in tubular lysosomes observed in nsif-1 mutants under PA14 infection was suppressed in the nsif-1;elt-3 double mutants, where tubular lysosomes, vesicular lysosomes were restored to levels comparable to elt-3 single mutants (Figs 7B and S22D). Additionally, the lysosomal diameter in nsif-1; elt-3 double mutants exhibited an enlarged lysosome diameter similar to that of elt-3 single mutants (Figs 7B and S22E). This indicates that the morphological changes caused by nsif-1 mutation are dependent on ELT-3.

Secondly, we measured the lysosomal acidification. In nsif-1 mutants, lysosomes exhibited enhanced acidification, as indicated by reduced NUC-1::pHTomato fluorescence intensity (Fig 7C). However, in nsif-1;elt-3 double mutants, lysosomal acidification was diminished, resembling the phenotype of elt-3 mutants (Fig 7C). This demonstrates that the enhanced acidification caused by nsif-1 loss is dependent on ELT-3. Thirdly, lysosomal degradation activity, measured by NUC-1::CHERRY cleavage, was elevated in nsif-1 mutants but returned to elt-3 single mutant levels in the nsif-1;elt-3 double mutants under both PA14 infection and normal feeding conditions (Figs 7D, 7E and S22F). This further supports that ELT-3 is necessary for the increased degradation activity observed in the absence of nsif-1.

Finally, we monitored lysosomal maturation following heat shock and found that lysosomes in nsif-1 mutants matured within 6 h, whereas lysosomes in nsif-1; elt-3 double mutants displayed delayed maturation, with persistent colocalization between GFP and CHERRY signals (Fig 7F, 7G). This indicates that accelerated lysosomal maturation in nsif-1 mutants also requires ELT-3.

Taken together, these findings demonstrate that NSIF-1 impairs lysosomal function by inhibiting ELT-3. Loss of nsif-1 leads to increased nuclear localization of ELT-3, which subsequently enhances lysosomal acidification, degradation capacity, and maturation. Therefore, ELT-3 functions downstream of NSIF-1 and is both necessary and sufficient for mediating the lysosomal improvements observed in nsif-1 mutants.

NSIF-1 damages the cuticle by inhibiting ELT-3

Our RNA-seq analysis comparing wild-type N2 and nsif-1(ok1337) mutants, revealed that nsif-1 mutants exhibit elevated expression of collagen-related genes (Fig 8A; S3 Table). This upregulation likely contributes to the observed densification of the cuticle structure in nsif-1(ok1337) mutants (Figs 3D and 8B).

Given that (i) ELT-3, a transcription factor, is known to activate lysosomal function and regulate collagen organization, and (ii) nsif-1 mutation enhances the nuclear localization of ELT-3, we hypothesized that the restoration of cuticle collagen organization observed in nsif-1 mutants under PA14 infection depends on ELT-3.

Under normal OP50 conditions, elt-3 mutants display fragmented and disorganized annular structures in the cuticle, and nsif-1; elt-3 double mutants exhibit similar cuticle features to elt-3 single mutants (Fig 8B, 8C). This observation indicates that ELT-3 functions downstream of NSIF-1 in maintaining proper collagen organization. During PA14 infection, the disrupted collagen organization seen in wild-type worms is significantly alleviated in nsif-1 mutants. However, in nsif-1; elt-3 double mutants, the dense furrows seen in nsif-1 mutants is lost, replaced by a fragmented furrows pattern similar to that observed in elt-3 mutants (Fig 8B, 8C). This finding demonstrates that the maintenance of cuticle collagen organization in nsif-1 mutants under PA14 infection is dependent on ELT-3. Moreover, the loss of ELT-3 renders nsif-1 mutants as susceptible to PA14 infection as elt-3 mutants (Fig 8D). Collectively, our data indicate that PA14-stimulated NSIF-1 impairs epidermal cuticle collagen by inhibiting ELT-3, thereby facilitating PA14 infection.

In summary, we identify NSIF-1 as a neurosecretory inhibitory factor released in response to PA14 infection. Upon secretion, NSIF-1 is taken up by epidermis, where NSIF-1 disrupts lysosomal function by inhibiting ELT-3 activity, thereby impairing cuticle collagen organization (Fig 8E).

Neuronal regulation of epidermal lysosomal function

Lysosomes are central to ECM remodeling and barrier maintenance, with their activity tightly regulated during development and stress responses. Previous studies in C. elegans demonstrated that lysosomal activation is essential for cuticle collagen turnover during molting [18]. Our work extends these findings by showing that pathogens like PA14 disrupt this process through neuronal signaling. NSIF-1, secreted from ASI/ASJ neurons in response to infection, inhibits epidermal lysosomal acidification, degradation, and maturation. This mechanism mirrors strategies employed by intracellular pathogens such as Mycobacterium tuberculosis and Salmonella, which impair lysosomal function to evade host defenses [47–50]. However, our study uniquely highlights neurons as initiators of epidermal lysosomal dysfunction during infection, a pathway not previously described in metazoans.

In our model, we propose that neuronal NSIF-1 is secreted and subsequently taken up by hypodermal cells. Because the signal peptide is co-translationally removed during biogenesis in the neuron, the internalized protein in the hypodermal cell is already the mature (SP-cleaved) form, yet retains its intact internal NLS. Once inside the hypodermal cell, this mature protein may escape from endosomal compartments into the cytosol and then translocate to the nucleus via NLS-mediated import. However, the precise molecular mechanism by which NSIF-1 is diverted from the secretory pathway to the nucleus remains to be determined.

The nuclear co-localization of ΔSP NSIF-1 with ELT-3 and the restoration of lysosomal defects in nsif-1;elt-3 double mutants reveal that NSIF-1 acts by antagonizing ELT-3, a transcription factor known to enhance lysosomal activity and collagen organization. This interaction aligns with reports that ELT-3 regulates stress-responsive genes in the epidermis [8,69], but our discovery of its suppression by a neuronal derived factor adds a new layer of complexity to neuro-epidermal crosstalk. Notably, the dual role of NSIF-1 in both lysosomal and collagen regulation suggests that pathogens target centralized regulatory nodes to maximize tissue damage—a strategy analogous to bacterial toxins that disrupt multiple host pathways simultaneously.

Collagen remodeling regulated by lysosomes as a host–pathogen battleground

Collagen disruption is a common feature in infections ranging from Staphylococcus aureus-induced skin lesions to Leishmania-associated cutaneous ulcers [70–73]. In C. elegans, PA14-induced cuticle damage activates epidermal immunity (Fig 1), consistent with previous findings that defects in epidermal or cuticle collagen trigger compensatory immune responses [38,74]. While earlier studies have shown that disruption of the attachments between the cuticle, epidermis, and basement membrane activates lysosomal activity in the epidermis [18], our work reveals a paradox: although PA14 damages collagen to breach the host barrier, the nematode’s own neuronal response—mediated by NSIF-1—further exacerbates this damage by suppressing lysosomal-dependent repair mechanisms.

Under normal conditions, epidermal damage activates lysosomal pathways to degrade damaged ECM components and promote the synthesis of new cuticle, thereby maintaining tissue homeostasis [18]. This process is analogous to the lysosomal activation observed during the molting period, where new collagen is synthesized. However, upon PA14 infection, the pathogen exploits host neuronal signals by inducing the secretion of NSIF-1 to suppress epidermal lysosomal activity. This suppression hinders the normal repair and remodeling of the ECM, thus facilitating pathogen invasion. Our findings suggest that PA14 hijacks host factors regulating ECM and collagen integrity as a virulence strategy, disrupting tissue repair to facilitate infection. Therefore, our findings underscore that the modulation of lysosomal function represents a critical battleground in host-pathogen interactions, with potential implications for therapeutic interventions in chronic infectious and wound-healing disorders.

Implications for host-directed therapies

Our findings suggest that targeting neuro-immune signaling could mitigate pathogen-induced tissue damage. For instance, blocking NSIF-1 secretion or enhancing ELT-3 activity might restore lysosomal function and collagen integrity during infection. This approach aligns with emerging therapies for fibrosis and chronic infections, where lysosomal reactivation (e.g., via TRPML1 agonists) shows promise [75]. Although the role of neuronal secreted NSIF-1 in disrupting epidermal lysosomal function is specific to C. elegans, the evolutionary conservation of ELT-3/GATA transcription factors across species raises the possibility that similar neuro-epidermal pathways operate in mammals, offering translational relevance.

In summary, we delineate a pathway where PA14 exploits neuronal signaling to impair lysosomal function and disrupt collagen. By linking neurosecretion, lysosomal regulation, and ECM dynamics, this work advances our understanding of host-pathogen interactions and highlights neuronal signaling as a vulnerable target for therapeutic intervention.

Limitations of the study

While our findings reveal a neuro-epidermal pathway linking neuronal signaling, lysosomal dysfunction, and collagen disorganization, several limitations should be acknowledged.

First, although we demonstrate that NSIF-1 disrupts lysosomal function via ELT-3, the precise mechanism by which NSIF-1 inhibits ELT-3 nuclear localization remains unresolved. Similarly, the intracellular itinerary of NSIF-1—specifically, how it escapes the endosomal compartment and translocates to the nucleus—is still unknown. Identifying the receptor(s) responsible for NSIF-1 uptake and the machinery governing endosomal escape will be important directions for future research. Furthermore, whether SP cleavage efficiency or alternative trafficking routes contribute to NSIF-1 nuclear localization in vivo during infection warrants further investigation.

