Project Summary/Abstract Bacterial diseases constitute a major global burden, primarily due to respiratory, peritoneal, and blood infections. Our lab and others have demonstrated that investigating host-derived innate immune responses may elucidate pathways important for combating bacterial threats. Myeloid cells, such as tissue-resident alveolar macrophages, serve as an essential first line of defense. Because phagocytosis of bacteria and subsequent microbial lysis in the acidified phagolysosome are an integral part of their response, targets that regulate the phagolysosome are of major physiological significance and could possibly lead to points of clinical intervention during infection. Interestingly, chloride anion (Cl-) governs a range of biological functions, including intracellular vesicular acidification and cell signaling. Over the past four years, a new Cl- channel, proton-activated chloride channel 1 (PACC1), has been discovered and structurally characterized. However, the main biological role of PACC1 remains poorly defined, and its relevance to bacterial immunity is entirely unknown. Our key conceptual contribution to the nascent PACC1 biology field is that PACC1 may play a critical function in the highly acidic and chloride-packed phagolysosome in myeloid cells. To explore this, we have generated PACC1-deficient (PACC1- /-) mice, which our data suggest have defects in phagolysosomal acidification when challenged with bacteria. Accordingly, this defect is associated with increased susceptibility to S. pneumoniae infection in the lung and E. coli infection in the peritoneum and blood, compared to wildtype mice. Dysregulated hyperinflammation and myeloid cell recruitment in PACC1-/- mice are unable to clear infection. In this proposal, we will test our hypothesis that PACC1 provides protection by promoting myeloid cell responses during bacterial infection, particularly in alveolar macrophages, by supporting phagolysosomal acidification and bacterial killing. Our innovative approach will leverage multiple disease models (pneumonia and sepsis), novel mouse strains, and transcriptomics, to uncover the role of PACC1. In Aim 1, we will test if global PACC1 loss in PACC1-/- mice impairs phagolysosomal and effector functions in myeloid cells in vitro (Aim 1.1); and impair bacterial killing by myeloid cells in vitro and by alveolar macrophages in vivo (Aim 1.2). In Aim 2, we will study the myeloid cell-specific role of PACC1 during infection in vivo. We will infect novel myeloid cell conditional PACC1 knockout mice with S. pneumoniae to study clinical severity and immunologic responses, and probe mechanisms using phagolysosomal proton pump inhibitors and neutrophil-depleting antibodies (Aim 2.1). We will also test if PACC1 loss in alveolar macrophages and neutrophils results in deleterious transcriptomic perturbations during pneumococcal pneumonia via RNA- sequencing (Aim 2.2). Finally, we will test if novel myeloid cell conditional PACC1 over...