https://orcid.org/0009-0006-2834-3488
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Figures
Abstract
Distinct microbial environments exert diverse effects on the physiology and survival of the nematode Caenorhabditis elegans. Here, we show that C. elegans grown on two Escherichia coli strains exhibit different survival dynamics. Wild-type C. elegans on the B type OP50 exhibit more early deaths compared to C. elegans on K-12 type CS180. These early deaths on OP50 are characterized by swollen pharynges (P-deaths) due to bacterial accumulation within the tissue. In contrast, animals on CS180 are more resistant to P-deaths. These bacteria-dependent differences in P-deaths depend on bacterial lipopolysaccharide structures and the activities of the C. elegans neuropeptide neuromedin U receptor NMUR-1, which reduces P-deaths on OP50, but not on CS180. Surprisingly, however, NMUR-1 promotes the opposite response when the insulin receptor DAF-2 has reduced function—where NMUR-1 now stimulates P-deaths on OP50, but again with no effect on CS180. We also find that NMUR-1 acts in sensory neurons to promote its bi-directional effects on longevity, which depend on the FOXO transcription factor DAF-16. In addition, NMUR-1 downregulates the expression of the insulin-like peptide daf-28, but only when DAF-2 function is not reduced. This suggests a regulatory mechanism through which NMUR-1 maintains insulin receptor DAF-2 signaling at a suitable level. Thus, our studies reveal that NMUR-1 serves to buffer the dynamic range of DAF-2 receptor signaling, thereby optimizing pharyngeal health and survival in response to specific bacteria.
Author summary
Host-microbial interactions influence animals’ survival dynamics. A key pathway that regulates survival is insulin receptor signaling, which responds to bacterial-derived cues. Because too high or too low insulin receptor signaling can be harmful, its activity must be kept within a certain optimal range. However, how animals maintain this optimal range during large changes in their environment remains an important question. To address this, we used the nematode C. elegans to examine how its bacterial diet affects insulin receptor activity. We find that the neuropeptide neuromedin U receptor NMUR-1 buffers large fluctuations in insulin receptor signaling in response to specific bacterial cues, such as the lipopolysaccharide structure. NMUR-1 acts in sensory neurons to increase or decrease insulin receptor activity, which depends on the state of the receptor. NMUR-1 increases insulin receptor signaling through a mechanism that is distinct from how it decreases insulin receptor signaling. Thus, this work provides insight into how neuropeptide receptors fine-tune key metabolic pathways to support health and survival.
Citation: Sifoglu D, Pereira B, DeGregory C, Shah R, Maier W, Guan J, et al. (2026) The neuropeptide neuromedin U receptor NMUR-1 buffers insulin receptor signaling in bacteria-dependent C. elegans survival. PLoS Genet 22(6): e1012190. https://doi.org/10.1371/journal.pgen.1012190
Editor: Laura Bianchi, University of Miami, UNITED STATES OF AMERICA
Received: August 18, 2025; Accepted: May 28, 2026; Published: June 11, 2026
Copyright: © 2026 Sifoglu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: QC was funded by the ERC Starting Investigator Grant #NeuroAge 242666 and the Research Councils UK Fellowship for this work. JA was funded by the Novartis Research Foundation, Swiss National Science Foundation (31003A_134958), Wayne State University, the Alcedo family and NIH (R01 GM108962) for this work. The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Bacteria are the major dietary source for some animals [1,2], provide metabolites as part of the intestinal microbiome [3–5], or act as pathogens in other situations [3,6]. As these three major bacterial functions impact and shape an animal’s life history traits, animals must adapt and respond to their microbial environment for optimal health and survival.
In the nematode worm C. elegans, bacteria serve as its primary food source [1], supplying nutrients that stimulate differential gene expression to affect physiology and survival [7–13]. Since C. elegans eat different kinds of bacteria, its genetic tractability and the ease of studying its physiology have also made it a useful model for microbiome-derived metabolite studies [5,14]. At the same time, several bacteria have been shown to pose a threat to C. elegans [6]. To isolate the contributions of these bacterial functions, C. elegans physiology can be dissected when grown on specific bacterial types—such as wild-type versus mutant bacteria or live versus dead bacteria or different bacterial species [5–13]. These studies reveal that some bacteria are a source of nutrients, metabolites, and/or infection [10,11,13,15].
In the laboratory, C. elegans usually feeds on a diet of the B-type E. coli OP50 [16], which is not part of the animal’s native microbiome [5]. E. coli OP50 has also been shown to be pathogenic to the worm [15,17]. Colonization of C. elegans pharynges by live, proliferating OP50 leads to swelling of the pharynx, ultimately killing the animals—a type of death known as P-death [15,17]. Presently, the mechanism(s) underlying this type of death remain unclear. For example, the OP50 bacteria-derived cue(s) that promote P-deaths are unknown. Previously, we have shown that worms grown on E. coli OP50 live shorter than worms grown on a K-12 type of E. coli, CS180, and that this lifespan difference is at least partly dependent on the E. coli lipopolysaccharide (LPS) structure [7]. Here we show that LPS structure also mediates OP50-dependent P-deaths in C. elegans.
In the host, only a few C. elegans genes have been implicated in modulating P-deaths [17–19], which include regulators of innate immunity. Of particular interest is the neuropeptide neuromedin U receptor nmur-1, which elicits distinct responses to different pathogenic bacteria [20]. nmur-1 promotes survival against the pathogen Enterococcus faecalis, limits survival on Salmonella enterica, and has no effect on Pseudomonas aeuruginosa [20]. Interestingly, nmur-1, which is expressed in several sensory neurons and in subsets of interneurons and motor neurons [7,21], has also been shown to mediate the E. coli OP50-dependent effects on mitochondrial function and longevity in C. elegans [7,11]. However, the nmur-1 deletion allele, ok1387, used in these studies is also tightly linked to a second mutation, ot611, which is located in the gene filamin-2 (fln-2), whose gene product promotes P-deaths [18]. To dissect the effects of nmur-1 on OP50-dependent P-deaths, we recombined fln-2(ot611) away from nmur-1(ok1387).
