〔Abstract〕 Abstract Objective To identify autophagy-related genes involved in pulmonary infection caused by the highly drug-resistant and virulent methicillin-resistant Staphylococcus aureus strain USA300 (USA300), and to explore the underlying molecular mechanisms, thereby providing potential targets for immunotherapy. Methods The GSE220943 dataset of a USA300-induced pulmonary infection mouse model was obtained from the GEO database. Differentially expressed genes (DEGs) were identified using the DESeq2 package. Autophagy-related genes (ARGs) were retrieved from the MSigDB and Autophagy databases. Weighted gene co-expression network analysis (WGCNA) was performed to construct gene co-expression modules. Genes overlapping among DEGs, ARGs, and WGCNA modules were identified as autophagy-related DEGs. Gene Ontology (GO) enrichment analysis was conducted using the clusterProfiler R package, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed via the Metascape platform. Immune cell infiltration was analyzed using the ImmuCellAI-mouse website. A protein–protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified through topological analysis in Cytoscape. ROC curves were plotted via the website https://www.bioinformatics.com.cn. Finally, key gene expression was validated in mouse lung tissues by real-time quantitative reverse transcription PCR (RT-qPCR).Results A total of 6 135, 4 075, 3 680, and 2 342 differentially expressed genes (DEGs) were identified at 12, 24, 48, and 96 hours post-infection, respectively. By integrating DEGs,autophagy-related genes (ARGs), and WGCNA modules, 19 autophagy-related DEGs were identified. GO and KEGG enrichment analyses indicated that these genes were mainly involved in CD4⁺ T cell activation and regulation, innate immune responses, and autophagosome membrane formation. Immune infiltration analysis revealed that innate immune cells such as neutrophils and dendritic cells predominated during the early phase of infection, while γδ T cells and M2 macrophages became more prominent in the later stages. PPI network analysis identified 12 hub autophagy-related genes, among which three upregulated key genes (Eif2ak2, Ikbke, and Nfkbiz) were further confirmed. The area under the ROC curve (AUC) for all three genes was 1.0.RT-qPCR validation demonstrated significantly elevated expression of these three genes in lung tissues at 24 hours post-infection (all P<0.05). Conclusion Eif2ak2, Ikbke, and Nfkbiz may be involved in the pulmonary infection caused by USA300 by promoting autophagy and hold promise as potential targets for immunotherapy.