Second, our study does not fully distinguish whether the observed lysosomal dysfunction primarily affects collagen degradation, synthesis, or recycling. Addressing these mechanistic gaps could provide deeper insights into how pathogens exploit neuro-epidermal ECM pathways and may inform potential therapeutic strategies for combating chronic infections and promoting tissue repair.

C. elegans strains and maintenance

1) The following strain/alleles was obtained from Caenorhabditis Genetics Center (CGC) and SunyBiotech (Fuzhou, China):

N2 Bristol (wild type strain);

VC798: tag-293/nsif-1(ok1337);

KU25: pmk-1(km25);

VC143: elt-3(gk121);

IG274: frIs7 [P_nlp-29GFP + P_col-12DsRed];

AU78: agIs219[P_sysm-1GFP:: unc-54 3’UTR + P_ttx-3GFP + unc54 3’UTR];

OP559: unc-119(tm4063);wgIs559[elt-3::TY1::EGFP::3×FLAG + unc-119(+)]

DV3327: pmk-1(re170)[pmk-1::mNeonGreen::3xFlag];

DR96: unc-76(e911);

SD1809: elt-3(vp1);

HZ108: cup-5(bp510);

JDW389: bli-1(wrd84)[bli-1::linker::mNeonGreen::3xFLAG(internal)::linker];

JDW461: noah-2(wrd120)[noah-2::mNeonGreen::3xFLAG];

JDW655: cut-2(wrd233)[cut-2::mNeonGreen::3xFLAG].

BHX9508: nsif-1(syb9508)[nsif-1::mNeonGreen::3xFLAG knock-in], (This strain was generated by SunyBiotech.

2) The following strain/alleles was obtained from Dr. Xiaochen Wang lab or Dr. Chonglin Yang lab:

XW5399: qxIs257[P_ced-1NUC-1::CHERRY] [76];

XW17096: qxIs383[P_hyp-7NUC-1::CHERRY] [18,51];

XW13734: qxIs612[P_hspNUC-1::sfGFP::CHERRY] [18,51];

XW18042: qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion] [18];

XW19180: qxIs750 [P_hspNUC-1::pHTomato] [18,51];

XW17147: cup-5(bp510);qxIs383[P_hyp-7NUC-1::CHERRY]

3) The following strains were constructed by this study:

YNU6: unc-76(e911);tag-293/nsif-1(ok1337);

YNU210: elt-3(gk121);nsif-1(ok1337)

YNU467: nsif-1(ok1337);qxIs383[P_hyp-7NUC-1::CHERRY];

YNU470: nsif-1(ok1337); qxIs612[P_hspNUC-1::sfGFP::CHERRY];

YNU471: nsif-1(ok1337);qxIs750 [P_hspNUC-1::pHTomato];

YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion];

YNU474: nsif-1(ok1337);wgIs559[elt-3::TY1::EGFP::3 × FLAG + unc-119(+)];

YNU512: cup-5(bp510);nsif-1(ok1337);qxIs383;

YNU529: elt-3(vp1); qxIs383[P_hyp-7NUC-1::CHERRY];

YNU504: elt-3(gk121); qxIs750 [P_hspNUC-1::pHTomato];

YNU510: elt-3(gk121); qxIs612[P_hspNUC-1::sfGFP::CHERRY]

YNU534: elt-3(gk121); qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion]

YNU569: nsif-1(ok1337);elt-3(vp1); qxIs383[P_hyp-7NUC-1::CHERRY];

YNU527: nsif-1(ok1337);elt-3(vp1); qxIs612[P_hspNUC-1::sfGFP::CHERRY];

YNU541: nsif-1(ok1337);elt-3(vp1);qxIs750 [P_hspNUC-1::pHTomato];

YNU514: nsif-1(ok1337);elt-3(vp1); qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion];

YNU18: unc-76(e911);ylfEx17[P_nsif-1::GFP; unc-76(+)];

YNU22: unc-76(e911);ylfIs1[P_nsif-1NSIF-1::GFP; unc-76(+)];

YNU134: unc-76(e911);ylfIs4[P_nsif-1NSIF-1::3 × FLAG::GFP; unc-76(+)];

YNU138: ylfIs8[P_rgef-1NSIF-1::GFP + P_odr-1RFP];

YNU270: ylfIs22[P_vha-6NSIF-1::GFP + P_odr-1RFP];

YNU275: ylfIs27[P_myo-3NSIF-1::GFP + P_odr-1RFP];

YNU278: unc-76(e911);ylfIs30[P_nsif-1ΔSPNSIF-1::GFP + unc-76(+)];

YNU468: ylfEx261[P_col-12GBP::mKate2 + ylfIs8 + rol-6(su1006)];

YNU469: ylfEx262[P_rgef-1ΔSPNSIF-1::GFP;P_col-12GBP::mKate2;rol-6(su1006)];

YNU284: ylfEx151[P_sra-6mKate2 + P_nsif-1GFP];

YNU285: ylfEx152[P_trx-1mKate2 + P_nsif-1GFP];

YNU286: ylfEx153[P_tph-1mKate2 + P_nsif-1GFP];

YNU287: ylfEx154[P_ggr-2mKate2 + P_nsif-1GFP];

YNU554: ylfEx281[qxIs257; P_col-12NSIF-1::GFP + P_odr-1RFP];

YNU555: ylfEx282[qxIs257; P_col-12ΔSPNSIF-1::GFP + P_odr-1RFP];

YNU777: epic-1(ylf93)[epic-1::GFP::3xFLAG knock-in];

YNU738: nsif-1(ylf82);

YNU763: syb9508;qxIs383[P_hyp-7NUC-1::CHERRY];

YNU764: nsif-1(ylf82);qxIs383[P_hyp-7NUC-1::CHERRY];

YNU747: ylfIs52[P_col-12GBP::mKate2];

YNU765: ylfIs52[P_col-12GBP::mKate2];ylfIs8[P_rgef-1NSIF-1::GFP + P_odr-1RFP];

YNU556: ylfEx283[qxIs383;nsif-1(ok1337); P_trx-1NSIF-1::GFP + P_odr-1RFP];

YNU557: ylfEx284[qxIs383;nsif-1(ok1337); P_trx-1ΔSPNSIF-1 + P_odr-1RFP];

YNU558: ylfEx285[qxIs383;nsif-1(ok1337); P_str-3NSIF-1::GFP + P_odr-1RFP];

YNU559: ylfEx286[qxIs383;nsif-1(ok1337); P_str-3ΔSPNSIF-1 + P_odr-1RFP];

YNU755: ylfEx357[qxIs383;P_str-3EGL-1::3 × FLAG + P_odr-1RFP];

YNU756: ylfEx358[qxIs383;P_trx-1EGL-1::3 × FLAG+ P_odr-1RFP];

YNU757: ylfEx359[qxIs722;nsif-1(ok1337);P_str-3NSIF-1::mKate2 + P_odr-1GFP];

YNU758: ylfEx359[qxIs722;nsif-1(ok1337);P_str-3ΔSPNSIF-1::mKate2 + P_odr-1GFP];

YNU759: ylfEx361[qxIs722;nsif-1(ok1337);P_trx-1NSIF-1::mKate2 + P_odr-1GFP];

YNU760: ylfEx362[qxIs722;nsif-1(ok1337);P_trx-1ΔSPNSIF-1::mKate2 + P_odr-1GFP];

YNU761: qxIs383;nsif-1(ok1337);ylfIs1[P_nsif-1NSIF-1::GFP];

YNU762: qxIs383;nsif-1(ok1337);ylfIs30[P_nsif-1ΔSPNSIF-1::GFP];

YNU779: ylfEx363[qxIs722;nsif-1(ok1337);P_nsif-1NSIF-1::mKate2 + P_odr-1GFP];

YNU786: ylfEx370[qxIs722;nsif-1(ok1337);P_nsif-1ΔSPNSIF-1::mKate2 + P_odr-1GFP];

YNU778: sid-1(qt9);qxIs383[P_hyp-7NUC-1::CHERRY];

YNU780: ylfEx364 [sid-1(qt9);qxIs383; P_str-3SID-1::3 × FLAG+P_odr-1RFP];

YNU781: ylfEx365[sid-1(qt9);qxIs383;P_trx-1SID-1::3 × FLAG+P_odr-1RFP];

YNU782: ylfEx366[ylfIs52;ylfIs8 + P_str-3EGL-1::3 × FLAG+rol-6(su1006)];

YNU783: ylfEx367[ylfIs52;ylfIs8 + P_trx-1EGL-1::3 × FLAG+rol-6(su1006)];

YNU794: nsif-1(ylf82);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion];

YNU811: nsif-1(ylf82);bli-1(wrd84)[bli-1::linker::mNeonGreen::3xFLAG(internal)

::linker];

YNU813: nsif-1(ylf82); cut-2(wrd233)[cut-2::mNeonGreen::3xFLAG];

YNU814: nsif-1(ylf82); epic-1(ylf93)[epic-1::GFP::3xFLAG knock-in];

YNU815: nsif-1(ok1337);bli-1(wrd84)[bli-1::linker::mNeonGreen::3xFLAG(internal)

::linker];

YNU817: nsif-1(ok1337); cut-2(wrd233)[cut-2::mNeonGreen::3xFLAG];

YNU818: nsif-1(ok1337); epic-1(ylf93)[epic-1::GFP::3xFLAG knock-in];

YNU858: ylfEx411[ylfIs52;P_str-3NSIF-1::GFP + rol-6(su1006)];

YNU859: ylfEx412[ylfIs52;P_str-3ΔSPNSIF-1::GFP + rol-6(su1006)];

