Rv2520c Still unknown · low auto-curated

Annotation: from legacy to revised

Curation note: 2026-10-02 (mtbc/Rv2520c project, back-propagation P52.2, destination C1, status: published). This is one of six non-open-access articles that the full-text counter of this record (n_unverifiable = 6) cannot read; the dedicated project read all six on their PMC pages and each names Rv2520c (Mollenkopf 2004, Bertholet 2008, Coscolla 2015, Dubnau 2002, Mehaffy 2010, Rao 2008). Mollenkopf et al. chose Rv2520c among 36 DNA vaccine candidates because the gene is present in BCG while the protein was not detected in the BCG proteome on 2D gels, and call it a transmembrane protein of unknown function of 8.6 kDa. In mice its protection was intermediate and not significantly different from the vector control in most experiments (the text concludes that it failed to induce reliable protection), and as a boost it did not significantly add to BCG. It induced IFN-gamma at day 28 but not at day 35, and high IgG1 and IgG2 titres. A vaccine-candidate result in mice, not a function. 2026-10-02 (mtbc/Rv2520c project, back-propagation P52.2, destination C1, status: published). Bertholet et al. produced Rv2520c as one of 94 recombinant H37Rv proteins screened for human T-cell antigens (Table I): His-tagged in E. coli and purified to more than 90% homogeneity. The authors report that every antigen of the panel was recognised by at least 44% of PPD-positive donors (IFN-gamma recall, 18 donors); this floor belongs to the panel, not to Rv2520c, whose own response is only in a figure. The protein can be produced recombinantly and is seen by T cells of PPD-positive donors; nothing is said of its function. 2026-10-02 (mtbc/Rv2520c project, back-propagation P52.2, destination C1, status: in preparation, not peer-reviewed). BCG_2541c, the BCG orthologue of Rv2520c, is detected in the FT-MS proteome of M. bovis BCG of Zheng et al.: seven peptide identifications in Supplementary Table S3 (four distinct non-nested peptides, among them DQLAATIDSLAER and VDRDPNTIKQEIDQTR) covering 46 of 75 residues (61.3%, computed by the dedicated project; the article text does not name the gene). The BCG protein is identical to Rv2520c: RefSeq WP_003412942.1 is encoded in H37Rv, M. bovis AF2122/97 (Mb2549c) and the BCG substrains checked (Pasteur 1173P2, Tokyo 172, Mexico, Korea 1168P, Moreau, Russia), and Mycobrowser gives Mb2549c as 100.0% identical (the 98.7% in the orthologs layer is an alignment-based figure that does not match). A gel non-detection in BCG, the reason Rv2520c was chosen as a vaccine candidate in 2004 (Mollenkopf et al., above), therefore does not show that the protein is absent from BCG. Analysis of the dedicated project, not peer-reviewed. 2026-10-02 (mtbc/Rv2520c project, back-propagation P52.2, destination C1, status: published). Coscolla et al. list N74S in Rv2520c (H37Rv position 2,837,395, T to C) in their Table 2, one of 24 nonsynonymous mutations present in all Beijing Wat Tham Krabok (WTK) isolates and absent from other lineage 2 strains, in a study of the spread of a multidrug-resistant clone; no functional claim. Residue 74 lies in the C-terminal tail, outside the DUF3618 domain (3 to 48). In this atlas the change is carried by 27 strains, all lineage 2 (snapshot 2026-09-29), below the 0.1% allele-frequency floor of the conservation layer, which therefore does not count it. 2026-10-02 (mtbc/Rv2520c project, back-propagation P52.2, destination C1, status: published). Dubnau et al. tested Rv2520c by molecular-beacon RT-PCR in M. tuberculosis inside THP-1 macrophages, among candidate genes from their selection of macrophage-induced genes (the article reports 8 of 13 tested genes as induced more than twofold); the ratios to broth for Rv2520c are 2.2 +/- 1.3 at 24 h (1.1 +/- 0.1 after 2 h in RPMI) and 0.9 +/- 0.5 at 72 h (mean +/- standard error of triplicates). The error at 24 h is large and the value is back to baseline at 72 h: at most a weak, transient early induction. No function is proposed. 2026-10-02 (mtbc/Rv2520c project, back-propagation P52.2, destination C1, status: published). Mehaffy et al. detected Rv2520c in the cytosolic fraction of closely related clinical isolates (iTRAQ, Table 2 of proteins differing significantly between strains): ratios to isolate BE of 1.26 (CDC1551), 0.88 (C28) and 1.06 (H6), from one unique peptide, DQLAATIDSLAER (residues 18 to 30; protein-level probability 80.7%, 17.33% coverage). It is one of six tryptic peptides of this protein within the usual LC-MS/MS window, five of them N-terminal (count by the dedicated project, not from the article), so detection carries no information on topology. 'Cytosolic' names the fraction, not a localisation finding: the protein is also detected in a membrane fraction (Sanyal et al. 2026, localization entry below). No function. 