Here we show that NMUR-1 has complex effects on OP50-dependent C. elegans survival but has little or no effect on CS180-dependent worm survival. In the presence of wild-type insulin receptor DAF-2 signaling, the other known regulator of P-deaths [17,19], wild-type NMUR-1, NMUR-1(+), inhibits P-deaths on OP50. Intriguingly, NMUR-1(+) produces an opposite response on OP50 when DAF-2 insulin receptor function is reduced. In this context, NMUR-1(+) now increases P-deaths, as well as deaths that are not associated with swollen pharynges (non-P deaths). This interaction with DAF-2 suggests that NMUR-1(+), which acts in sensory neurons, adjusts the dynamic range of insulin receptor signaling, a pathway known to be important for survival (reviewed by [22]). We further find that NMUR-1(+) specifically regulates the expression of an insulin-like peptide (ILP) ligand, daf-28, when DAF-2 function is unimpaired, which suggests that NMUR-1(+) tunes insulin receptor signaling through defined ILPs. Thus, this mechanism should provide the physiological flexibility necessary in coping with diverse microbial environments.
Results
E. coli LPS structure modulates C. elegans survival dynamics
The E. coli B-type OP50 caused worms to live shorter than a K-12 type E. coli, CS180 (Fig 1A; Table 1 and S1 Table; [7]). Worms on OP50 also had a higher rate of early deaths compared to worms on CS180 (Fig 1A), which suggests the presence in OP50 of an early hazard that is absent from CS180. Early deaths on OP50 due to bacterial colonization can be visualized by swollen pharynges (P-deaths; compare Fig 1D to 1F; [15,17]). Since CS180 reduced deaths in early adulthood (Fig 1A; Table 1 and S1 Table), we tested if P-deaths contributed to the lifespan differences between wild-type worms on the two bacteria (see Materials and Methods on determination of P-deaths; S1 Fig; S2 Table). First, we observed that all wild-type P-deaths on OP50 or CS180 occurred by day 15 of adulthood (S2 Fig; S3 Table). Second, upon censoring all non-P deaths, we found that OP50-fed worms showed about 3 times more P-deaths, when compared to CS180-fed worms (Fig 1B and 1C; Table 1 and S1 Table), revealing that worms on CS180 were more resistant to P-deaths.
(A) Wild-type C. elegans fed E. coli OP50 had more early deaths compared to C. elegans fed E. coli CS180. (B) The early deaths depended on swollen pharynges (P-deaths) in worms fed OP50 compared to worms fed CS180. To generate the survival plots that only depict P-deaths in this figure and subsequent figures, we censored all non-P deaths. (C) The fraction of P-deaths out of 144 total deaths on OP50 or 164 total deaths on CS180. (D) DIC image of a non-swollen pharynx in a live one-day old adult worm. (E-F) DIC images of dead five-day old adult worms that either have a swollen pharynx (E) or a non-swollen pharynx (F). Scale bar is 10 µm. (G) LPS structures of OP50 and CS180. The red scissors indicate the LPS truncations that correspond to CS2198 and CS2429, which are derived from CS180. (H-I) All deaths (H) versus P-deaths (I) of wild-type C. elegans on CS180 and the LPS-truncated CS2198 and CS2429. (J) The fraction of P-deaths out of 64 deaths on CS180, 61 deaths on CS2429 and 65 deaths on CS2198. Chi-square analyses were carried out to determine significant differences between the fractions of P-deaths among the different groups of animals on the different bacteria in this figure and subsequent figures. *** denotes P < 0.001, whereas **** denotes P < 0.0001. See Table 1 for the rest of the statistical analyses that also pertain to this figure and subsequent figures.
Next, we asked what bacterial cues might contribute to the P-death differences between OP50-grown and CS180-grown worms. We previously showed that the E. coli LPS structure can modulate C. elegans longevity [7]. CS180 LPS truncation mutants, CS2198 and CS2429 (Fig 1G), have been shown to shorten wild-type worm lifespan [7]. Hence, we compared the number of P-deaths on both CS2198 and CS2429 to those on CS180. While we found that only the short LPS mutant E. coli CS2198 decreased worm lifespan in this study (Fig 1H; Table 1 and S1 Table), both E. coli strains with the shorter LPS produced more P-deaths than CS180 (Fig 1I and 1J; Table 1 and S1 Table). Thus, our results show that altering the core LPS structure is sufficient to promote E. coli colonization of the pharynx and increased P-deaths.
Opposing effects of nmur-1 on E. coli OP50 depends on the daf-2 insulin receptor
We then asked what host genetic factors influence the bacterial-dependent P-deaths. One candidate gene is the neuropeptide neuromedin U receptor nmur-1, which has been demonstrated to mediate bacteria-specific innate immune responses [20], some of which might depend on the LPS structure of E. coli [7]. The nmur-1(ok1387) deletion mutation used in these studies is tightly linked to the ot611 mutation present in the putative actin-binding scaffold protein gene fln-2, which also regulates P-deaths [18]. To address OP50-dependent P-death phenotypes that are specific to the nmur-1 deletion, we separated the fln-2(ot611) and nmur-1(ok1387) mutations.
