PROJECT SUMMARY Pneumonia accounts for more deaths than any other infectious disease worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of hospital-acquired pneumonia. The prevalence of MRSA is increasing worldwide. To confront the growing problem of MRSA, we require a greater understanding of the host-pathogen interactions during infection. This remains poorly understood partly due to the lack of in vivo models relevant to infections occurring in healthcare settings. Most research to date has focused on highly virulent and cytotoxic MRSA strains, despite the fact that many nosocomial infections are caused by MRSA isolates that exhibit low virulence in ex vivo models and in normal mice. This proposal aims to functionally dissect host- and bacterial-directed mechanisms that lead to mortality in nosocomial settings. The work proposed in this exploratory R21 is novel as it utilizes a new nosocomial murine model of antibiotic conditioning, which we found lowers the barrier to infection, mimicking hospitalized patients. Using this model, we will investigate how low cytotoxic strains, which are avirulent in healthy mice, are capable of inducing pneumonia. This study is also technically innovative, as Dual RNA-seq will be utilized for the first time in MRSA infected lungs to identify pathogen and host gene-networks important during nosocomial infections. Altogether, the proposed work can greatly impact the development of new treatment strategies against MRSA by discovering novel bacterial factors which may be exploited for therapeutic benefit, as well as host pathways that could be targeted by future immunotherapies.