nlp-29 encodes a small (73-residue) secreted precursor of the YARP (YGGW-amide related peptide) / NLP-CNC antimicrobial-peptide family in Caenorhabditis elegans. The precursor carries an N-terminal signal peptide and is predicted to be processed on pairs of basic residues into several short, glycine/tyrosine-rich, C-terminally amidated peptides (QWGYGGY-amide, GYGGYGGY-amide and repeated GMYGGY/GMYGGW-amide motifs). It is produced in the epidermis (hyp7 syncytium), where its transcription is strongly and rapidly induced by infection with the natural fungal pathogen Drechmeria coniospora, by bacterial infection (Serratia marcescens), by sterile wounding, and by osmotic stress. Infection- and wound-induced expression requires an epidermal signaling cassette (a GPCR/G-protein and PKCdelta/TPA-1 input converging on the TIR-1/SARM-NSY-1-SEK-1-PMK-1 p38 MAPK cascade and the STAT-like factor STA-2 with SNF-12 and the GATA factor ELT-3), whereas the osmotic response is largely p38-independent. nlp-29 lies in a rapidly evolving, positively selected multigene cluster (nlp-27 to nlp-34) of co-regulated antimicrobial peptides that contribute to antifungal host defense. Beyond its role as a putative immune effector, secreted NLP-29 also acts as a cross-tissue signaling ligand for the neuronal orphan G protein-coupled receptor NPR-12, promoting protective sleep after wounding or infection and driving age- and infection-associated dendrite degeneration of PVD and FLP sensory neurons.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: Localization to the extracellular region, mapped from the UniProt "Secreted" subcellular-location keyword. Consistent with the N-terminal signal peptide (residues 1-22) and with experimental evidence that epidermally produced NLP-29 acts non-cell-autonomously on distant neurons, implying it is secreted. Reason: NLP-29 is a signal-peptide-bearing secreted peptide; the SubCell-derived extracellular annotation is well supported. Secretion is corroborated experimentally because epidermally expressed NLP-29 must reach neurons expressing its receptor NPR-12 to signal. Supporting Evidence: PMID:29301098 direct access to the epidermis was required for NPR-12 to receive NLP-29 |
| GO:0003674 molecular_function | ND GO_REF:0000015 | REMOVE | Summary: Root molecular_function with the ND ("no biological data") evidence code, a WormBase placeholder recorded in 2019 indicating no molecular-function data were curated at that time. Reason: The ND root placeholder is uninformative and is now superseded: a specific molecular function (neuropeptide receptor binding, GO:0071855) is annotated and has since been experimentally corroborated by demonstration that NLP-29 is a ligand/agonist of the neuronal GPCR NPR-12. Per GO ND conventions, the root "no data" placeholder should be removed once informative molecular-function evidence exists. Supporting Evidence: PMID:29301098 we conclude that NPR-12 is the receptor for NLP-29 in mediating aging-associated PVD dendrite degeneration |
| GO:0005576 extracellular region | ISM PMID:11717458 Identification of neuropeptide-like protein gene families in... | ACCEPT | Summary: Sequence-model (ISM) prediction of extracellular localization, based on the preproprotein/signal-peptide architecture identified for the nlp genes. Redundant with the SubCell-derived IEA annotation but biologically valid. Reason: The prediction is sound: NLP-29 has a cleavable signal peptide and is a processed, secreted peptide, consistent with an extracellular location. Retained as a valid duplicate of the SubCell IEA annotation. Supporting Evidence: PMID:11717458 peptidergic neurotransmitters are encoded as preproproteins that are posttranslationally processed to yield bioactive neuropeptides |