2026-10-02 (mtbc/Rv2520c project, back-propagation P52.2, destination C1, status: in preparation, not peer-reviewed). Membrane association is observed, not only predicted: in Sanyal et al. (Mol Cell Proteomics 2026, Table S6) Rv2520c is among the proteins detected in all replicates of the membrane fraction of three strains (5 unique peptides, 48% coverage, 253 PSMs). This shows association with the fraction, not integral insertion or orientation, and the authors' own annotation columns for this protein read TM = 1 and Localization = Cytoplasmic. The boundaries of the single helix agree across tools: residues 52 to 69 (DeepTMHMM), 51 to 69 (Phobius), 51 to 69 (UniProt, automatic). Which side of the membrane carries the DUF3618 domain (residues 3 to 49) is NOT resolved: DeepTMHMM and Phobius predict opposite orientations, family-wide sequence statistics and translocation mechanics point in opposite directions, and the published datasets able to separate compartments (protease shaving, Lepe et al. 2025; APEX2 labelling and surface biotinylation, Jaisinghani et al. 2024; membrane fractions under secA1 and tatA knockdown, Sanyal et al.) do not assign it. No claim is made that the domain faces the cytoplasm or the surface. Analysis of the dedicated project, not peer-reviewed. 2026-10-02 (mtbc/Rv2520c project, back-propagation P52.2, destination C1, status: in preparation, not peer-reviewed). CRISPRi evidence beyond the H37Rv row served in the vulnerability field. (1) Bosch et al. 2021 give a second row for this gene in strain HN878: VI -5.32, interval -9.87 to -0.324 (it excludes zero, unlike the H37Rv interval), also certain = False, with crispr_ess = Essential in both strains. (2) Wang et al. (Nat Commun 2026), with a different guide library, call the gene Essential in 6 of 8 independent screens (Supplementary Data 2: parent mc2 6206 three times, five drug-resistant derivatives once each); the two screens without the call belong to one batch (ex08). Supplementary Data 3 flags it More Essential than the parent in two bedaquiline-resistant derivatives (BDQ-14, atpE A63P; BDQ-2, Rv0678 frameshift) and in none of the levofloxacin-, linezolid- or rifampicin-resistant ones; on day-14 log2 fold change (4 guides) the difference from the batch-matched parent is -1.76 and -1.53, against -0.33 to +0.86 for the other three. Each derivative was screened in one batch and the two bedaquiline strains share one, so strain and batch cannot be separated: a signal, not a demonstrated conditional vulnerability. Bosch and Wang calls are as published (Wang et al. do not name the locus in their text; the calls are in supplementary tables); the day-14 differences are the dedicated project's calculation, not peer-reviewed. 2026-10-02 (mtbc/Rv2520c project, back-propagation P52.2, destination C1, status: in preparation, not peer-reviewed). Reading of the full-text hit Talwar et al. (mSystems 2020), listed in the full-text field: Rv2520c is in its Table 3 of 136 genes with at least 60% of proline codons decoded by proT (100% here), and the article proposes that proT-rich genes are translationally repressed by the toxin VapC12 in cholesterol-induced persistence; it does not test this gene. Rv2520c has 3 proline codons, all CCC, so the 100% rests on a tiny denominator: with the genomic CCC frequency (29.4%, recomputed by the dedicated project; Talwar et al. report 36.4%) the chance of 3 of 3 is about 2.6%; 4 of 97 genes with exactly 3 proline codons reach 100% CCC (4.1%), and 24 of 3,881 genes reach 100%, 22 of them with 3 or fewer proline codons. The figure carries no weight on its own; membership in the 60% list is not contested. Calculation of the dedicated project, not peer-reviewed. 2026-10-02 (mtbc/Rv2520c project, back-propagation P52.2, destination C1, status: in preparation, not peer-reviewed). Reading of the full-text hit Ogbonna et al. (Microbiol Spectr 2023), listed in the full-text field, a ClpC1 interactome study in M. smegmatis. It names MSMEG_4752, the orthologue of Rv2520c, only in tables of co-purified proteins, at the detection floor (score 0.06 in the ClpC1 EQ-trap pull-down, rank 78 of 94 by score, 84 of 94 by spectral count), not in the wild-type dataset (so no enrichment ratio), and not retained by the authors, whose one validated substrate is MSMEI_3879. A low score is an absence of evidence of interaction, not evidence of absence. The dedicated project found no support for a ClpC1 link elsewhere: no curated IntAct interaction (checked 2026-10-02); under ecumicin (Barter et al. 2026) a fall shared by 124 of 2,542 proteins; in the M. smegmatis clpC1 knockdown (Chi et al. 2026) MSMEG_4752 decreases (0.505, q = 0.004) instead of accumulating, as in the clpX knockdown (0.649, q = 0.010), with a non-significant increase (1.57, q = 0.067) under an oxygen shift. The two-hybrid screen of Ziemski et al. (FEBS J 2021) is not open access and could not be read. Analysis of the dedicated project, not peer-reviewed.

