ECLIPSE: exploring the dark proteome of ESKAPE pathogens through the sequence similarity network of the Protein...

The accelerating crisis of antimicrobial resistance among the critical so-called ESKAPE pathogens demands the urgent identification of novel molecular targets. However, a substantial fraction of ESKAPE proteomes remains functionally uncharacterized, with many genes annotated as encoding hypothetical proteins. These protein sequences often lack significant similarity to known protein families when conventional homology-based annotation methods are used and thus remain "dark". This limits our ability to explore their roles in pathogenicity, and it is thus crucial to bridge this substantial gap in pathogen biology by developing new strategies to illuminate these "dark" regions of the ESKAPE pan-proteome.

We introduce ECLIPSE (ESKAPE Connectome Linkage and Inference for Proteome Sequence Exploration), a network-based computational framework that systematically identifies and prioritizes functionally dark protein families in ESKAPE pan-proteomes. ECLIPSE embeds target ESKAPE pathogen proteomes within the global sequence similarity network of the Protein Universe Atlas. It detects connected components composed entirely of unannotated proteins, called the "dark proteome." As a case study, we applied ECLIPSE to a pan-proteome of 3 460 657 protein sequences from 635 strains of Pseudomonas aeruginosa (PA). ECLIPSE identified 120 985 proteins (4%) residing in completely dark connected components. Furthermore, we have performed a taxonomic diversity analysis using normalized Shannon indices to characterize each dark component by its enrichment in ESKAPE pathogens. The analysis utilized the evenness (E) value (see Methods 2.1), which distinguishes Pseudomonas-specific (target-specific) from ESKAPE-enriched dark components. We then developed the Dark Proteome Prioritization Score (DPPS), a composite multidimensional scoring framework (see Methods 2.5). It ranks these dark components by biological relevance across four orthogonal axes: (i) functional darkness, (ii) P. aeruginosa proportion in the Atlas, (iii) AMR-clade taxonomic restriction, and (iv) conservation across the 635 P. aeruginosa strains. This framework outputs a robust four-tier scoring system; the prioritized Tier I components were validated by weight sensitivity analysis and remained stable across 500 Monte Carlo weight perturbations. Structural characterization of one of the top-ranked ESKAPE-enriched dark components revealed that it belongs to the beta-barrel fold DUF1302 (PF06980) family, for which no experimentally solved three-dimensional structure exists in the PDB. The genomic context analysis indicates that it is co-localized with a LuxR-type transcriptional regulator. Collectively, ECLIPSE identifies evolutionarily conserved, structurally defined, and functionally dark proteins enriched across ESKAPE pathogens; these dark proteins can further be utilized as alternative antimicrobial targets for experimental characterization.

The source code and dataset are available for free at: Github: https://github.com/surabhilata/ECLIPSE.git, Zenodo: DOI: 10.5281/zenodo.21064323.