YNU860: ylfEx413[ylfIs52;P_trx-1NSIF-1::GFP + rol-6(su1006)];

YNU861: ylfEx414[ylfIs52;P_trx-1ΔSPNSIF-1::GFP + rol-6(su1006)];

YNU862: ylfEx415[qxIs383;nsif-1(ok1337)+P_col-12NSIF-1::GFP + P_odr-1RFP];

YNU863: ylfEx416[qxIs383;nsif-1(ok1337)+P_col-12ΔSPNSIF-1::GFP + P_odr-1RFP];

YNU864: ylfEx417[qxIs383 + P_nsif-1NSIF-1::GFP + P_odr-1RFP];

YNU865: ylfEx418[qxIs383 + P_nsif-1ΔSPNSIF-1::GFP + P_odr-1RFP];

YNU868: ylfEx421[qxIs722 + P_nsif-1NSIF-1::mKate2 + P_odr-1GFP];

YNU869: ylfEx422[qxIs722 + P_nsif-1ΔSPNSIF-1::mKate2 P_odr-1GFP];

YNU870: ylfEx423[qxIs722 + P_col-12NSIF-1:: mKate2 + P_odr-1GFP];

YNU871: ylfEx424[qxIs722 + P_col-12ΔSPNSIF-1:: mKate2 P_odr-1GFP];

YNU872: ylfEx425[qxIs722;nsif-1(ok1337)+P_myo-3NSIF-1::mKate2 + P_odr-1GFP];

YNU873: ylfEx426[qxIs722;nsif-1(ok1337)+P_vha-6NSIF-1:: mKate2 P_odr-1GFP];

YNU874: ylfEx427[qxIs722;nsif-1(ok1337)+P_col-12NSIF-1:: mKate2 + P_odr-1GFP];

YNU875: ylfEx428[qxIs722;nsif-1(ok1337)+P_col-12ΔSPNSIF-1:: mKate2 + P_odr-1GFP];

YNU876: ylfEx429[syb9508 + P_col-12::mKate2];

YNU879: ylfEx432[sid-1(qt9);qxIs383 + P_str-3NSIF-1::GFP + P_str-3SID-1::3 × FLAG+P_odr-1RFP];

YNU880: ylfEx433[sid-1(qt9);qxIs383 + P_trx-1NSIF-1::GFP + P_trx-1SID-1::3 × FLAG+P_odr-1RFP];

YNU881: ylfEx435[qxIs383 + P_str-3NSIF-1::GFP + P_odr-1RFP];

YNU882: ylfEx434[qxIs383 + P_str-3NSIF-1::GFP + P_str-3EGL-1::3 × FLAG+ P_odr-1RFP];

YNU883: ylfEx437[qxIs383 + P_trx-1NSIF-1::GFP + P_odr-1RFP];

YNU884: ylfEx436[qxIs383 + P_trx-1NSIF-1::GFP + P_trx-1EGL-1::3 × FLAG+ P_odr-1RFP];

YNU885: qxIs383;nsif-1(ok1337);ylfIs22[P_vha-6NSIF-1::GFP + P_odr-1RFP];

YNU886: qxIs383;nsif-1(ok1337);ylfIs27[P_myo-3NSIF-1::GFP + P_odr-1RFP];

YNU887: ylfEx438[ylfIs1 + P_str-3mKate2];

YNU888: ylfEx439[ylfIs1 + P_trx-1mKate2];

YNU889: ylfEx440[ylfIs1 + P_tph-1mKate2];

YNU890: ylfEx441[ylfIs1 + P_ggr-2mKate2].

Bacterial strains

E. coli OP50, E. coli HT115, and P. aeruginosa PA14 were cultured in LB medium at 37°C. Overnight bacterial cultures were seeded onto Nematode Growth Medium (NGM) plates for subsequent experiments.