This approach enabled us to dissect the complex effects of the isolated nmur-1(ok1387) mutation. First, nmur-1(ok1387) produced a short lifespan on OP50 but not on CS180, whereas the fln-2(ot611) mutant lived long only on OP50 (Fig 2A; Table 1 and S4 Table). While fln-2(ot611) mutants also had fewer P-deaths (Fig 2B and 2C; Table 1 and S4 Table), the nmur-1(ok1387) mutant had more P-deaths on OP50 but not on CS180 (Fig 2D and 2E; Table 1 and S4 Table). These results were recapitulated in a second independent deletion allele of nmur-1, lst1672 ([21]; Fig 2F and 2G; Table 1 and S4 Table). Loss of nmur-1 affected survival largely through pharynx-dependent deaths (S3A to S3B Fig; S5 and S6 Tables). When we only counted deaths that are characterized by unswollen pharynges (non-P deaths; S3C Fig; S5 and S6 Tables), this time censoring all P-deaths, the survival of nmur-1 mutants is more similar to wild-type survival. In this context, nmur-1(+) acts to protect C. elegans from P-deaths in a bacterial-dependent manner.
(A) On OP50, the nmur-1(ok1387) single mutant shortened lifespan whereas the fln-2(ot611) single mutant extended lifespan. On CS180, nmur-1 mutants lived slightly longer than wild type, but fln-2 mutants lived like wild type. (B) fln-2(ot611) had fewer P-deaths on OP50. (C) The fraction of P-deaths on OP50 out of 138 deaths for wild type and 162 deaths for fln-2(ot611). (D) nmur-1(ok1387) had more P-deaths on OP50 but not on CS180. (E) The fraction of P-deaths on OP50 out of 203 deaths for wild type and 231 deaths for nmur-1(ok1387). (F) A second allele of nmur-1, lst1672, also shortened lifespan and increased P deaths on OP50. (G) The fraction of P-deaths on OP50 out of 257 deaths for wild type and 269 deaths for nmur-1(lst1672). The following symbols denote: **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.
Intriguingly, the effect of nmur-1 deletion on OP50-dependent deaths is altered by the presence of mutations that reduce DAF-2 protein activity. The wild-type insulin receptor DAF-2 promotes deaths caused by bacterial colonization and pharyngeal swelling [17,19]. In insects and mammals, neuromedin U signaling influences insulin signaling by suppressing insulin secretion under certain contexts [23–27], which led us to test whether the P-death phenotype of nmur-1 mutations would be daf-2-dependent. Unexpectedly, the daf-2(e1368) mutation, which decreases receptor protein function, not only lengthened lifespan and suppressed P-deaths but also revealed that the nmur-1 mutations have bi-directional effects on lifespan and P-deaths. Unlike animals with wild-type DAF-2 function (Fig 2A and 2D to 2G; Table 1 and S4 Table), nmur-1(ok1387) and nmur-1(lst1672) now led to fewer P-deaths in daf-2 reduction-of-function mutant backgrounds (e1368 or mu150) on OP50 (Fig 3; Table 1 and S7 Table), but not on CS180 (Fig 3A; Table 1; S7 Table). Thus, deletion of nmur-1 further extends the long lifespan of daf-2 mutants in a bacteria-dependent manner (Fig 3; Table 1 and S7 Table).
(A) In the daf-2(e1368) reduction-of-function mutant background, nmur-1(ok1387) had an opposing effect on lifespan on OP50—nmur-1(ok1387) increased longevity by decreasing P-deaths. Again, nmur-1(ok1387) had little effect on CS180. (B) A second allele of nmur-1, lst1672, also increased lifespan and reduced P-deaths in the daf-2(e1368) mutant background. (C) nmur-1(ok1387) also enhanced the lifespan of another reduction-of-function allele of daf-2, mu150, which led to fewer P-deaths. (D) The fraction of P-deaths on OP50 out of 203 deaths for wild type, 231 deaths for nmur-1(ok1387) single mutants, 182 deaths for daf-2(e1368) single mutants and 206 deaths for daf-2(e1368); nmur-1(ok1387) double mutants out of 2 trials. (E) The fraction of P-deaths on OP50 out of 165 deaths for wild type, 160 deaths for nmur-1(lst1672) single mutants, 105 deaths for daf-2(e1368) single mutants and 156 deaths for daf-2(e1368); nmur-1(lst1672) double mutants out of 2 trials. (F) Left panel: The difference in the median time of all deaths between wild type and daf-2 reduction-of-function mutants in the presence (n = 6 trials) or absence of nmur-1 (n = 6 trials). Right panel: The difference in the median time of P-deaths between wild type and daf-2 reduction-of-function mutants in the presence (n = 9 trials) or absence of nmur-1 (n = 10 trials). Significance in median differences is determined by the Mann-Whitney test. The following symbols denote: *, P < 0.05; **, P < 0.01.
In contrast to animals with wild-type daf-2, the nmur-1 mutation modulated both P-deaths and non-P deaths in a daf-2 reduction-of-function mutant background (S3D to S3F, S4A and S4C Figs; S5, S6 and S8 Tables). This suggests that nmur-1(+) affects survival through other mechanisms besides pharyngeal colonization. Importantly, the opposing nmur-1 mutant phenotypes in the wild-type daf-2 versus mutant daf-2 backgrounds suggest that NMUR-1(+) adjusts and buffers insulin receptor protein activity. Specifically, loss of nmur-1 enhances the impact of daf-2 mutations on lifespan: it increases the difference in the median time of death between wild type and daf-2 reduction-of-function mutants, when considering either total deaths or P-deaths (Fig 3F; S7 Table). This increase in the dynamic range of median lifespan between wild-type and daf-2 mutants implies that loss of nmur-1 leads to an animal’s greater sensitivity to DAF-2 protein activity levels. Together, these results suggest a role for wild-type NMUR-1 in buffering the impact of DAF-2 receptor activity on lifespan.
nmur-1 promotes opposing effects on survival by acting in sensory neurons in response to LPS structure
To address the mechanisms through which nmur-1 exerts its multiple activities, we first verified its role through rescue experiments. Expression of nmur-1(+) from its own promoter [21] rescued the nmur-1 short-lived single mutant phenotype (Fig 4A; Table 1 and S9 Table). When daf-2 activity is reduced, extrachromosomal expression of nmur-1(+) from its own promoter also rescued the longer life phenotype due to the nmur-1 mutation (Fig 4C; Table 1 and S9 Table). The same construct rescued the P-death (Fig 4C; Table 1 and S9 Table) and non-P death phenotypes caused by nmur-1(ok1387) in the daf-2(e1368) background (S4A and S4B Fig; S5 and S8 Tables).