| GO:0071855 neuropeptide receptor binding | ISM PMID:11717458 Identification of neuropeptide-like protein gene families in... | ACCEPT | Summary: Neuropeptide receptor binding, originally a family-based sequence-model prediction (NLP-29 was named for its similarity to YGGWamide neuropeptides). This prediction is now directly corroborated: synthetic NLP-29 activates the neuronal orphan GPCR NPR-12 (calcium mobilization in NPR-12-transfected cells, specific relative to the related peptide NLP-31 and to NPR-32/beta2-AR), and genetic epistasis places NLP-29 upstream of NPR-12 in both protective-sleep and dendrite-degeneration signaling. Reason: This is the single molecular activity of NLP-29 with direct experimental support and represents a core function. The ISM annotation remains formally correct, and later work identifies NPR-12 as a bona fide NLP-29 receptor, upgrading confidence in the term without changing it. Retained as core. Supporting Evidence: PMID:29301098 induced a significant elevation of calcium, while the solvent-only control did not cause any changes PMID:33259791 it acts through the neuropeptide receptor NPR-12 in locomotion-controlling neurons that are presynaptic to RIS and that depolarize this neuron to induce sleep |
| GO:0050832 defense response to fungus | IEP PMID:18636113 Anti-fungal innate immunity in C. elegans is enhanced by evo... | NEW | Summary: nlp-29 is strongly and specifically induced in the epidermis by the natural fungal pathogen Drechmeria coniospora and belongs to a co-regulated antimicrobial-peptide cluster whose overexpression increases resistance to D. coniospora, supporting involvement in antifungal defense. This antifungal role is the flagship, most-studied aspect of nlp-29 biology and is captured by UniProt only as a keyword-derived IEA. Reason: A defense-response-to-fungus annotation for nlp-29 is well motivated by infection-induced expression plus cluster-level gain-of-function resistance, and matches the UniProt keyword annotation. Added as an expression/genetics-based (IEP) annotation. Important caveat: the nlp-29 single-gene null shows no marked resistance phenotype, so this is an involvement, not a demonstration of individual necessity (see knowledge gaps). Supporting Evidence: PMID:18636113 These results indicate that the nlp genes can contribute in vivo to increased resistance to fungal infection, but probably not to osmotic stress. PMID:15048112 We report here the identification of infection-inducible antimicrobial peptides in Caenorhabditis elegans. |
| GO:0007218 neuropeptide signaling pathway | IMP PMID:33259791 Innate Immunity Promotes Sleep through Epidermal Antimicrobi... | NEW | Summary: NLP-29 acts as a secreted neuropeptide-like ligand in a cross-tissue signaling pathway: it activates the neuronal GPCR NPR-12, and nlp-29/npr-12 genetic epistasis places NLP-29 upstream of NPR-12 in sleep-promoting and dendrite-degeneration signaling. Reason: The neuropeptide-signaling role is now experimentally supported (direct NPR-12 activation by synthetic NLP-29 and loss-of-function/epistasis phenotypes for sleep and dendrite degeneration), and matches the UniProt keyword-derived annotation. Added to capture the demonstrated signaling function alongside the antimicrobial-effector role. Supporting Evidence: PMID:33259791 it acts through the neuropeptide receptor NPR-12 in locomotion-controlling neurons that are presynaptic to RIS and that depolarize this neuron to induce sleep PMID:29301098 we conclude that NPR-12 is the receptor for NLP-29 in mediating aging-associated PVD dendrite degeneration |
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Download this section (compressed HTML)Q: Do the individual mature amidated NLP-29 peptides have intrinsic microbicidal activity against D. coniospora or S. marcescens in vitro, and if so by what mechanism?
Suggested experts: Nathalie Pujol, Jonathan J. Ewbank
Q: Is any single nlp gene in the nlp-29 cluster individually required for pathogen resistance, or is the effector function fully redundant?
Suggested experts: Nathalie Pujol, Jonathan J. Ewbank
Experiment: Synthesize each predicted amidated NLP-29 peptide and the full-length mature peptide, and test dose-dependent inhibition of D. coniospora hyphal growth and S. marcescens viability in standard microbicidal/MIC assays, with membrane-permeabilization and nucleic-acid-binding readouts.
Hypothesis: The mature NLP-29 peptides directly inhibit or kill fungi/bacteria.
Type: in vitro antimicrobial assay
Experiment: Generate precise single and higher-order CRISPR deletions across the nlp-27 to nlp-34 cluster and compare survival after D. coniospora infection, distinguishing single-gene necessity from cluster-level redundancy.
Hypothesis: nlp-29 contributes non-redundantly to antifungal survival only in combination with its cluster paralogs.