Generation of transgene strains

1. 1). To construct the C. elegans plasmid for expression of P_nsif-1::GFP, 490 bp promoter of nsif-1 was inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_nsif-1::GFP (20 ng/µl) and unc-76(+) (p76-16B)(50 ng/µl) was injected into the gonads of adult unc-76(e911).
2. 2). To construct the C. elegans plasmid for expression of P_nsif-1NSIF-1::GFP, 490 bp promoter and 881 bp of nsif-1 genomic DNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_nsif-1NSIF-1::GFP (20 ng/µl) and unc-76(+) (p76-16B)(50 ng/µl) was injected into the gonads of adult unc-76(e911).
3. 3). To construct the C. elegans plasmid for expression of P_nsif-1ΔSPNSIF-1::GFP, 490 bp promoter and 824 bp of ΔSPnsif-1 genomic DNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_nsif-1ΔSPNSIF-1::GFP (20 ng/µl) and unc-76(+) (p76-16B)(50 ng/µl) was injected into the gonads of adult unc-76(e911).
4. 4). To construct the C. elegans plasmid for expression of P_nsif-1NSIF-1::3 × FLAG::GFP, 490 bp promoter, 881 bp of nsif-1 genomic DNA and 3 × FLAG were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_nsif-1NSIF-1::3 × FLAG::GFP (20 ng/µl) and unc-76(+) (p76-16B)(50 ng/µl) was injected into the gonads of adult unc-76(e911).
5. 5). To construct the C. elegans plasmid for expression of P_rgef-1NSIF-1::GFP, 3057 bp promoter of rgef-1 and 881 bp of nsif-1 genomic DNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_rgef-1NSIF-1::GFP(20 ng/µl) and P_odr-1RFP(50 ng/µl) was injected into the gonads of adult wild type N2.
6. 6). To construct the C. elegans plasmid for expression of P_myo-3NSIF-1::GFP, 2403 bp promoter of myo-3 and 881 bp of nsif-1 genomic DNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_myo-3NSIF-1::GFP(20 ng/µl) and P_odr-1RFP(50 ng/µl) was injected into the gonads of adult wild type N2.
7. 7). To construct the C. elegans plasmid for expression of P_vha-6NSIF-1::GFP, 1593 bp promoter of vha-6 and 881 bp of nsif-1 genomic DNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_vha-6NSIF-1::GFP(20 ng/µl) and P_odr-1RFP(50 ng/µl) was injected into the gonads of adult wild type N2.
8. 8). To construct the C. elegans plasmid for expression of P_col-12GBP::mKate, 2, 613 bp promoter of col-12 and 345 bp of GBP DNA sequence were inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_col-12GBP::mKate2 (20 ng/µl) and rol-6(su1006) (50 ng/µl) was injected into the gonads of adult YNU138: ylfIs8[Prgef-1NSIF-1::GFP+ Podr-1RFP].
9. 9). To construct the C. elegans plasmid for expression of P_rgef-1ΔSPNSIF-1::GFP, 3057 bp promoter of rgef-1 and 824 bp of ΔSPnsif-1 genomic DNA sequence were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_rgef-1ΔSPNSIF-1::GFP (20 ng/µl), P_col-12GBP::mKate2 (20 ng/µl) and rol-6(su1006) (50 ng/µl) was injected into the gonads of wild type N2.
10. 10). To construct the C. elegans plasmid for expression of P_sra-6mKate2, 2932 bp promoter of sra-6 was inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_sra-6mKate2(20 ng/µl) and P_nsif-1::GFP(20 ng/µl) was injected into the gonads of adult wild type N2.
11. 11). To construct the C. elegans plasmid for expression of P_trx-1mKate2, 496 bp promoter of trx-1 was inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_trx-1mKate2(20 ng/µl) and P_nsif-1::GFP(20 ng/µl) was injected into the gonads of adult wild type N2.
12. 12). To construct the C. elegans plasmid for expression of P_tph-1mKate2, 2023 bp promoter of tph-1 was inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_tph-1mKate2(20 ng/µl) and P_nsif-1::GFP(20 ng/µl) was injected into the gonads of adult wild type N2.
13. 13). To construct the C. elegans plasmid for expression of P_ggr-2mKate2, 2115 bp promoter of ggr-2 was inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_ggr-2mKate2(20 ng/µl) and P_nsif-1::GFP(20 ng/µl) was injected into the gonads of adult wild type N2.
14. 14). To construct the C. elegans plasmid for expression of P_col-12NSIF-1::GFP, 613 bp promoter of col-12 and 881 bp of nsif-1 genomic DNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_col-12NSIF-1::GFP(20 ng/µl) and P_odr-1RFP(50 ng/µl) was injected into the gonads of adult XW5399: qxIs257[P_ced-1NUC-1::CHERRY].
15. 15). To construct the C. elegans plasmid for expression of P_col-12ΔSPNSIF-1::GFP, 613 bp promoter of col-12 and 824 bp of ΔSPnsif-1 genomic DNA was inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_col-12ΔSPNSIF-1::GFP(20 ng/µl) and P_odr-1RFP(50 ng/µl) was injected into the gonads of adult XW5399: qxIs257[P_ced-1NUC-1::CHERRY].
16. 16). To construct the C. elegans plasmid for expression of P_trx-1NSIF-1::GFP, 496 bp promoter of trx-1 and 881 bp of nsif-1 genomic DNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_trx-1NSIF-1::GFP (20 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult YNU467: nsif-1(ok1337);qxIs383[P_hyp-7NUC-1::CHERRY].
17. 17). To construct the C. elegans plasmid for expression of P_trx-1ΔSPNSIF-1::GFP, 496 bp promoter of trx-1 and 824 bp of ΔSPnsif-1 genomic DNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_trx-1ΔSPNSIF-1::GFP (20 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult YNU467: nsif-1(ok1337);qxIs383[P_hyp-7NUC-1::CHERRY].
18. 18). To construct the C. elegans plasmid for expression of P_str-3NSIF-1::GFP, 259 bp promoter of str-3 and 881 bp of nsif-1 genomic DNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_str-3NSIF-1::GFP (20 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult YNU467: nsif-1(ok1337);qxIs383[P_hyp-7NUC-1::CHERRY].
19. 19). To construct the C. elegans plasmid for expression of P_str-3ΔSPNSIF-1::GFP, 259 bp promoter of str-3 and 824 bp of ΔSPnsif-1 genomic DNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_str-3ΔSPNSIF-1::GFP (20 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult YNU467: nsif-1(ok1337);qxIs383[P_hyp-7NUC-1::CHERRY].
20. 20). To construct the C. elegans plasmid for expression of P_str-3EGL-1::3 × FLAG, 259 bp promoter of str-3 and 875 bp of EGL-1 genomic DNA were inserted into the pPD49.26−3 × FLAG vector. DNA plasmid mixture containing P_str-3EGL-1::3 × FLAG (25 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult XW17096:qxIs383[P_hyp-7NUC-1::CHERRY].
21. 21). To construct the C. elegans plasmid for expression of P_trx-1EGL-1::3 × FLAG, 496 bp promoter of trx-1 and 875 bp of EGL-1 genomic DNA were inserted into the pPD49.26−3 × FLAG vector. DNA plasmid mixture containing P_trx-1EGL-1::3 × FLAG (25 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult XW17096: qxIs383[P_hyp-7NUC-1::CHERRY].
22. 22). To construct the C. elegans plasmid for expression of P_str-3NSIF-1::mKate2, 259 bp promoter of str-3 and 881 bp of nsif-1 genomic DNA were inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_str-3NSIF-1::mKate2 (20 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
23. 23). To construct the C. elegans plasmid for expression of P_str-3ΔSPNSIF-1::mKate2, 259 bp promoter of str-3 and 824 bp of nsif-1 genomic DNA were inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_str-3ΔSPNSIF-1::mKate2 (20 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
24. 24). To construct the C. elegans plasmid for expression of P_trx-1NSIF-1::mKate2, 496 bp promoter of trx-1 and 881 bp of nsif-1 genomic DNA were inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_trx-1NSIF-1::mKate2 (20 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
25. 25). To construct the C. elegans plasmid for expression of P_trx-1ΔSPNSIF-1:: mKate2, 496 bp promoter of trx-1 and 824 bp of nsif-1 genomic DNA were inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_trx-1ΔSPNSIF-1::mKate2 (20 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
26. 26). To construct the C. elegans plasmid for expression of P_nsif-1NSIF-1::mKate2, 490 bp promoter of nsif-1 and 881 bp of nsif-1 genomic DNA were inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_nsif-1NSIF-1::mKate2 (20 ng/µl) and P_odr-1GFP(50 ng/µl) was injected into the gonads of adult YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
27. 27). To construct the C. elegans plasmid for expression of P_nsif-1ΔSPNSIF-1::mKate2, 490 bp promoter of nsif-1 and 824 bp of nsif-1 genomic DNA were inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_nsif-1NSIF-1::mKate2 (20 ng/µl) and P_odr-1GFP(50 ng/µl) was injected into the gonads of adult YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
28. 28). 28) To construct the C. elegans plasmid for expression of P_str-3SID-1::3 × FLAG, 259 bp promoter of str-3 and 2331 bp of sid-1 cDNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_str-3SID-1::3 × FLAG (20 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult YNU778: sid-1(qt9);qxIs383[P_hyp-7NUC-1::CHERRY].
29. 29). To construct the C. elegans plasmid for expression of P_trx-1SID-1::3 × FLAG, 496 bp promoter of trx-1 and 2331 bp of sid-1 cDNA were inserted into the pPD49.26-GFP vector. DNA plasmid mixture containing P_trx-1SID-1::3 × FLAG (20 ng/µl) andP_odr-1RFP (50 ng/µl) was injected into the gonads of adult YNU778: sid-1(qt9);qxIs383[P_hyp-7NUC-1::CHERRY].
30. 30). To construct the C. elegans plasmid for expression of P_str-3EGL-1::3 × FLAG, 259 bp promoter of str-3 and 875 bp of EGL-1 genomic DNA were inserted into the pPD49.26−3 × FLAG vector. DNA plasmid mixture containing P_str-3EGL-1::3 × FLAG (25 ng/µl) and rol-6(su1006) (50 ng/µl) was injected into the gonads of adult YNU765: ylfIs52[P_col-12GBP::mKate2];ylfIs8[P_rgef-1NSIF-1::GFP + P_odr-1RFP].
31. 31). To construct the C. elegans plasmid for expression of P_trx-1EGL-1::3 × FLAG, 496 bp promoter of trx-1 and 875 bp of EGL-1 genomic DNA were inserted into the pPD49.26−3 × FLAG vector. DNA plasmid mixture containing P_trx-1EGL-1::3 × FLAG (25 ng/µl) and rol-6(su1006) (50 ng/µl) was injected into the gonads of adult YNU765: ylfIs52[P_col-12GBP::mKate2];ylfIs8[P_rgef-1NSIF-1::GFP + P_odr-1RFP].
32. 32). To construct the C. elegans expression of P_str-3NSIF-1::GFP in YNU747: ylfIs52[P_col-12GBP::mKate2], a DNA plasmid mixture containing P_str-3NSIF-1::GFP (25 ng/µl) and rol-6(su1006) (50 ng/µl) was injected into the gonads of adult YNU747:ylfIs52[P_col-12GBP::mKate2].
33. 33). To construct the C. elegans expression of P_str-3ΔSPNSIF-1::GFP in YNU747: ylfIs52[P_col-12GBP::mKate2], DNA plasmid mixture containing P_str-3ΔSPNSIF-1::GFP (25 ng/µl) and rol-6(su1006) (50 ng/µl) was injected into the gonads of adult YNU747:ylfIs52[P_col-12GBP::mKate2].
34. 34). To construct the C. elegans expression of P_trx-1NSIF-1::GFP in YNU747: ylfIs52[P_col-12GBP::mKate2], a DNA plasmid mixture containing P_trx-1NSIF-1::GFP (25 ng/µl) and rol-6(su1006) (50 ng/µl) was injected into the gonads of adult YNU747:ylfIs52[P_col-12GBP::mKate2].
35. 35). To construct the C. elegans expression of P_trx-1ΔSPNSIF-1::GFP in YNU747: ylfIs52[P_col-12GBP::mKate2], DNA plasmid mixture containing P_trx-1ΔSPNSIF-1::GFP (25 ng/µl) and rol-6(su1006) (50 ng/µl) was injected into the gonads of adult YNU747:ylfIs52[P_col-12GBP::mKate2].
36. 36). To construct the C. elegans expression of P_col-12NSIF-1::GFP in YNU467: nsif-1(ok1337);qxIs383[P_hyp-7NUC-1::CHERRY], DNA plasmid mixture containing P_col-12NSIF-1::GFP (25 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult YNU467:nsif-1(ok1337);qxIs383[P_hyp-7NUC-1::CHERRY].
37. 37). To construct the C. elegans expression of P_col-12ΔSPNSIF-1::GFP in YNU467: nsif-1(ok1337);qxIs383[P_hyp-7NUC-1::CHERRY], DNA plasmid mixture containing P_col-12ΔSPNSIF-1::GFP (25 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult YNU467:nsif-1(ok1337);qxIs383[P_hyp-7NUC-1::CHERRY].
38. 38). To construct the C. elegans expression of P_nsif-1NSIF-1::GFP in XW17096: qxIs383[P_hyp-7NUC-1::CHERRY], DNA plasmid mixture containing P_nsif-1NSIF-1::GFP (25 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult XW17096:qxIs383[P_hyp-7NUC-1::CHERRY].
39. 39). To construct the C. elegans expression of P_nsif-1ΔSPNSIF-1::GFP in XW17096: qxIs383[P_hyp-7NUC-1::CHERRY], DNA plasmid mixture containing P_nsif-1ΔSPNSIF-1::GFP (25 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult XW17096:qxIs383[P_hyp-7NUC-1::CHERRY].
40. 40). To construct the C. elegans expression of P_nsif-1NSIF-1::mKate2 in XW18042: qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion], DNA plasmid mixture containing P_nsif-1NSIF-1:: mKate2 (25 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult XW18042: qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
41. 41). To construct the C. elegans expression of P_nsif-1ΔSPNSIF-1:: mKate2 in XW18042: qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion], DNA plasmid mixture containing P_nsif-1ΔSPNSIF-1::mKate2 (25 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult XW18042: qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
42. 42). To construct the C. elegans expression of P_col-12NSIF-1::mKate2 in XW18042: qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion], DNA plasmid mixture containing P_col-12NSIF-1::mKate2 (25 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult XW18042: qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
43. 43). To construct the C. elegans expression of P_col-12ΔSPNSIF-1:: mKate2 in XW18042: qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion], DNA plasmid mixture containing P_col-12ΔSPNSIF-1:: mKate2 (25 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult XW18042: qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
44. 44). To construct the C. elegans plasmid for expression of P_myo-3NSIF-1::mKate2, 2403 bp promoter of myo-3 and 881 bp of nsif-1 genomic DNA was inserted into the pPD49.26-mKate2 vector, DNA plasmid mixture containing P_myo-3NSIF-1::mKate2 (25 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
45. 45). To construct the C. elegans plasmid for expression of P_vha-6NSIF-1::mKate2, 1593 bp promoter of myo-3 and 881 bp of nsif-1 genomic DNA was inserted into the pPD49.26-mKate2 vector, DNA plasmid mixture containing P_vha-6NSIF-1::mKate2 (25 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
46. 46). To construct the C. elegans expression of P_col-12NSIF-1::mKate2 in YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion], DNA plasmid mixture containing P_col-12NSIF-1::mKate2 (25 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
47. 47). To construct the C. elegans expression of P_col-12ΔSPNSIF-1:: mKate2 in YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion], single-copy insertion], DNA plasmid mixture containing P_col-12ΔSPNSIF-1:: mKate2 (25 ng/µl) and P_odr-1GFP (50 ng/µl) was injected into the gonads of adult YNU473: nsif-1(ok1337);qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion].
48. 48). To construct the C. elegans plasmid for expression of P_col-12mKate2, 613 bp promoter of col-12 was inserted into the pPD49.26-mKate2 vector, DNA plasmid mixture containing P_col-12mKate2 (25 ng/µl) was injected into the gonads of adult BHX9508: nsif-1(syb9508)[nsif-1::mNeonGreen::3xFLAG knock-in].
49. 49). To construct the C. elegans plasmid for expression of P_str-3SID-1::3 × FLAG and P_str-3NSIF-1::GFP into YNU778: sid-1(qt9);qxIs383[P_hyp-7NUC-1::CHERRY], DNA plasmid mixture containing P_str-3SID-1::3 × FLAG (20 ng/µl), P_str-3NSIF-1::GFP (20 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult YNU778: sid-1(qt9);qxIs383[P_hyp-7NUC-1::CHERRY].
50. 50). To construct the C. elegans plasmid for expression of P_trx-1SID-1::3 × FLAG and P_trx-1NSIF-1::GFP into YNU778: sid-1(qt9);qxIs383[P_hyp-7NUC-1::CHERRY], DNA plasmid mixture containing P_trx-1SID-1::3 × FLAG (20 ng/µl), P_trx-1NSIF-1::GFP (20 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult YNU778: sid-1(qt9);qxIs383[P_hyp-7NUC-1::CHERRY].
51. 51). To construct the C. elegans expression of P_str-3NSIF-1::GFP in XW17096: qxIs383[P_hyp-7NUC-1::CHERRY], DNA plasmid mixture containing P_str-3NSIF-1::GFP (25 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult XW17096: qxIs383[P_hyp-7NUC-1::CHERRY].
52. 52). To construct the C. elegans expression of P_str-3EGL-1::3 × FLAG and P_str-3NSIF-1::GFP in XW17096: qxIs383[P_hyp-7NUC-1::CHERRY], DNA plasmid mixture containing P_str-3EGL-1::3 × FLAG (25 ng/µl), P_str-3NSIF-1::GFP (25 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult XW17096: qxIs383[P_hyp-7NUC-1::CHERRY].
53. 53). To construct the C. elegans expression of P_trx-1NSIF-1::GFP in XW17096: qxIs383[P_hyp-7NUC-1::CHERRY], DNA plasmid mixture containing P_trx-1NSIF-1::GFP (25 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult XW17096: qxIs383[P_hyp-7NUC-1::CHERRY].
54. 54). To construct the C. elegans expression of P_trx-1EGL-1::3 × FLAG and P_trx-1NSIF-1::GFP in XW17096: qxIs383[P_hyp-7NUC-1::CHERRY], DNA plasmid mixture containing P_trx-1EGL-1::3 × FLAG (25 ng/µl), P_trx-1NSIF-1::GFP (25 ng/µl) and P_odr-1RFP (50 ng/µl) was injected into the gonads of adult XW17096: qxIs383[P_hyp-7NUC-1::CHERRY].
55. 55). To construct the C. elegans plasmid for expression of P_str-3mKate2 in YNU22: unc-76(e911);ylfIs1[P_nsif-1NSIF-1::GFP; unc-76(+)], 259 bp promoter of str-3 was inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_str-3mKate2 (20 ng/µl) was injected into the gonads of adult YNU22: unc-76(e911);ylfIs1[P_nsif-1NSIF-1::GFP; unc-76(+)].
56. 56). To construct the C. elegans plasmid for expression of P_trx-1mKate2 in YNU22: unc-76(e911);ylfIs1[P_nsif-1NSIF-1::GFP; unc-76(+)], 496 bp promoter of trx-1 was inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_trx-1mKate2 (20 ng/µl) was injected into the gonads of adult YNU22: unc-76(e911);ylfIs1[P_nsif-1NSIF-1::GFP; unc-76(+)],.
57. 57). To construct the C. elegans plasmid for expression of P_tph-1mKate2 in YNU22: unc-76(e911);ylfIs1[P_nsif-1NSIF-1::GFP; unc-76(+)], 2023 bp promoter of tph-1 was inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_tph-1mKate2 (20 ng/µl) was injected into the gonads of adult YNU22: unc-76(e911);ylfIs1[P_nsif-1NSIF-1::GFP; unc-76(+)].
58. 58). To construct the C. elegans plasmid for expression of P_ggr-2mKate2 in YNU22: unc-76(e911);ylfIs1[P_nsif-1NSIF-1::GFP; unc-76(+)], 2115 bp promoter of ggr-2 was inserted into the pPD49.26-mKate2 vector. DNA plasmid mixture containing P_ggr-2mKate2 (20 ng/µl) was injected into the gonads of adult YNU22: unc-76(e911);ylfIs1[P_nsif-1NSIF-1::GFP; unc-76(+)].