(A-B) The longevity and P-death phenotypes of nmur-1(ok1387) single mutants that were rescued in nmur-1-expressing cells (A) or in sensory neurons alone (B). (C-D) The longevity and P-death phenotypes of daf-2(e1368); nmur-1(ok1387) double mutants, where nmur-1 was rescued in nmur-1-expressing cells (C) or in sensory neurons alone (D).
Next, we sought to determine where nmur-1(+) acts to influence C. elegans survival. Expression of nmur-1(+) from the sensory neuron-specific promoter osm-6p [21] rescued the nmur-1 mutant survival phenotypes in wild-type and mutant daf-2 backgrounds (Fig 4B and 4D, S4C and S4D Fig; Table 1, S5, S8 and S9 Tables). This result suggests that NMUR-1(+) in sensory neurons inhibits P-deaths when DAF-2 protein activity is wild type, although it has less of an effect on P-deaths when DAF-2 receptor activity is reduced (Fig 4B and 4D; Table 1 and S9 Table). Interestingly, NMUR-1(+) in sensory neurons rescued the non-P death phenotype of daf-2(e1368); nmur-1(ok1387) double mutants (S4C and S4D Fig; S5 and S8 Tables) more robustly than the P-death phenotype of these animals (Fig 4D; Table 1 and S9 Table). Together these data suggest that wild-type NMUR-1 acts in sensory neurons to exert its multiple, context-dependent effects on C. elegans survival.
We also wanted to test whether LPS structure influences nmur-1 activity to modulate P-deaths. E. coli CS180 has little effect on the nmur-1 mutant phenotype in wild-type or mutant daf-2 background (Fig 5A; Table 1 and S10 Table). However, a truncation of the LPS structure in E. coli CS2429 recapitulated the nmur-1 mutant phenotypes on OP50: (i) an increase in P-deaths and shortening of lifespan when DAF-2 is wild type; and (ii) a decrease in P-deaths and lengthening of lifespan when DAF-2 receptor activity is reduced (Table 1 and S10). Thus, these findings suggest that LPS structure plays a role in the NMUR-1(+)-modulation of infection-dependent P-deaths that is mediated by DAF-2 receptor protein activity.
(A-B) The longevity and P-death phenotypes of nmur-1(ok1387) single mutants and daf-2(e1368); nmur-1(ok1387) double mutants on CS180 (A) versus CS2429 (B).
nmur-1 regulates the sensory neuron expression of the insulin-like peptide daf-28 in a context-dependent manner
The complex interactions between nmur-1 and the daf-2 insulin receptor motivated us to determine if they act in the same pathway. To test whether NMUR-1 acts upstream of the DAF-2 receptor, we determined if loss of nmur-1 affects the expression of some ILPs. C. elegans has forty ILPs that are organized into an ILP-to-ILP network, where some ILPs have been proposed to act as agonists or antagonists of DAF-2 [28]. We focused on two ILPs, ins-6 and daf-28, which encode potential DAF-2 agonists with known roles in lifespan and whose expression are modulated by bacteria-derived cues [29–32]. Because ins-6 and daf-28 overlap in expression [29,32,33] with nmur-1 in the sensory neuron ASJ [34,35], we compared the expression of the two ILPs in the ASJ neurons of control animals versus nmur-1 loss-of-function mutants.
While deletion of nmur-1 had no effect on ins-6 expression in ASJ (Fig 6A; S11 Table), it significantly increased daf-28 expression in these neurons (Fig 6B and 6C; S11 Table). This raises the possibility that nmur-1 alters the expression of ILPs, such as daf-28, to exert its effects on survival. Consistent with this possibility, a deletion of daf-28 decreased the number of P-deaths (Fig 6D; Table 1 and S11 Table). This suggests that NMUR-1(+) inhibits daf-28 expression, which can subsequently suppress wild-type DAF-2 receptor function and promote survival. However, the inhibitory effect of NMUR-1(+) on daf-28 expression only occurs when DAF-2 function is wild type (Fig 6B and 6C; S11 Table), which suggests that wild-type NMUR-1 regulates the expression of certain ILPs within the context of DAF-2 receptor function. Thus, NMUR-1(+) might regulate other ILPs when the DAF-2 receptor has reduced activity.
(A-B) The effects of an nmur-1 deletion on the ASJ neuron expression of the ins-6p::mCherry transcriptional reporter drcSi68 [A; n = 45, wild type; n = 41, nmur-1(ok1387)] and the daf-28p::mCherry transcriptional reporter drcSi98 [B; n = 46, wild type; n = 44, nmur-1(ok1387)] at mid-L4 on OP50. (C) The effects of the loss of nmur-1 on daf-28p::mCherry expression in ASJ neurons of mid-L4 larvae that have wild-type (black versus red circles) or reduced (grey versus blue circles) DAF-2 function [n = 49, wild type; n = 52, nmur-1(ok1387); n = 49, daf-2(e1368); n = 54, daf-2(1368); nmur-1(ok1387)] on OP50. (D) The OP50-dependent P-death phenotypes of animals that have wild-type daf-28 or a deletion in the daf-28 gene. ** indicates P value ≤ 0.01.