Auto-curated: this verdict and function were generated by rules from PGAP + Pfam + Foldseek and have not been hand-reviewed.

In the literature (TB corpus sweep) named only in full text

No TB publication mentions this locus tag in its title or abstract (sweep of a ~330k-abstract PubMed TB corpus). A full-text search does however find 4 paper(s) naming this gene in their body text — see the full-text field below. The gene remains dark on function, but it is not unmentioned, and this page no longer claims that it is.

Scope of the sweep: What was actually swept: the H37Rv locus tag and the ortholog locus tags (M. bovis Mb…, M. marinum MMAR_…, M. smegmatis MSMEG_…, M. leprae ML…, M. abscessus MAB_…), plus the gene name where one exists. NOT swept: protein and enzyme names, including family shorthands such as Ldt(Mt4) or VapC28, and their orthographic variants. An absent locus-tag mention is therefore evidence about the TAG, and only indirect evidence about the protein (P18.14).

An antigen status or a virulence phenotype is NOT a molecular function: the verdict stays unchanged. This layer adds the missing context, it does not requalify the gene. This distinguishes a gene that is dark because nobody has looked from one that is dark despite having been studied. Source: PubMed (whole), MULTI-ALIAS sweep of title+abstract: the H37Rv locus tag AND every ortholog identifier (M. bovis Mb…, M. marinum MMAR_…, M. smegmatis MSMEG_…, M. leprae ML…, M. abscessus MAB_…), each hit VERIFIED against the abstract text (word-boundary regex). phase73, 2026-07-13.

Binding-pocket screen (P2Rank, geometric prediction) detector blind at this length

Read with care. This protein (75 aa) is below the size where this detector has meaningful power: on proven enzymes, only 3.8% (1/26) under 200 aa reach this layer's P2Rank confidence threshold of best_probability >= 0.70, versus 60.5% (75/124) above it (P16.3b calibration, negative control EsxA/EsxB-scale panel). Both figures are recall AT 0.70, which is also the best separation this detector reaches on that panel (Youden J = 0.416) and whose false-positive rate above 200 aa is 18.9% (81.1% specificity); the looser 0.50 that appears elsewhere in this repository would raise recall above 200 aa to 74.2% and drop specificity to 64.9%. A negative or weak pocket result here should NOT be read as evidence against a ligand-binding role -- the test essentially has no power at this length, not that the protein lacks a site. The same holds in the other direction, and that half was missing here: a STRONG score at this length is not a result either. The same calibration panel does carry that other arm below 200 aa, and the two do not separate: the 3.8% above is matched by 6.6% (5/76) of proteins annotated as non-catalytic clearing 0.70 (Fisher p = 1.00; 15.4% against 11.8% at 0.50, p = 0.73). At this length the score therefore carries no measured information in either direction -- which is a stronger statement than untested power, and the reason a strong score here is not a result. P16.3e, calibration phase72b_pocket_calibration.py, 2026-08-03.

Conditional expression context (iModulons)

Member of 1 independently-modulated gene set(s): Unc_4.

iModulon membership (independently-modulated gene sets from a 647-sample RNA-seq compendium): the conditional co-expression context. Co-expression is a regulatory context, NOT a molecular function. Source: iModulonDB / modulome_mtb (Yoo 2022).

CRISPRi vulnerability

Vulnerability index -8.08 (95% CI -16.05 to 0.38). A more negative index = more vulnerable to knockdown (better drug-target quality); indicative threshold VI ≤ -6 = highly vulnerable.

Quantitative CRISPRi knockdown, graded (finer than binary Tn-seq essentiality). Source: CRISPRi vulnerability index (Bosch 2021, supplementary Table S2 'mmc3.xlsx', bulk import via Europe PMC -- supersedes the per-gene pebble.rockefeller.edu scrape of phase46_crispri_vulnerability.py).