Generating knock-out mutants using CRISPR

Generation of EPIC-1::GFP knock-in strain using CRISPR/Cas9

Generation of transgene strains by TMP/UV

Stable transgenic C. elegans lines were generated using UV/trimethylpsoralen (TMP)-mediated integration. Synchronized L4-stage transgenic worms were first treated with TMP (50 µg/mL) to enhance UV sensitivity, followed by UV irradiation (350–400 J/m²) to induce DNA damage and facilitate genomic integration of extrachromosomal arrays. After recovery and expansion, single-worm lines were screened for stable inheritance of both the co-marker and target plasmid expression. Positive integrations were subsequently outcrossed with wild-type N2 worms at least four times to remove background mutations.

C. elegans RNAi feeding strategy

RNAi by feeding was performed using bacterial clones from the MRC RNAi library [78] or the ORF-RNAi Library [79]. Synchronized C. elegans (L1-stage larvae) were transferred onto RNAi NGM plates seeded with E. coli HT115 expressing the corresponding double-stranded RNA. Worms were maintained at 20 °C and allowed to develop either to the L4 larval stage or the next generation, depending on the specific experimental requirements.

C. elegans tissue-specific RNAi feeding strategy

To achieve tissue-specific RNA interference, we cross sid-1(qt9) mutant into the background (qxIs383) to abolish systemic RNAi. The sid-1 gene was then selectively restored in either ASI or ASJ neurons by expressing sid-1 under the control of neuron-specific promoters (P_str-3SID-1::3 × FLAG for ASI and P_trx-1SID-1::3 × FLAG for ASJ).

In these transgenic backgrounds, feeding RNAi was performed to knock down target genes. Briefly, worms were transferred onto NGM plates seeded with RNAi bacteria and incubated at 20 °C until the indicated developmental stages. The L4440 empty vector strain was used as a negative control.

Pseudomonas aeruginosa PA14 survival assay

The slow-killing assay was performed as previously described [80]. Overnight cultures of Pseudomonas aeruginosa PA14 were inoculated onto modified NGM agar plates (0.35% peptone, 35 mm in diameter). The inoculated plates were air-dried at room temperature and subsequently incubated at 37 °C for 24 h, followed by an additional incubation at 25 °C for 8−24 h. Before initiating the assay, plates were equilibrated at room temperature for at least 1 h. Synchronized L4-stage C. elegans were transferred onto PA14-seeded slow-killing plates and maintained at 25 °C. Survival was assessed at 12-h intervals, with mortality determined by the absence of response to mechanical stimulation of both the anterior and posterior regions using an eyebrow hair. To suppress progeny production and prevent "bagging," 5-fluoro-2′-deoxyuridine (FUdR, 50 mg/mL; Sigma) was added to the assay medium. Worms exhibiting vulval rupture or those that migrated off the agar surface were excluded from analysis. All survival assays were conducted in triplicate to ensure statistical robustness.

Quantitative RT-PCR

RNA-seq preparation and analysis

Fluorescent microscopy

Worms were anesthetized using 1 mM levamisole and mounted on 3% agarose pads for imaging. Fluorescence microscopy was performed using either an upright Leica DM6 B microscope equipped with a 10× objective lens or a Zeiss LSM 900 confocal laser scanning microscope with a 60× oil immersion objective. Fluorescence signals were detected using the following filter sets: GFP (excitation: 488 nm, emission: 510–550 nm) and RFP/CHERRY/mKate2 (excitation: 561 nm, emission: 570–620 nm). Images were acquired and processed using ImageJ (NIH) or ZEN imaging software v3.4 (Carl Zeiss).

Protein extraction from C. elegans

Worms were collected and washed three times with M9 buffer to remove residual bacteria. The worm pellet was then resuspended in protein lysis buffer (50 mM Tris-HCl, pH 8.0; 50 mM NaCl; 0.5% deoxycholate; 10% glycerol; 1% NP-40) supplemented with PMSF (Sangon Biotech), Protease Inhibitor Cocktail (MCE, HY-K0011), and Phosphatase Inhibitor Cocktail I (MCE, HY-K0021). Worms were homogenized and lysed, followed by centrifugation at 12,000 rpm for 10 min at 4 °C. The supernatant was collected, and protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher, 23227).