Wild-type NMUR-1 modulates the activity of a mutant DAF-2 receptor in a daf-16-dependent manner
The FOXO transcription factor DAF-16 is the downstream effector of many DAF-2 functions that include longevity (reviewed by [22]), leading us to test if nmur-1 mutant phenotypes are also daf-16-dependent. As with prior work [19], we found that loss of daf-16 increased the number of all deaths, including P-deaths (Fig 7A; Table 1 and S12 Table), and suppressed the effect of daf-2 mutations on all types of deaths (Fig 7B; Table 1 and S12 Table). Animals that lack nmur-1 in wild-type and mutant daf-2 backgrounds also lived as short as daf-16 single mutants when daf-16 was deleted in all these animals (Fig 7A and 7B; Table 1 and S12 Table). This raises the possibility that wild-type NMUR-1 acts with DAF-16 in the same pathway to modulate survival.
(A-B) The daf-16-dependence of the longevity and P-death phenotypes of nmur-1(ok1387) single mutants (A) and of daf-2(e1368); nmur-1(ok1387) double mutants (B). (C) The effects of an nmur-1 deletion on the expression of the DAF-16 target sod-3p::GFP, muIs84 [36], in wild-type or mutant daf-2 background. The quantification of sod-3p::GFP in the procorpus and anterior pharyngeal bulbs of one-day old animals on OP50 are shown [n = 34, wild type; n = 34, nmur-1(ok1387); n = 32, daf-2(e1368); n = 36, daf-2(1368); nmur-1(ok1387)]. * indicates P value ≤ 0.05, whereas “au” means arbitrary units.
To test whether NMUR-1(+) modulates DAF-16 activity, we measured the effect of the nmur-1 mutation on the expression of a DAF-16 target gene, the manganese superoxide dismutase sod-3, using an integrated sod-3p::GFP reporter [36]. While loss of nmur-1 alone in the presence of wild-type DAF-2 had no effect on sod-3p::GFP, animals that are mutant for both daf-2 and nmur-1 had significantly higher sod-3p::GFP expression than animals that are mutant for daf-2 alone (Fig 7C; S12 Table). Together these findings suggest that wild-type NMUR-1 modulates mutant DAF-2 receptor signaling by decreasing DAF-16 activity, whereas wild-type NMUR-1 might inhibit wild-type DAF-2 signaling in parallel to DAF-16 (Fig 8; see Discussion below).
See text for details.
Discussion
Bacteria can modulate insulin signaling as pathogens, food, or part of the microbiome ([37]; reviewed by [38,39]), thereby influencing key physiological processes that are important for survival. Here we used C. elegans genetics to dissect the contributions of these interactions on lifespan. By stratifying early and late deaths due to different bacteria-host interactions in a population, we reveal how wild-type NMUR-1 contributes to the overall survival dynamics under normal and reduced insulin signaling. Through systematic analyses of gene-gene and gene-environment interactions, our findings reveal a new role for the neuromedin U pathway in buffering the effects of perturbations to insulin signaling during bacteria-host interactions.
Neuromedin U receptor NMUR-1 modulates survival dynamics
The survival curve of C. elegans is produced primarily by early deaths due to bacterial accumulation in the pharynx that are analogous to infection [15,17] and late deaths due to other causes. Here we implicate the neuropeptide neuromedin U receptor NMUR-1 as a modulator of both early and late deaths by acting from sensory neurons. While the NMUR-1 effects on early deaths are consistent with a role in pathogen-specific innate immune responses, its effects on late deaths suggest additional role(s).
We also show that the NMUR-1 effect on early deaths occurs in response to the bacterial LPS structure. Furthermore, NMUR-1 can exert opposing effects on lifespan depending on DAF-2 receptor protein activity. More importantly, our findings on the bi-directional effects of NMUR-1 on pharyngeal-dependent survival suggest a model where NMUR-1 adjusts the dynamic range of insulin receptor signaling to promote tissue health and longevity (Fig 8).
Bacterial LPS structure interacts with nmur-1 to influence early death
We show that the bacterial LPS structure determines the frequency of early deaths caused by bacterial colonization of the pharynx (Figs 1E, 1G to 1J and 5; Table 1, S1 and S10 Tables). The LPS might affect C. elegans pharyngeal integrity by changing pharyngeal pumping rates [7]. However, this possibility is not supported by our previous findings that wild-type animals have similar pumping rates on E. coli OP50, CS180 or CS2429, which have different LPS structures ([7]; and references therein). Alternatively, LPS structure might affect bacterial adherence to the pharyngeal tissues, where LPS acts as an important stimulator for the host immune system [40–43]. Some E. coli strains have an O-antigen that promotes adherence to tissues, an important step in pathogenesis [44]; but the strains used here lack an O-antigen (Fig 1G; [7]]. Unlike the O-antigen, the bacterial core LPS has been shown to be less adhesive, although the core LPS may regulate the expression of adherence proteins [45–47]. For example, the truncated LPS core of E. coli CS2198, CS2429 and OP50 might stimulate or hinder specific immune responses in C. elegans.
Here we find that LPS structure modulates the two activities of the C. elegans neuropeptide receptor NMUR-1 in altering pharynx-dependent deaths (Fig 5; Table 1 and S10 Table). Sun and colleagues have recently shown that nmur-1 regulates different immune responses to specific bacterial pathogens [20]. Wild-type nmur-1 promotes resistance to Enterococcus faecalis, inhibits resistance to Salmonella enterica and has no effect on survival on Pseudomonas aeruginosa [20]. While bacterial LPS has not been directly implicated in these differing responses, all three bacteria have different cell wall and LPS compositions: E. faecalis is a Gram-positive bacterium, which likely lacks an LPS, similar to many Gram-positive bacteria [48,49], whereas S. enterica and P. aeruginosa are both Gram-negative bacteria with different LPS structures [50, 51]. Interestingly, the intestinal accumulation of E. faecalis in nmur-1 mutants [20] is reminiscent of the bacterial colonization of pharynges on the truncated LPS mutant CS2429 (Fig 5; Table 1 and S10 Table).
