The rise in multidrug resistant (MDR) Klebsiella pneumoniae, the third leading cause of hospital-acquired pneumonia in adults, has resulted in limited treatment options for K. pneumoniae-related infections. Therefore, development of alternative therapeutic strategies, such as those enhancing host immune responses, would greatly benefit affected patient populations. Given sex differences observed in host responses to other bacterial pathogens, consideration of such differences in K. pneumoniae infection is essential for developing broadly effective immunotherapies targeting this pathogen. Our preliminary data indicate that in response to the MDR K. pneumoniae strain ST258, female mice have a sixfold higher mortality rate compared to males upon infection. In addition, the lungs of healthy female mice have more T cells compared to males, including CD4+ helper T lymphocytes, which help coordinate the immune response by stimulating macrophages, B lymphocytes, and CD8 T lymphocytes. Primary infection with K. pneumoniae is controlled via lymphoid activation, including natural killer (NK) cells, gamma-delta T cells, and innate lymphoid cells (ILCs). Given previous studies demonstrating that females elicit stronger innate and adaptive immune responses than males, it is possible that increases in lymphoid populations (including T cells, NK cells, and ILCs) contribute to immune-induced pathologies and a higher mortality rate from K. pneumoniae infection in female versus male mice. Defining the mechanisms underlying sex differences in the immune response to MDR K. pneumoniae would enable development of treatments with sex-based precision efficacy. The objectives of this Research Project are to elucidate the mechanisms that contribute to greater susceptibility of female versus male mice to MDR ST258 K. pneumoniae pathogenesis and characterize sex differences in immune response to a new K. pneumoniae vaccine. The central hypothesis is that estrogen-dependent increases in lung lymphocytes (T cells, NK cells, and ILCs) in healthy females lead to greater inflammation upon ST258 primary infection and more robust immune responses to a tissue resident memory CD4+ T cell-eliciting vaccine. Specific Aim 1 will test the hypothesis that estrogens mediate the inflammatory immune responses in K. pneumoniae-infected female mice. Specific Aim 2 will test the hypothesis that estrogens produce a stronger immune response to a mucosal K. pneumoniae vaccine in female compared to male mice. Host immune responses to infection and vaccination will be evaluated using male, female, and ovariectomized female mice. In addition, single-cell RNAseq will define estrogen-related genes and pathways that contribute to sex differences in host response to infection. Completion of the proposed research, which is aligned with the Tulane COBRE in Sex-Based Precision Medicine (SPM) focus, will elucidate the pathways that drive sex differences in response to infection and vaccination, ultimately facilitating development of sex-based precision therapies to treat K. pneumoniae infection in humans.