Western blot

Protein samples were prepared by adding 5× SDS loading buffer to the quantified protein at a 1:4 ratio. Samples were then heated at 95 °C for 10 min to denature the proteins. Proteins were separated via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane for subsequent analysis.

To assess CHERRY expression, the membrane was incubated with anti-CHERRY antibody (1:3000; Proteintech, 26765-1-AP), with anti-β-actin (1:3000; Proteintech, 66009-1-lg) as a reference protein. Following primary antibody incubation, the membrane was washed and incubated with appropriate secondary antibodies. Protein bands were visualized using chemiluminescence detection.

Isolation of secreted proteins from C. elegans

Synchronized L1-stage worms were cultured in 500 μL of M9 buffer, with or without the addition of 5 μL Pseudomonas aeruginosa PA14 (OD600 = 1) or E. coli OP50, in a 1.5 mL tube. The samples were gently shaken for 4 h. Following incubation, worms were pelleted by centrifugation at 50 × g for 1 min, and the supernatant was collected.

The collected supernatant was filtered through a 0.22 μm filter unit to isolate secreted proteins. The resulting protein samples were then subjected to mass spectrometry analysis using an Orbitrap hybrid mass spectrometer (Orbitrap Exploris 480, Thermo Scientific).

We selected synchronized L1-stage animals for the secretome analysis because bleach‑synchronized L1s have never been exposed to any bacterial food source, and therefore lack any prior innate‑immune history. When these naïve L1s are subsequently challenged with P. aeruginosa PA14, the secreted proteins they produce should reflect a direct response to PA14 itself, uncontaminated by adaptive effects resulting from a previous diet of E. coli OP50. In contrast, L4‑stage animals have been reared on E. coli for approximately 48 h and may have already developed a baseline level of innate‑immune adaptation, which could attenuate or otherwise modify their response upon transfer to PA14.

Quantification of lysosomal morphology

Lysosome maturation process

L4-stage worms were subjected to heat shock at 34 °C for 30 min to induce transient expression of NUC-1::sfGFP::CHERRY strain: XW13734: qxIs612[P_hspNUC-1::sfGFP::CHERRY], followed by recovery at 20 °C for 1, 3, 6, and 12 h. At each time point, different worms were selected and examined using confocal fluorescence microscopy (Carl Zeiss LSM900). To assess lysosome maturation under various conditions, worms were heat-shocked and analyzed at different time points post-recovery. The colocalization of CHERRY and GFP was quantified using Pearson’s correlation coefficient in the ImageJ colocalization tool.

Quantification of NUC-1::pHTomato fluorescence intensity

Different C. elegans strains were crossed with the NUC-1::pHTomato strain XW19180: qxIs750 [P_hspNUC-1::pHTomato] to generate homozygous lines. Synchronized worms were cultured to the L4 stage and subjected to heat shock at 34 °C for 30 min. Immediately after heat shock, worms were transferred to a 20 °C incubator for a 12-h recovery period, followed by fluorescence imaging using a confocal microscope. For Pseudomonas aeruginosa PA14 treatment, L4-stage worms were transferred to NGM plates seeded with PA14 before undergoing the same heat shock and recovery protocol. To ensure consistency, laser intensity and exposure time were maintained uniformly during imaging. Fluorescence intensity was quantified by measuring the 150 lysosomes from 20 worms per strain

Each lysosome's fluorescence intensity was divided by the mean fluorescence intensity of all control-group lysosomes. Specifically, for the control group, each lysosome's value was normalized to the control mean (yielding a distribution centered around 1.0). For the mutant group, each lysosome's value was likewise divided by the control mean, so that the mutant values can be read as fold-change relative to control.

Quantitative analysis of ELT-3 fluorescence intensity in nuclear

ELT-3::sfGFP-expressing C. elegans (OP559: wgIs559) were imaged using confocal fluorescence microscopy (Carl Zeiss LSM900). Nuclear-localized ELT-3::sfGFP fluorescence intensity was quantified using ZEN Blue v3.4 software. To ensure reliability, at least 20 animals per strain were analyzed at different developmental stages. Fluorescence intensity within the nucleus was measured and compared to evaluate ELT-3::sfGFP expression levels across samples.

Quantification of lysosomal degradation activity

L4-stage C. elegans expressing NUC-1::CHERRY in the epidermis [XW5399: qxIs257(P_ced-1NUC-1::CHERRY) and XW17096:qxIs383(P_hyp-7NUC-1::CHERRY)] were synchronized and collected 24 h post-L4 (~100 worms per stage or condition) into 1.5 mL tubes. Protein samples were mixed with 5 × SDS loading buffer at a 1:4 ratio and heated at 95 °C for 10 min to denature the proteins. Worm lysates were then analyzed by western blot using anti-Cherry (1:3000; Proteintech, 26765-1-AP) and anti-β-actin (1:3000; Proteintech, 66009-1-lg) antibodies. The %NUC-1::CHERRY cleavage = Intensity of CHERRY/(Intensity of NUC-1::CHERRY+CHERRY)/Fold change of reference (Control = 1).

Quantification of cuticle organization density and defects

To evaluate cuticle monomer density, XW18042: qxIs722[P_dpy-7DPY-7::sfGFP, single-copy insertion] worms at the same developmental stage were imaged using a Zeiss LSM900 confocal microscope. The clearest cuticle layer was selected for each worm, and images were captured at a consistent magnification (63 × oil objective). Image analysis was performed using Zeiss ZEN Blue 3.4 software, applying region selection and counting tools to quantify monomer structures within a defined area. A minimum of 20 worms per strain were analyzed, with each experiment repeated three times. To assess cuticle structural defects, worms at the same developmental stage were imaged and analyzed in real-time using an Olympus BX53 upright fluorescence microscope. Images were captured at a consistent magnification (40× objective). At least 20 worms were examined per strain, and the proportion of worms displaying cuticle damage was quantified.

Hoechst staining assay

Hoechst staining was performed as previously described [37]. Briefly, synchronized young adult worms were incubated in M9 buffer containing 1 mg/ml Hoechst 33342 (Wako, Japan) at room temperature for 15 min, followed by several washes with M9 buffer to remove residual dye. Fluorescent images were acquired under identical imaging conditions, and nuclear fluorescence in the tail and head region was quantified using ImageJ. The intestinal area was excluded to avoid interference from autofluorescence. Specifically, the head was defined as the broader, rounded anterior end of the animal and was readily recognized by the autofluorescence of the pharynx. The tail was defined as the tapered posterior end and was identified by the autofluorescence near the anal opening. All confocal images were acquired at a resolution of 1,024 × 1,024. For fluorescence quantification, identical ROIs (Region of Interest) (78 μm × 60 μm) were positioned in the head and tail regions using these anatomical landmarks and applied consistently across all animals analyzed.

Quantification and statistical analysis

Fluorescence intensity of worms was quantified using ImageJ software. Data are presented as the mean ± SEM and were analyzed with GraphPad Prism 10. Survival curves were generated using GraphPad Prism 10, and the significant difference of two groups of samples was analyzed by Log rank (Mantel-Cox) test. Statistical analyses were performed using a Mann–Whitney's nonparametric test for comparisons two sample groups that were not normally distributed. Statistical significance was determined as follows: */^P < 0.05, **/^^P < 0.01, ***/^^^P < 0.001, The symbols "*" and "^" denote statistical comparisons with different groups, as specified in the figure legend. All experiments were performed independently at least three times.

S1 Fig. PA14 infection does not cause significant developmental retardation.

(A) Confocal microscopy images of the vulva of animals in L4 stage. Scale bars: 10 µm. (B) Confocal microscopy images of the body length of L4 animals after 24-h OP50 or PA14 exposure. Scale bars: 100 µm. (C) Confocal microscopy images of the egg morphology of L4 animals after 24-h OP50 or PA14 exposure. Scale bars: 20 µm.

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S2 Fig. PA14-induced cuticle damage is not confined to molting cycles but occurs progressively in post-molt, intact cuticles.

(A) Schematic diagram of the timelapse microscopy method for observing cuticle morphological changes in wild type (WT) animals from the L4 stage upon PA14 infection. The molting status and the DPY-7::sfGFP was observed after transferring L4 animals to OP50 or PA14 for 0, 3, 6, 12, 24 h. (B) Confocal microscopy images of the molting status and the DPY-7::sfGFP of L4 animals after 0, 3, 6, 12, and 24 h OP50 or PA14 exposure. Scale bars: 10 µm. (C-E) Quantitative analysis of the worms with abnormal DPY-7::sfGFP pattern on OP50(C) and PA14(D) in different stage. And quantitative analysis of the worms with abnormal DPY-7::sfGFP pattern percentage between OP50 and PA14 in the same stage (E). n = 90 animals. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test (C, D). **p < 0.01, ***p < 0.001, n.s., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S3 Fig. PA14 induces cuticle damage during the adult stage.

(A) Schematic diagram of the timelapse microscopy method for observing cuticle morphological changes in wild type (WT) animals from L4 + 24h(adult) stage upon PA14 infection. DPY-7::sfGFP was observed after transferring L4 + 24h(adult) stage animals to OP50 or PA14 for 0, 3, 6, 12, and 24 h. (B) Confocal microscopy images DPY-7::sfGFP of L4 + 24h(adult) animals after 0, 3, 6, 12, and 24 h OP50 or PA14 exposure. Scale bars: 10 µm. (C-E) Quantitative analysis of the worms with abnormal DPY-7::sfGFP pattern on OP50(C) and PA14(D) in different stage. And quantitative analysis of the worms with abnormal DPY-7::sfGFP pattern percentage between OP50 and PA14 in the same stage(E). n = 90 animals. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test (C, D). *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. All experiments were performed independently at least three times. The data underlying this Figure can be found in S2 Data.