In rodents, LPS-induced responses are also modulated by the neuromedin U (NMU) peptide, a ligand of mammalian NMUR [52–54]. LPS exposure increases the production of the inflammatory cytokine interleukin IL-6 from peritoneal macrophages, which is abolished by the loss of the NMU peptide [53]. In this study [53], the presence of NMU promotes inflammation and LPS-induced mortality. However, in another study [54], NMU is shown to be protective against LPS-induced neuronal death, where NMU promotes the production of the neuroprotective brain-derived neurotrophic factor, BDNF, but has no effect on interleukins. While it is unclear whether the two studies used the same LPS isolate [53,54], the NMU/NMUR signaling pathway has differing responses to LPS in both C. elegans and rodents. Since the NMU signaling pathway mediates LPS responses in mammals, and LPS has also been shown to affect mammalian insulin activity [55,56], we propose that the differing NMUR-1 responses in C. elegans depend on the levels of insulin receptor activity (Fig 8), as we discuss below.
nmur-1 buffers insulin receptor signaling levels to maintain health
The insulin signaling pathway regulates C. elegans immune responses (reviewed by [57]), pharyngeal health, and survival [17,19]. Severe reduction or hyperactivation of insulin receptor activity is deleterious to the animal. Insulin receptor daf-2 null mutants exhibit lethality or embryonic and larval arrest [58], whereas a gain-of-function mutation in daf-2 results in short-lived animals that are vulnerable to stressors [59,60]. These studies suggest the importance of maintaining insulin receptor signaling levels at an optimal level. Modulators provide a mechanism for fine-tuning insulin receptor activity in fluctuating environments [61].
Wild-type nmur-1, which is co-expressed with the daf-2 insulin receptor and/or its ILP ligands in neurons [7,21,34,62], can serve as a potential modulator of DAF-2 receptor activity in regulating pharyngeal health (Fig 8). Here we show that wild-type NMUR-1 promotes healthy pharynges and prevents death (Fig 2; Table 1 and S4 Table), presumably by decreasing wild-type DAF-2 receptor signaling (Fig 8, condition 1). In contrast, when DAF-2 has reduced activity because of a mutation, NMUR-1 decreases the number of healthy pharynges and increases deaths (Fig 3; Table 1 and S7 Table), this time by potentially upregulating DAF-2 signaling (Fig 8, condition 2). These bi-directional effects of NMUR-1(+) on pharyngeal health and survival are features of neuromodulators, which ensure that cells and tissues signal within an optimal range to function appropriately across different environments [61,63]. Here we propose that NMUR-1(+) modulates tissue and cell activities by buffering and preventing large fluctuations in DAF-2 signaling. This is supported by how NMUR-1 limits the differences in median survival between wild type and daf-2 reduction-of-function mutants (Fig 3F; S7 Table).
NMU signaling suppresses insulin secretion from Drosophila insulin-producing cells [23] and mammalian pancreatic β-cells in some [24–27] but not all contexts [64,65]. In C. elegans, NMUR-1(+) may ensure that the insulin receptor signals appropriately by regulating the expression of specific ILP ligands. Here we show that NMUR-1(+) specifically suppresses the expression of the ILP daf-28, but not of ins-6 (Fig 6A to 6C; S11 Table), when DAF-2 function is wild type. Because wild-type DAF-28 increases P-deaths (Fig 6D; Table 1 and S11 Table), this suggests that NMUR-1(+) limits wild-type DAF-2 receptor signaling by downregulating its agonist ligand DAF-28 (Fig 8, condition 1). Intriguingly, in this context, NMUR-1(+) does not increase DAF-16 activity, as demonstrated by its lack of effect on the DAF-16 target gene sod-3 (Fig 7C; S12 Table). This suggests that NMUR-1(+) might inhibit wild-type and daf-28-dependent DAF-2 signaling through a mechanism parallel to DAF-16 (Fig 8, condition 1). However, DAF-16 has multiple isoforms, not all of which activate sod-3 transcription [66]. Since the deletion mutation we used here, mu86 (Fig 7A and 7B), removes all functional isoforms of the DAF-16 protein [66], it remains a possibility that NMUR-1(+) impedes wild-type DAF-2 signaling through a specific DAF-16 isoform.
On the other hand, when DAF-2 has a mutation-induced reduction in receptor function, NMUR-1(+) now increases its activity (Fig 8, condition 2) in a daf-16-dependent manner, but without altering the expression of the ILP daf-28 (Fig 6C; S11 Table). Considering that the worm has 40 ILPs with different functions [28,29,33,67], it is possible that NMUR-1(+) will regulate other ILPs under this scenario.
How then does wild-type NMUR-1 determine when to promote or inhibit DAF-2 signaling? One possibility is that some DAF-2/DAF-16 targets signal back to NMUR-1 and/or its ligands. Through such a feedback mechanism, NMUR-1(+) can buffer and modulate the levels of insulin receptor signaling. Thus, in the presence of NMUR-1(+), the C. elegans insulin receptor is neither hyperactive nor hypoactive in response to the bacteria in the animal’s environment (Fig 8). This model highlights a mechanism that ultimately prevents large deviations in insulin pathway activity (Fig 8), which is necessary in optimizing pharyngeal health and survival.
