Staphylococcus aureus is an opportunistic bacterial pathogen that causes acute and chronic infections responsible for a broad spectrum of human diseases. The high prevalence of antibiotic-resistant strains in clinical context and the spreading of these strains in the community, make S. aureus infections a leading cause of morbidity and mortality in developed countries. A better understanding of the factors contributing to bacteria resistance and host-pathogen interaction is essential to counteract S. aureus infection and, ultimately, to develop more effective therapeutic approaches. MicroRNAs (miRNAs) are genome-encoded small non-coding RNAs, which play a pervasive role in the post-transcriptional control of eukaryotic gene expression. Emerging evidence supports a determinant role for miRNAs in the interplay between bacterial pathogens and host cells, as part of the immune response, or as a molecular strategy exploited by pathogens to subvert host cellular functions. However, a comprehensive analysis of host miRNAs function during infection by S. aureus is still missing. The main goal of this project is to identify and characterize host miRNAs, which are critical for S. aureus-host interaction at the cellular level. This will be achieved through interdisciplinary and innovative strategies, such as large-scale genome-wide miRNAs screenings and high-content analysis. To increase the biological relevance of the study, the effect of selected miRNAs will be validated in S. aureus clinical isolates obtained from osteomyelitis patients. The identification and characterization of miRNA molecular targets will be based on computational and experimental approaches. Through the application of cutting-edge techniques, this project will reveal previously unappreciated factors/pathways essential for S. aureus infection, providing insights into the complex host-pathogen interplay. Additionally, the identified novel factors/pathways may be explored for therapeutic intervention.