Type: combinatorial genetics / survival assay
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Whether the mature amidated NLP-29 peptides themselves directly kill or inhibit microbes, and by what mechanism (membrane permeabilization, DNA binding as reported for the paralog NLP-31, or another target), has not been demonstrated. Every C. elegans study uses nlp-29 as an induced-expression readout, and the in-vivo antifungal evidence comes from overexpression of the whole nlp-29 cluster rather than a purified-peptide killing assay for NLP-29.
OPEN BIOLOGYONTOLOGY MF_DARK
What is known: nlp-29 is a bona fide secreted AMP-family gene, strongly induced in the epidermis by fungal/bacterial infection and wounding, and overexpression of the nlp-29 cluster increases resistance to D. coniospora. Its one biochemically demonstrated molecular activity is as a ligand/agonist of the neuronal GPCR NPR-12, not a microbicidal activity.
Significance: This is why nlp-29 carries no molecular-function annotation for antimicrobial activity: such activity is both unproven for NLP-29 itself and lacks an adequate GO molecular-function term. Closing it would define the effector arm of the flagship C. elegans epidermal immune pathway.
What would resolve it: In vitro microbicidal, membrane-permeabilization and nucleic-acid-binding assays with the individual synthetic mature NLP-29 peptides against D. coniospora and S. marcescens, plus structural characterization.
Provenance (the field's own admissions):
Proposed term (ontology gap):
Gap: The specific contribution of nlp-29 alone to pathogen resistance and survival is undetermined: the nlp-29(tm1931) null shows no marked change in resistance to D. coniospora, consistent with functional redundancy within the nlp-27 to nlp-34 cluster, so single-gene loss-of-function has not established necessity.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: Loss of upstream regulators (e.g. tir-1, pmk-1, sta-2) abolishes nlp-29 induction and reduces antifungal resistance, and cluster overexpression increases resistance; but these manipulate the signaling pathway or the whole AMP cluster, not nlp-29 individually.
Significance: Determines whether nlp-29 is an individually important effector or a redundant member of a buffered AMP battery, which is central to interpreting the widely used Pnlp-29 immune reporter.
What would resolve it: Higher-order combinatorial deletions across the cluster and quantitative survival assays of precise single and compound nlp mutants under D. coniospora and S. marcescens challenge.
Provenance (the field's own admissions):
Gap: It is unknown which of the several distinct amidated peptides cleaved from the NLP-29 precursor mediate antimicrobial activity versus which engage NPR-12, and the predicted proprotein processing and C-terminal amidation have not been directly demonstrated for NLP-29.
OPEN BIOLOGY MF_DARK
What is known: The precursor sequence, signal peptide, predicted cleavage on paired basic residues and the Tyr/Trp-amidation motifs are annotated by UniProt sequence analysis; synthetic full-length NLP-29 peptide activates NPR-12 and induces dendrite degeneration, whereas the related NLP-31 does not.
Significance: Distinguishing effector peptides from signaling peptides within a single precursor would clarify whether the antimicrobial and neuroimmune functions of NLP-29 are molecularly separable.
What would resolve it: Mass-spectrometric identification of the endogenous mature peptides and structure-activity assays of individual synthetic peptides against microbes and against NPR-12.
Provenance (the field's own admissions):
Gap: Whether NLP-29's antimicrobial action is receptor-mediated or a direct physicochemical effect on microbes is unresolved. NPR-12 is the only demonstrated receptor and mediates host neuroimmune signaling (sleep, dendrite degeneration), not microbial killing, so any receptor or target underlying an effector function is unidentified.
OPEN BIOLOGY MF_DARK
What is known: NPR-12 is a validated NLP-29 receptor for cross-tissue host signaling and is expressed in neurons, not microbes; no microbial or host target for a direct antimicrobial effect is known.
Significance: Clarifies whether the same peptide uses distinct molecular mechanisms for host signaling versus pathogen defense.
What would resolve it: Target-identification (pulldown/photo-crosslinking) with labeled NLP-29 peptides on microbial and host membranes, and testing whether antimicrobial protection requires any host receptor.
Provenance (the field's own admissions):
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