The C. elegans pharynx also resembles the mammalian heart both structurally and mechanistically [68], whose health is susceptible not only to diet [69] but also to bacterial infections [70–72]. Moreover, mammalian insulin signaling plays a role in promoting cardiac health versus disease states [73–75]. Because of the high degree of conservation between C. elegans and mammals, we speculate that the NMUR-1-mediated buffering of insulin receptor signaling in C. elegans might also exist in higher animals.
Materials and methods
C. elegans strains and growth conditions
All C. elegans mutants used in this study were backcrossed at least three times to wild type. Mutants that were used in the survival assays are reported with their genotypes in Table 1 and in S5 Table. All experiments were carried out at 25°C. However, all worms were grown for at least two generations at 20oC on the specified bacteria, before they were shifted to 25oC past the dauer larval arrest decision stage, including animals carrying the daf-2(e1368) or daf-2(mu150) mutation [58,76,77]. The temperature 20oC is permissive for growth for daf-2 mutants, which prevents dauer entry, whereas 25°C is non-permissive for these animals [58].
Bacterial strains and growth conditions
The bacterial strains that were used in the study are E. coli OP50, E. coli CS180, E. coli CS2198, and E. coli CS2429 (see [7]; and references therein). Bacterial strains were grown from single colonies in Luria-Bertani media at 37°C until the log-phase, with an optical density (OD) of ~0.6 at 600 nm. For the experimental assays, 6-cm Nematode-Growth (NG) agar plates [16] were seeded with approximately 250 μl of bacteria and streaked to cover the entire plate (full-lawn bacterial plates). We used full-lawn plates during the lifespan assays and ILP and sod-3p::GFP imaging to prevent the confounding factor of worms avoiding the bacterial lawns [78]. Plates were incubated at 25°C overnight before they were used for any experiment.
Recombining nmur-1(ok1387) away from fln-2(ot611)
To recombine nmur-1(ok1387) away from fln-2(ot611), which is about 420 kilobases away on chromosome X of the QZ58 C. elegans strain, QZ58 was crossed to wild type. Among the subsequent progeny of the nmur-1 fln-2/+ + cross-progeny, we identified 2 recombination events out of 206 chromosomes: one progeny was homozygous for the fln-2(ot611) mutation and heterozygous for nmur-1(ok1387); another animal was homozygous for nmur-1(ok1387), but not for fln-2(ot611). These animals were allowed to reproduce to isolate the nmur-1 single mutant and the fln-2 single mutant. The mutations were detected by PCR.
The ok1387 deletion was detected by using the primers: ok1387 fw (5’-ATA AGT GTC ATA GAT ACA GG-3’); ok1387 rv (5’-AAT ACA TAT ACT GAT TGA CC-3’); and ok1387 int rv (5’-AAT GCT ATG GCA GAG AAG TG-3’). The mutant was detected as a 441-bp band, whereas wild type was detected as a 602-bp band.
The ot611 point mutation was detected by using a forward primer whose 3’ end is complementary to the adenine point mutation and generates a 253-bp band with the ot611 reverse primer, 5’-CCT GTC ACA TGA GCA CTA ATG TC-3’. The wild-type allele of fln-2 was detected by using a forward primer whose 3’ end is complementary to cytosine and generates a 253-bp band with the ot611 reverse primer. The presence or absence of the wild-type and ot611 alleles were further confirmed by sequencing. We used the ot611_F primer, 5’-GTC ACT ATA ATA GAC GCC GTA ATG C-3’, and the ot611 reverse primer to generate a 536-bp fragment that was sequenced to determine whether position 301 of the fragment is a C or an A.
Lifespan assays
Worms were picked for all experiments at the late L4 stage at 25oC and were transferred onto full lawns of the specified bacteria daily for the first 6 days of adulthood, thereby preventing the mixing of subjects with their progeny. The details of the censoring during experiments are explained in the legend of Table 1. Kaplan-Meier estimates were done using the JMP 8.0.1 software (SAS). P values of both Wilcoxon and log-rank tests are reported in the data tables. The Wilcoxon test is the better measure of statistical significance when hazard ratios are not constant throughout an assay [7,79], which is the case for most of our survival comparisons. For animals that carry the extrachromosomal array ofm-1p::GFP in Fig 6D, late L4 larvae were selected under blue light to visualize the green fluorescence.
Necropsy analysis to determine P-deaths versus non-P deaths
The pharynges of all the dead animals in survival assays were imaged using a Nikon Eclipse Ni-U microscope and a Photometrics Coolsnap ES2 camera at 400x magnification. The surface area of the terminal pharyngeal bulb (see Fig 1C to 1E) was measured using the NIS-Elements software (Nikon Instruments, Inc). The surface area of the terminal bulb was then divided by the diameter of the body of the same animal at the region of the terminal bulb, which is also known as the grinder (areaP/diameterG). This normalization addressed the possibility that the general size of the animals affected the pharyngeal surface area.
Through a principal component analysis of dead wild-type animals on OP50 (n = 387) from 8 independent survival assays, we initially separated these animals into two clusters—one with swollen pharynges (P-deaths) and one without swollen pharynges (non-P deaths). Since P-deaths happen early in the lifespan of the population [15], we used areaP/diameterG and the age of death as variables. The principal component analysis was carried out in the R 4.4.2 software [80], where we plotted the data (S1 Fig) using ggplot2 [81] and ggfortify [82]. From S1 Fig, we determined the threshold areaP/diameterG that would separate the two clusters, which was a ratio value of 27. This threshold value was then used to categorize animals that died with significant pharyngeal swelling (P-deaths) or with no pharyngeal swelling (non-P deaths) in all experiments. To assess the amount of P deaths only, all non-P deaths were censored in the survival assays. To assess the amount of non-P deaths, all P deaths were in turn censored.