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S4 Fig. PA14 induces cuticle damage during the adult stage.

(A) Confocal fluorescence images and quantitative analysis of Hoechst 33,342 fluorescence intensity in young adult animals after 24h PA14 exposure (infection initiated at L4 stage). RNAi knockdown of dpy-7 and bus-8 act as positive control. n = 20 animals, Scale bars: 10 µm. (B) Representative fluorescence microscopy images and quantification of P_nlp-29::GFP intensity in adult wild-type worms subjected to RNAi targeting dpy-7, sqt-3, col-142, dyn-1, and syx-5, compared to control RNAi. n = 24 animals, Scale bars: 100 µm. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ***p < 0.001,all experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S5 Fig. PA14-dependent dysregulation of lysosomal architecture and cuticle integrity requires functional virulence factor expression.

(A) Confocal fluorescence imaging and quantitative analysis were performed on the epidermis of wild-type (WT) expressing NUC-1::CHERRY after 24h PA14 and PA14’s key virulence regulators deficient strain ΔgacA, ΔlasR, ΔmvfR, ΔpqsF exposure. The number of tubular lysosomes was quantified by counting within 35 × 25(µm²) unit areas per individual worm, with a total of 20 worms analyzed (n = 20 animals). Scale bars: 10 µm. (B) Confocal fluorescence imaging and quantitative analysis of abnormal DPY-7::sfGFP pattern in wild-type (WT) L4-stage animals following 24-h exposure to PA14 and PA14’s key virulence regulators deficient strain ΔgacA, ΔlasR, ΔmvfR, ΔpqsF. n = 300 animals. Scale bars: 10 µm. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. *Indicates comparison with the OP50, ^ indicates comparison with the PA14; ***/^^^p < 0.001, ns., not significant. All experiments were performed independently at least three times. The data underlying this Figure can be found in S2 Data.

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S6 Fig. Screen of PA14 induced neuronal secretion proteins which modulates lysosomal function and susceptibility to PA14.

(A) List of secreted proteins induced by PA14 infection, as identified by mass spectrometry. Candidate proteins were screened for signal peptides using SignalP-5.0 and evaluated for neuronal expression patterns via CeNGEN analysis. (B) Confocal imaging and quantification of NUC-1::pHTomato fluorescence in hypodermal lysosomes of wild-type animals following RNAi targeting rla-5, dao-2, nlp-77, tag-293, and lec-5. tag-293 RNAi significantly reduced lysosomal fluorescence intensity compared to control RNAi (150 lysosomes analyzed from 30 worms). n = 30 animals. Scale bars: 10 µm. (C) Immunoblot detection of secreted TAG-293 in liquid culture medium after 6-h PA14 exposure using TAG-293::3xFLAG::GFP animals. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ***p < 0.001, ns., not significant. All experiments were performed independently at least three times. The data underlying this Figure can be found in S2 Data.

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S7 Fig. NSIF-1 disrupts collagen integrity.

(A) Schematic of the nsif-1 gene and NSIF-1 protein. Exons are shown as filled boxes and introns as connecting lines. The positions of the ok1337 deletion and the ylf82 point mutation are indicated. The signal peptide (SP) and SHKT domains of NSIF-1 are shown. (B-D) Confocal fluorescence images and quantitative analysis of BLI-1::mNeonGreen (B), CUT-2::mNeonGreen (C) and EPIC-1::GFP (D) in wild-type (WT), nsif-1(ok1337) and nsif-1(ylf82) following 24-h PA14 exposure (infection initiated at L4 stage) or OP50. Quantification of cuticle furrows density were performed on 30 worms (n = 30). And the quantitative analysis of abnormal pattern after 24 h PA14 infection were performed on 100 worms with 3 times repeats (n = 300 animals). Scale bar: 5 µm. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. **p < 0.01, ***p < 0.001. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S8 Fig. NSIF-1 enhances susceptibility to PA14.

Survival analysis of wild-type N2, nsif-1(ok1337) mutants, and N2 animals overexpressing full-length NSIF-1 driven by its own native promoter (Pnsif-1) in the PA14 slow-killing assay. Data are representative of three independent biological replicates. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using Log rank (Mantel-Cox) test. * Indicates comparison with the wild-type, ^ indicates comparison with the nsif-1(ok1337) mutant. */^p < 0.05, ***/^^^p < 0.001; ns., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S9 Fig. NSIF-1 expression pattern.

(A) Confocal fluorescence imaging of transgenic animals expressing NSIF-1::GFP under the native nsif-1 promoter (P_nsif-1NSIF-1::GFP). GFP expression was observed in various neurons, muscle cells, and the intestine. Scale bars: 5 µm. (B) Confocal fluorescence imaging of the CRISPR knock-in strain NSIF-1::mNeonGreen::3 × FLAG (syb9508), showing that the tagged protein colocalizes with the epidermis, which is marked by P_col-12::mKate2. Scale bars: 100 µm (in whole animals) and 10 µm (in epidermis).

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S10 Fig. PA14-induced upregulation and lysosomal localization of NSIF-1 are specific responses to active virulence.

(A) Confocal fluorescence images and quantification of NSIF-1::mNeonGreen::3 × FLAG(syb9508) knock-in worms following 24-h exposure to wild-type PA14 or the virulence regulator-deficient mutants ΔgacA, ΔlasR, ΔmvfR, and ΔpqsF. n = 30 animals. Scale bars: 10 µm. (B) Confocal fluorescence imaging of NSIF-1::mNeonGreen::3 × FLAG and NUC-1::CHERRY following 24-h exposure to wild-type PA14 or the PA14 virulence regulator-deficient mutants ΔgacA, ΔlasR, ΔmvfR, and ΔpqsF. Scale bars: 10 µm. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. *Indicates comparison with the OP50, ^indicates comparison with the PA14. ***/^^^p < 0.001. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S11 Fig. PA14 virulence regulators are required for neuronal NSIF-1 secretion to the epidermis.

Confocal fluorescence images and quantification of neuronally secreted NSIF-1::GFP detected in the epidermis of transgenic animals expressing P_rgef-1NSIF-1::GFP (neuronal) and P_col-12GBP::mKate2 (epidermal) following 24 h of exposure to wild-type PA14 or the virulence-regulator-deficient mutants ΔgacA, ΔlasR, ΔmvfR, and ΔpqsF. n = 20 animals per condition. Scale bars: 10 µm. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. *Indicates comparison with the OP50, ^indicates comparison with the PA14. ***/^^^p < 0.001. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S12 Fig. Collagen related genes disruption does not impair NSIF-1 secretion to the epidermis.

Confocal fluorescence images and quantification of epidermal NSIF-1::GFP fluorescence in P_rgef-1NSIF-1::GFP;P_col-12GBP::mKate2 worms following col-124, dpy-7, dyn-1, sqt-3, or syx-5 RNAi. No significant difference in epidermal NSIF-1::GFP fluorescence was observed compared with the control RNAi group. n = 30 animals. Scale bars: 10 µm. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ns., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S13 Fig. NSIF-1 release occurs through a noncanonical pathway independent of traditional neuropeptide processing mechanisms.

Confocal fluorescence images and quantification of epidermal NSIF-1::GFP fluorescence in P_rgef-1NSIF-1::GFP;P_col-12GBP::mKate2 worms following 24-h exposure to OP50 or PA14 with unc-31 or egl-3 RNAi. Neither unc-31 nor egl-3 RNAi significantly affected epidermal NSIF-1::GFP fluorescence. n = 30 animals. Scale bars: 10 µm. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ns., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S14 Fig. Co-localization analysis of NSIF-1 transcriptional and translational reporters with ASI, ASJ, ADF, and RIS neurons.

(A) Localization of the NSIF-1 transcriptional reporter (P_nsif-1::GFP) in ASI (P_str-3::mKate2), ASJ (P_trx-1::mKate2), ADF (P_tph-1::mKate2), and RIS (P_ggr-2::mKate2) neurons. Scale bars: whole animal, 100 µm; others, 20 µm. (B) Localization of the NSIF-1 translational reporter (P_nsif-1NSIF-1::GFP) in ASI (P_str-3::mKate2), ASJ (P_trx-1::mKate2), ADF (P_tph-1::mKate2), and RIS (P_ggr-2::mKate2) neurons. Scale bars: whole animal, 100 µm; others, 10 µm.

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S15 Fig. Loss of nsif-1 upregulates lysosome-associated gene expression.

(A) KEGG pathway analysis of differentially upregulated genes in nsif-1(ok1337) relative to wild-type (WT) from RNA-seq. Upregulated genes for KEGG pathway analysis were defined as those with a false discovery rate (FDR) < 0.05 and a fold change≥1.5. (B) Functional categorization of 54 lysosomal genes upregulated in nsif-1(ok1337) (identified in A) from RNA-seq. (C) qRT-PCR validation of lysosomal gene expression changes in WT and adult nsif-1(ok1337), grouped by functional class: membrane proteins, lipid hydrolases, proteases, nonproteases, and v-ATPase components. For all quantification, data are presented as mean±SEM. Statistical comparisons were performed using Mann–Whitney U-test. *p < 0.05, **p < 0.01, ***p < 0.001; ns., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S16 Fig. Functional analysis of multi-tissue rescue of NSIF-1 in regulating lysosomal homeostasis and cuticle integrity.

