A novel hypothetical protein (SAUSA300_1684) confers excellent protection against multi-drug-resistant Staphylococcus aureus infection in the murine model.

Staphylococcus aureus is a leading cause of nosocomial infections, including sepsis, bacteraemia, pneumonia, and endocarditis, and continues to pose a major public health challenge due to escalating multidrug resistance and the absence of an effective vaccine. To address this unmet clinical need, we employed a machine learning-guided reverse vaccinology approach to systematically interrogate the S. aureus proteome for novel vaccine antigens. This strategy led to the identification of eight previously uncharacterized hypothetical proteins with predicted immunogenic properties. Among these, SAUSA300_1684 protein (designated IMTS8) was prioritized for detailed translational evaluation. IMTS8 was found to be highly conserved across major clinical S. aureus lineages and expressed throughout growth phases of the methicillin-resistant strain USA300-FPR3757. Analysis of the surface-shaved proteome revealed that IMTS8 is likely exposed on the bacterial surface, a finding further substantiated by immunofluorescence microscopy. Immunization of mice with recombinant adjuvanted IMTS8 elicited strong antigen-specific IgG responses and conferred near-complete protection in a murine staphylococcal infection model. Collectively, these findings identify IMTS8 as a promising protein subunit vaccine candidate for the prevention of multidrug-resistant Staphylococcus aureus infections. In addition, this study highlights the translational utility of machine learning-based reverse vaccinology as an effective platform for accelerating antigen discovery against antimicrobial-resistant bacterial pathogens.