Imaging ILP::mCherry expression
Generation of the ILP::mCherry transcriptional reporter. The generation of the ins-6p::mCherry reporter drcSi68 is as previously described [33]. The daf-28p::mCherry drcSi98 was generated by flanking the mCherry gene with 3.3-kb sequences upstream of the daf-28 start codon and 4.7-kb sequences downstream of the daf-28 stop codon. Both 5’ and 3’ cis regulatory sequences of daf-28 were amplified from YAC Y116F11 with Phusion DNA polymerase and then cloned into the pCR-Blunt vector, which was sequenced for confirmation. The subsequent reporter was next cloned into a MosSCI vector for integration (pQL184) at the ttTi4348 site of chromosome I.
Live imaging of worms. Animals were grown on full lawns of OP50 at 20oC, before they were shifted to 25oC at the second larval stage (L2). Worms were then imaged at 1000x magnification, once they reached the mid-L4 stage at 25oC, using a Nikon Eclipse Ni-U microscope and a Photometrics Coolsnap ES2 cooled digital camera. We quantified fluorescence intensities using a built-in fluorescence quantification algorithm (NIS-Elements, Nikon Instruments, Inc). The Student’s t-test was used to compare each ILP expression between wild type and nmur-1(ok1387) single mutants in Fig 6A and 6B. To compare daf-28p::mCherry expression in wild type, nmur-1(ok1387) single mutants, daf-2(e1368) single mutants and daf-2(e1368); nmur-1(ok1387) double mutants, which were imaged in parallel (Fig 6C), one-way ANOVA and Tukey’s correction were used.
Imaging sod-3p::GFP expression
Animals that have an integrated sod-3p::GFP transgene, muIs84 [36], were also grown on full lawns of OP50 at 20oC, before they were shifted to 25oC at the L2 stage. Worms were then imaged at 400x and 100x magnification, once they reached the first day of adulthood at 25oC, using a Nikon Eclipse Ni-U microscope and a Photometrics Coolsnap ES2 camera. Using the same NIS-Elements algorithm as above, we quantified the fluorescence intensities in the procorpus and the anterior pharyngeal bulb at 400x magnification (Fig 7C), since these tissues have the brightest fluorescence in all the animals imaged. Statistical comparisons across the multiple groups of animals were determined by one-way ANOVA, followed by Tukey’s correction.
Statistical analyses
Statistical analyses were performed using JMP 8.0.1 (SAS) for all survival assays; GraphPad Prism 8 software for the ILP and sod-3p::GFP imaging measurements; and R 4.4.2 for the principal component analyses of the swollen pharynx-dependent deaths. For more details, refer to above and the figure and table legends.
Supporting information
S1 Fig. Principal component analysis (PCA) for necropsy analysis.
PCA analysis performed on areaP/diameterG and age of death (days) values of 387 wild-type animals from 8 separate experiments on OP50. Numerical values of datapoints represent the areaP/diameterG of each dead animal. Animals are separated into two clusters—probability ellipses shown in red (left side) and in blue (right side). The red cluster represents animals with swollen pharynges at death (P-deaths). The blue cluster represents animals with non-swollen pharynges at death (non-P deaths).
https://doi.org/10.1371/journal.pgen.1012190.s001
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S2 Fig. Distribution of the total P-deaths over the course of wild-type lifespan on different bacteria.
(A-B) P-deaths were no longer observed after day 15 of adulthood on OP50 (A) and on CS180 (B). The number of P deaths on OP50 was 395 out of 1070 deaths (number of trials, 10). The number of P deaths on CS180 was 55 out of 528 deaths (number of trials, 6).
https://doi.org/10.1371/journal.pgen.1012190.s002
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S3 Fig. nmur-1 has multiple and complex effects on OP50-dependent deaths.
(A-C) The survival curves of wild type and nmur-1(ok1387) single mutants (cumulative of 7 independent trials from Figs 2, 3 and 7), when all types of deaths (A) or only P-deaths (B) are included or when P-deaths are excluded (C). (D-F) The survival curves of daf-2(e1368) single mutants and daf-2(e1368); nmur-1(ok1387) double mutants (cumulative of 6 independent trials from Figs 3 and 7), when all types of deaths (D) or only P-deaths (E) are included or when P-deaths are excluded (F). See S5 Table for the statistical analyses of these data.
https://doi.org/10.1371/journal.pgen.1012190.s003
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S4 Fig. nmur-1 acts from sensory neurons to modulate non-P deaths when daf-2 activity is reduced.
(A-D) The non-P death phenotypes of daf-2(e1368) single mutants versus daf-2(e1368); nmur-1(ok1387) double mutants (A, C), where nmur-1 was rescued in nmur-1-expressing cells (B) or in sensory neurons alone (D). See S5 Table for the statistical analyses of these data.
https://doi.org/10.1371/journal.pgen.1012190.s004
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S5 Table. nmur-1-dependent P-deaths versus non-P deaths on E. coli OP50.
Cumulative statistics of all types of deaths of the indicated C. elegans strains. P values that are significant (P ≤ 0.05) are italicized and in bold face. If the test population lived longer or had fewer P-deaths than the population to which it is compared, the P values are also underlined. The superscripts indicate the population to which the test population is compared. ok1387* indicates the genotype daf-2(e1368); nmur-1(ok1387). The symbol ** denotes that the pharyngeal sizes of late deaths were left unmeasured.
https://doi.org/10.1371/journal.pgen.1012190.s009
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Acknowledgments
We thank the Caenorhabditis Genetics Center (funded by NIH P40 OD010440), I. Beets, C. Kenyon and J. Watteyne for strains used in this study. We also thank A. Caballero, D. A. Fernandes de Abreu and C. Liu for technical support in generating the drcSi98 strain, M. Friedrich for comments on the manuscript, and E. Gourgou for supporting B.P. during the revision experiments for this manuscript.
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