Caveat on this estimate. Bosch et al. flag this gene's vulnerability estimate as NOT certain, and here that call has a mechanical cause visible in the library itself: every guide targeting this gene is >=21 nt (str_span=0.403 on a 0-1 scale, near the genome floor), and guide repression strength saturates from 21 nt on -- so the library never sampled a partial knockdown for this gene, only the near-maximal repression plateau. The dose-response fit (and this VI) is therefore essentially unconstrained by data, not merely uncertain -- a limitation of the CRISPRi library design for the non-essential class (Bosch et al. 2021), not a defect in this specific measurement. n_guides=5 is descriptive only and does not predict this. Do not cite this VI as a standalone vulnerability claim.

Orthologues (reciprocal best hits across mycobacteria)

Reciprocal-best-hit orthologues (DIAMOND) against the Mycobrowser reference proteomes. A missing species is informative: e.g. a gene absent from M. leprae was likely lost in its reductive genome evolution. Locus tags link to Mycobrowser.

Curated reference (UniProt)

Functional vocabulary (eggNOG-mapper, orthology transfer)

Orthology-based transfer (eggNOG 5.0.2, diamond). EC/KO/GO/CAZy are computed annotations, not manual curation; cross-check against the primary literature before treating a specific reaction as established.

Conservation & selection (intra-MTBC, 142 601 strains, snapshot of 2026-09-29)

pN/pS from segregating SNPs (singletons removed) normalised by possible sites. Low pN/pS = purifying selection (a strong signal that a "hypothetical" is a real, constrained gene). A high pN/pS is ambiguous: relaxed constraint or positive selection (drug resistance, antigenic variation) inflate it; e.g. rpoB/katG/pncA score high here for resistance, not loss of function. A clonal disruption (one allele over a clade) suggests lineage pseudogenisation; a convergent one (many independent alleles) is typical of resistance loss-of-function.

Outgroup conservation (beyond the MTBC) Corynebacteriales

Two orthogonal outgroup signals. M. canettii (the immediate outgroup) gives a deep-divergence dN/dS (a low value confirms a constrained, real gene; shown as confident only at ≥8 substitutions, else flagged low-power). Genus-wide presence/absence (tblastn vs assembled non-MTBC genomes) places the gene on the ancient-core ↔ MTBC-specific axis: a gene absent even from the closest MTBAP relatives is a candidate MTBC-specific innovation (possible host-adaptation factor, to confirm by synteny). The phylostratum extends that axis outside the genus (tblastn vs 13 reference genomes spanning Mycobacteriaceae → Corynebacteriales → Actinomycetia → outside the phylum): it is the deepest clade in which a homolog is still detected, i.e. a proxy for gene age. Read it with the null model in mind: a shallow (young) stratum can also reflect homology-detection failure for short or fast-evolving ORFs, so it is a descriptive axis, not a proof of novelty.

Essentiality (transposon mutagenesis)

Genome-wide Himar1 transposon essentiality in H37Rv (DeJesus 2017). An essential call (ES/ESD/GD) is strong, independent evidence that a "hypothetical" locus encodes a functional, selectively required gene — orthogonal to intra-species conservation.

Proteomics (mass spectrometry) detected

Detection by mass spectrometry is direct, experimental evidence that the protein product exists — orthogonal to sequence conservation and to Tn-seq essentiality, and especially decisive for a "hypothetical" locus. Read the dataset count as abundance, not as replication. Measured across this atlas (3,491 detected proteins), the number of datasets a protein appears in tracks its abundance almost monotonically: median 1.2 ppm at 1 dataset of 16, 27 ppm at 10, 392 ppm at 15, 1,325 ppm at 16 — three orders of magnitude. A high count therefore says the protein is abundant more than it says several independent experiments confirmed it. What the count does establish, at any value, is that the product exists.

Predicted localisation (DeepTMHMM + lipobox)

Transmembrane topology and signal peptide from DeepTMHMM (deep-learning reference predictor); lipoproteins from a (myco)bacterial lipobox motif. A sequence-based prediction of subcellular context.

Physico-chemical properties (computed, ProtParam)

Computed from the ancestral MTBC0 sequence with the ExPASy ProtParam method (Biopython). Descriptive biophysical context: a positive GRAVY flags a hydrophobic (often membrane) protein, a high instability index (>40) predicts a short in-vitro half-life, an extreme pI hints at compartment or binding partner.

