Integrative Transcriptomic and Network Analysis Reveals Small Open Reading Frames as Regulators of DNA Methylation-Linked Pathogenicity and Adaptability 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 in Leptospira interrogans serovar Manilae strain UP-MMC-NIID-LP using RNA-seq–based transcriptomic profiling integrated with weighted gene co-expression network analysis (WGCNA) following 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 39 with significant differential expression (FDR < 0.05, |log₂FC| ≥ 1) across wild-type, mutant, and complemented strains. Gene co-expression networks constructed using WGCNA and interrogated with Cytoscape tools (MCODE, CytoHubba, ClueGO) revealed ten differentially expressed sORFs (FDR-adjusted p < 0.05); eight upregulated and two downregulated, across the three pairwise comparisons of wild-type, mutant, and complemented strains. These sORFs were enriched in pathways related to flagellar assembly, DNA recombination, and transcriptional regulation, which are core processes supporting genome stability and adaptive stress responses. Several previously uncharacterized sORFs occupied hub-like positions within co-expression modules, highlighting their integrative 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 central mediators that link epigenetic control with bacterial pathogenicity and adaptability. These findings provide a foundation for future antimicrobial and synthetic biology strategies targeting sORF-mediated regulation. Importance Small open reading frames (sORFs) are emerging as important regulators of bacterial gene expression, yet their roles in pathogenic species remain largely unexplored. This study provides the first genome-wide framework linking methylation-driven sORF regulation to virulence and adaptive processes in Leptospira interrogans. We identify previously uncharacterized sORFs occupying central positions in regulatory networks, connecting epigenetic control to motility, adhesion, and stress adaptation. Understanding these mechanisms in a neglected tropical disease pathogen has implications for improving public health and informs future antimicrobial and synthetic biology strategies.