ECLIPSE: Exploring the dark proteome of ESKAPE pathogens through the sequence similarity network of the Protein Universe Atlas

Motivation The accelerating crisis of antimicrobial resistance among the critical, so-called ESKAPE bacterial 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 using conventional homology-based annotation methods and thus remain “dark”. This limits our ability to explore their role in pathogenicity, and it is thus crucial to bridge this substantial gap in pathogen biology by developing novel strategies to illuminate these “dark” regions of the ESKAPE pan-proteomes. Results We introduce ECLIPSE (ESKAPE Connectome Linkage and Inference for Proteome Sequence Exploration), a network-based computational framework that systematically identifies and prioritises 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 (Durairaj et al . 2023). 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 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 multi-dimensional 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 component revealed that it belongs to the □-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 candidates can further facilitate the experimental characterization of dark proteins as an alternative antimicrobial target. Availability and implementation The source code and dataset are available for free at: Github: https://github.com/surabhilata/ECLIPSE.git Zenodo: DOI: https://doi.org/10.5281/zenodo.21064323