Integrative transcriptomic and network analysis reveals small open reading frames associated with cellular adaptive responsees and methylation-linked pathogenicity in Leptospira interrogans.

Small open reading frames (sORFs) are increasingly recognized as crucial regulators in bacterial gene expression, yet their biological roles remain largely unexplored in pathogenic species. Here, we investigated the genome-wide regulatory landscape of sORFs inLeptospira interrogans serovar Manilae strain UP-MMC-NIID-LP using RNA-seq-based transcriptomic profiling integrated with weighted gene co-expression network analysis (WGCNA). This study followed the targeted disruption of lomA, a gene mediating 4-methylcytosine (4mC) DNA modification. Loss of 4mC was associated with broad transcriptional dysregulation and phenotypic impairments, including reduced motility, adhesion, and virulence. Analysis of 363 predicted sORFs identified 31 with significant differential expression (FDR < 0.05, |log₂FC| ≥ 1) across wild-type, mutant, and complemented strains. Gene co-expression networks constructed using WGCNA and prioritized via topological ranking algorithms in Cytoscape. This analysis revealed seven high-confidence putative hub-like sORFs, which were significantly enriched in pathways related to flagellar assembly, DNA recombination, and transcriptional regulation. These candidates appear to function as core components supporting genome stability and adaptive stress responses. Several previously uncharacterized sORFs occupied central positions within co-expression modules, highlighting their potential roles in metabolic and regulatory networks. To our knowledge, this represents the first genome-wide integration of methylation-driven sORF regulation in Leptospira, revealing small proteins as associated with the link between epigenetic control bacterial pathogenicity and adaptability. These findings provide a foundation for future strategies targeting sORF-mediated regulation in pathogenic spirochetes.