Genome-Wide Discovery and Characterization of Putative Antimicrobial Resistance-Associated Small Open Reading Frames (sORFs) in the Staphylococcus aureus Pan-Genome

Advancements in molecular biology and genomics have uncovered a significant number of small open reading frames (sORFs) present in various transcripts, which were previously overlooked within genomes. In recent scenarios, it is widely recognised as a concealed reservoir of functional components being acknowledged for its significance in stress response and virulence, as well as aiding in comprehending therapeutic resistance and targets. The global escalation of antibiotic resistance presents a significant challenge, with methicillin-resistant Staphylococcus aureus showing a resistance rate of 35% across 76 countries, as reported by the WHO in 2022, underscoring the urgency for research into novel resistance mechanisms, including our investigation into the role of orf genes. This study employs pan-genomic and transcriptomic approaches to analyze the extensive and unexplored sORFs in S. aureus. Analyzing a large dataset of 964 genome assemblies and transcriptomic responses to antibiotic adjuvants, we uncovered a previously hidden layer of genetic diversity, including tens of thousands of unique sORFs. The outcome reveals that the S. aureus pan-genome is highly plastic, dominated by strain-specific genes, with a substantial proportion of 72.6% displaying significant sequence divergence from known references. These sORFs were predicted to be enriched in functional domains related to nucleotide binding and enzymatic functions, linking them to stress adaptation. Notably, we highlight that their expression is dynamically regulated under antimicrobial treatment, positioning sORFs as potential key players in the pathogen’s response to therapeutics. These findings position sORFs as key players in S. aureus’s adaptive toolkit, potentially contributing to antibiotic evasion and host-pathogen interactions. Our computational framework bridges genomic diversity with transcriptional dynamics, offering a roadmap for the experimental validation of high-priority sORFs. This work not only expands the functional annotation of S. aureus but also highlights sORFs as under-explored targets for combating antimicrobial resistance, paving the way for innovative therapeutic strategies.