Domains (Pfam, hmmscan --cut_ga)

HHpred profile-profile (no confident hit): Already assessed and rejected in P7.1g-lot1 (same HIG1-domain hit at 90.3%, E-value non-significant, generic short helical-membrane-repeat promiscuity) -- reconfirmed here, not re-derived. Remains dark. top hits 2LOM_A/2LON_A/9I6F_N HIG1 domain family (83.7-90.3%, E>=1.7). Remote homology search on the deep UniRef30 profile (MPI Toolkit), run for the residual dark set.

3D structure (interactive — Mol*/pdbe-molstar)

No experimental structure is deposited for this gene: showing the AlphaFold DB predicted model (mean pLDDT 76.1, confident), coloured by per-residue confidence (dark blue = very high, orange/red = low — treat low-confidence regions as unreliable, especially termini and loops). Drag to rotate, scroll to zoom.

Genomic context (neighbours & predicted operon)

Neighbours from the H37Rv annotation (- strand). The operon is predicted by co-directional intergenic distance (same strand, gaps ≤50 bp) — a transcription-unit hypothesis, not a mapped TSS. For a "hypothetical", co-transcription with a characterised operon is a concrete functional lead (complements the STRING neighborhood channel below).

Published operon evidence (two independent sources)

Two independent published operon maps, cited alongside the distance heuristic above: an association corroborating or contradicting the heuristic, never proof of shared function. REMap's RNA-seq coverage continuity is the most direct of the three signals; Roback's logistic regression and the intergenic-distance heuristic are statistical proxies.

Node colour = verdict, dashed = still uncharacterised; edge colour = evidence (green experimental, orange genomic-context, grey co-expression), width ∝ score. Click a partner to open its page; "Explore full network" to walk the graph.

Closest characterised functional partner: bcp (peroxiredoxin), high confidence (score 784, all STRING channels except text-mining; the partner is chosen on its genomic-context evidence). Read this as a lead toward a shared pathway or operon, not as evidence of a physical complex: when this atlas co-folded 98 such context-only pairs, their predicted interfaces were no more confident than those of length-matched control pairs.

Showing the 15 strongest of this gene's 82 partners at medium confidence (17 at high confidence). The table is an extract, not a neighbourhood: across the proteome the real median degree is 34 while the served median is 15, so no hub, degree distribution or centrality may be read on this table. None of the 82 partners rests on an interaction measured in M. tuberculosis itself.

STRING combines evidence channels (neighborhood, fusion, cooccurrence, coexpression, experimental, database, text-mining) into a 0–1000 score. The ctx badge marks edges carried by the genomic-context channels (conserved neighborhood, fusion, phylogenetic co-occurrence), which are independent of orthology and structure and the strongest signal for an unknown gene. The no text-mining column recomputes the score from data alone, so a link that does not depend on the literature is visible. Association is a function hypothesis, not proof: corroborate with the operon context and the primary literature before assigning a function.

Where the experimental evidence comes from, and why it matters here. STRING's experimental channel fuses interactions measured in M. tuberculosis with interactions measured in another organism and transferred to this pair by orthology. The two are not equivalent for this atlas, whose standing rule is that better studied elsewhere is not a function established in M. tuberculosis. Measured on the served network: of the 38 229 partner pairs shown across the atlas, 13 483 partner rows carry a non-zero experimental channel, and only 106 pairs rest on an interaction measured in M. tuberculosis itself — 0.3 % of the network. Those 106 are long-established complexes (ESX-1/ESAT-6, RNA polymerase with CarD and RbpA, the Pup–Mpa proteasome, gyrase, Emb, GarA with PknB and PknG, the Rel modules); no still-uncharacterised gene is among them. The reason is not a gap in this atlas but in the literature: there is almost no experimental interactome of this bacterium, IntAct holding 134 curated protein pairs for a ~4 000-gene proteome. A exp (transferred) badge is therefore a lead, never a measurement, and an absence of badge is not evidence that the pair does not interact.

Evidence

• Legacy H37Rv annotation: membrane protein
• MTBC0 PGAP product: DUF3618 domain-containing protein
• Pfam (hmmscan --cut_ga): DUF3618 PF12277.14 (E=3e-18)
• (auto-curated by rules from PGAP + Pfam + Foldseek; not hand-reviewed)

Ancestral MTBC0 protein sequence

>mtbc0_002684|Rv2520c|
MVDRDPNTIKQEIDQTRDQLAATIDSLAERANPRRLADDAKTRVIAFLRKPIVTVSLVGIGSVVVVVVIHKIRNR