(A) Confocal fluorescence imaging and quantitative analysis of DPY-7::sfGFP marked cuticle in wild-type(WT), nsfi-1(ok1337), nsfi-1(ok1337);P_myo-3NSIF-1::mKate2, nsfi-1(ok1337);P_vha-6NSIF-1::mKate2 animals following 24-h PA14 exposure (infection initiated at the L4 stage). Quantification of abnormal DPY-7::sfGFP pattern performed on 300 worms (n = 300 animals). Scale bars: 10 µm. (B) Confocal fluorescence imaging and quantitative analysis of NUC-1::CHERRY marked lysosome in wild-type(WT), nsfi-1(ok1337), nsfi-1(ok1337);P_myo-3NSIF-1::mKate2, nsfi-1(ok1337);P_vha-6NSIF-1::mKate2 animals following 24-h PA14 exposure (infection initiated at the L4 stage). Quantification of tubular lysosomes were quantified by counting three 35 × 25 µm² regions per worm (n = 20 worms). Scale bars: 10 µm. (C) Confocal fluorescence imaging and quantitative analysis of DPY-7::sfGFP marked cuticle in wild-type(WT), nsfi-1(ok1337), nsfi-1(ok1337);P_col-12NSIF-1::mKate2, nsfi-1(ok1337);P_col-12ΔSPNSIF-1::mKate2 animals following 24-h PA14 exposure (infection initiated at the L4 stage). Quantification of abnormal DPY-7::sfGFP pattern performed on 300 worms (n = 300 animals). Scale bars: 10 µm. (D) Confocal fluorescence imaging and quantitative analysis of NUC-1::CHERRY marked lysosome in wild-type(WT), nsfi-1(ok1337), nsfi-1(ok1337);P_col-12NSIF-1::mKate2, nsfi-1(ok1337);P_col-12ΔSPNSIF-1::mKate2 animals following 24-h PA14 exposure (infection initiated at the L4 stage). Quantification of tubular lysosomes were quantified by counting three 35 × 25 µm² regions per worm (n = 20 worms). Scale bars: 10 µm. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. *Indicates comparison with the wild-type, ^ indicates comparison with the nsif-1(ok1337) mutant; **/^^ p < 0.01, ***/^^^ p < 0.001; ns., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S17 Fig. Neuronal NSIF-1 in ASI/ASJ neurons is sufficient to impact lysosomal homeostasis and cuticle integrity.

(A) Confocal fluorescence images and quantitative analysis of NUC-1::CHERRY-labeled lysosomes in wild-type (WT), nsif-1(ok1337) mutants, and nsif-1(ok1337) mutants expressing full-length NSIF-1::GFP or the signal-peptide-deleted variant (ΔSP-NSIF-1::GFP) under the control of the endogenous nsif-1 promoter (Pnsif-1), the ASI promoter (P_ASI), or the ASJ promoter (P_ASJ) following 24 h of PA14 exposure (initiated at the L4 larval stage). Quantification of tubular lysosomes were quantified by counting three 35 × 25 µm² regions per worm (n = 20 worms). Scale bars: 10 µm. (B) Confocal fluorescence imaging and quantitative analysis of DPY-7::sfGFP marked cuticle in wild-type (WT), nsif-1(ok1337) mutants, and nsif-1(ok1337) mutants expressing full-length NSIF-1::GFP or the signal-peptide-deleted variant (ΔSP-NSIF-1::GFP) under the control of the endogenous nsif-1 promoter (Pnsif-1), the ASI promoter (P_ASI), or the ASJ promoter (P_ASJ) following 24 h of PA14 exposure (initiated at the L4 larval stage). Quantification of abnormal DPY-7::sfGFP pattern performed on 300 worms (n = 300). Scale bars: 10 µm. Note: the data shown in (A-B) for the wild-type (WT), nsif-1(ok1337) mutants, and nsif-1(ok1337) mutants expressing full-length NSIF-1::GFP (driven by the endogenous nsif-1 promoter /Pnsif-1, the ASI promoter /P_ASI, or the ASJ promoter/P_ASJ) are the same as those presented in Fig 6C, 6D. The S17 Fig extends these results by showing the corresponding data for nsif-1(ok1337) mutants expressing the signal-peptide-deleted variant (ΔSP-NSIF-1::GFP) under the control of the endogenous nsif-1 promoter (Pnsif-1), the ASI promoter (P_ASI), or the ASJ promoter (P_ASJ). For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. *Indicates comparison with the wild-type, ^ indicates comparison with the nsif-1(ok1337) mutant; **/^^ p < 0.01, ***/^^^ p < 0.001; ns., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S18 Fig. ASI/ASJ neuron ablation reduces NSIF-1 secretion to the epidermis and preserves lysosomal homeostasis during PA14 infection.

(A) Confocal microscopy images and quantification of ASI/ASJ neuron ablation efficiency and epidermal lysosomal morphology. EGL-1-mediated genetic ablation of ASI and ASJ neurons resulted in a significant reduction of NSIF-1::GFP fluorescence in these neurons. Following 24 h of PA14 exposure (initiated at the L4 larval stage), ablation of ASI/ASJ neurons prevented the PA14-induced reduction in tubular lysosomes. NSIF-1::GFP fluorescence intensity was quantified in 30 worms. Tubular lysosomes were quantified by counting three 35 × 25 µm² regions per worm (n = 20 worms). (B) Confocal microscopy images and quantification of epidermally captured NSIF-1::GFP in WT, ASI-ablated, or ASJ-ablated worms following 24-h PA14 exposure in transgenic animals expressing P_rgef-1NSIF-1::GFP + P_col-12GBP::mKate2 (infection initiated at the L4 stage) Compared with control worms, genetic ablation of either ASI or ASJ neurons significantly reduced the epidermal NSIF-1::GFP signal (n = 30 animals). Scale bars: 10 µm. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using Mann–Whitney U-test(A) and one-way ANOVA followed by Tukey’s multiple comparisons test(B). ***p < 0.001, ns., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S19 Fig. ASI/ASJ neuron ablation enhances epidermal lysosomal function.

(A) Confocal fluorescence imaging of NUC-1::CHERRY in wild-type (WT), nsfi-1(ok1337), and animals with ASI and ASJ neurons ablated, following 24 h of PA14 exposure (initiated at the L4 larval stage). Scale bars: 10 µm. (B) Quantitative analysis of the number of tubular lysosomes in wild-type (WT), nsfi-1(ok1337), and ASI/ASJ-ablated animals. The number of tubular lysosomes was quantified by counting within three 35 × 25 (µm²) unit areas per worm (n = 20 animals). For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ns., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S20 Fig. Epidermal expression of NSIF-1 is sufficient to impact lysosomal homeostasis and cuticle integrity.

(A) Confocal fluorescence imaging and quantification of NUC-1:: CHERRY-labeled lysosomes in wild-type (WT), nsif-1(ok1337) mutants, and WT animals expressing NSIF-1::mKate2 or the signal-peptide-deleted variant (ΔSP-NSIF-1::mKate2) under the control of the endogenous nsif-1 promoter (Pnsif-1) or the epidermal promoter (Pcol-12). Quantification was performed on 20 animals (n = 20). Scale bars: 10 µm. (B) Confocal fluorescence imaging and quantification of DPY-7::sfGFP-marked cuticle in WT, nsif-1(ok1337) mutants, and WT animals expressing NSIF-1::mKate2 or the signal-peptide-deleted variant (ΔSP-NSIF-1::mKate2) under the control of the endogenous nsif-1 promoter (Pnsif-1) or the epidermal promoter (Pcol-12). Quantification was performed on 300 animals (n = 300). Scale bars: 10 µm. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ***p < 0.001, ns., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

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S22 Fig. ELT-3 functions downstream of NSIF-1 to regulate epidermal lysosomal function.

(A) Confocal fluorescence imaging of localization of ELT-3::GFP and P_col-12ΔSPNSIF-1::mKate2::3 × HA in adult animals. Scale bars, 10 µm. (B) Transcription factor binding analysis identified ~200 ELT-3-regulated genes among transcripts upregulated in nsif-1 mutants. (C) qRT-PCR analysis of elt-3 expression changes in adult (L4 + 24 h) WT and nsif-1(ok1337) animals. Three biological replicates, each with three technical replicates, were analyzed for elt-3. (D-E) Analysis of hypodermal lysosomes in WT, nsif-1(ok1337), elt-3(vp1), and nsif-1(ok1337);elt-3(vp1) animals expressing NUC-1:: CHERRY following 24 h of PA14 exposure. The number of vesicular lysosomes (D) was quantified within three 35 × 25 µm² regions per worm (n = 20 animals). The diameter of vesicular lysosomes (E) was determined by measuring the six largest vesicles within a 35 × 25 µm² region per worm (n = 10 animals). (F) Immunoblots and quantification showing cleavage of CHERRY from NUC-1::CHERRY expressed in the epidermis of adult wild type(WT), nsif-1(ok1337), elt-3(gk121), nsif-1(ok1337);elt-3(vp1). N = 3 biological replicates. For all quantification, data are presented as mean ± SEM. Statistical comparisons were performed using Mann–Whitney U-test (C) and one-way ANOVA followed by Tukey’s multiple comparisons test (D-F). *Indicates comparison with the wild-type, ^ indicates comparison with the nsif-1(ok1337) mutant; */^p < 0.05, **/^^p < 0.01, ***/^^^p < 0.001; ns., not significant. All experiments were performed independently at least three times. The data underlying this figure can be found in S2 Data.

https://doi.org/10.1371/journal.pbio.3004016.s022

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