Project Summary Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality in patients of all ages, particularly the elderly. Streptococcus pneumoniae (pneumococcus) is a major cause of CAP. During CAP, pneumococcus releases pneumolysin (PLY) and cell wall components, which disrupt alveolar-capillary barrier. Pneumococcus invades bloodstream through the eroded vascular endothelium, causing bacteremia and sepsis in 25-30% of patients, with 5-7% fatality. Other complications include multi-organ dysfunction, empyema, respiratory failure, pericarditis, and adverse cardiac events. Regretfully, little is known about how pneumococcus regulates the release of PLY and cell wall components that disrupt alveolar-capillary barrier. Our long-term goal is to device effective therapy against pneumococcal infection. The immediate objectives of this proposal are to elucidate the role of competence-regulated allolyis in breaching the air-blood barrier and devise effective measures to thwart bacteremia and sepsis. Preliminarily, we have shown that induction of ComX, the master regulator of competence regulon by the competence stimulating pheromone peptide (CSP), upregulates the expression of allolytic enzymes LytA, CbpD and CibAB. Allolysis substantially enhances the release of PLY, and ultimately, facilitates pneumococcal escape from infected lung to cause systemic bacteremia. Worse yet, fluoroquinolone and clavulanate antibiotics widely prescribed to treat pneumococcal infection are capable of activating ComX-dependent allolysis. Significantly, we have synthesized dominant-negative competence stimulating peptides (dnCSPs) and shown they competitively inhibit the induction of ComX-dependent allolysis, PLY release and hemolysis, and reduce mouse mortality during bacteremic pneumonia. We propose 3 Specific Aims to test the hypothesis that dnCSPs will be effective in inhibiting the ComX-regulated allolysis during bacteremic pneumonia. In Aim 1, chemical synthesis and structural analysis will be used to derive the next generation high potency dnCSPs that will inhibit ComX-regulated allolysis. Then, both wild-type and dnCSPs will be utilized to identify the key structural features in CSP that are involved in both binding and activating of the histidine kinase receptor ComD. In Aim 2, we will examine if ComX-induced allolytic release of PLY and cell wall components during bacteremic pneumococcal pneumonia—in the presence or absence of treatment with fluoroquinolones—lead to air-blood barrier disruption, sepsis, and lung and cardiac dysfunction in mice. In Aim 3, we will determine the efficacy of dnCSPs in attenuating air-blood barrier disruption, bacteremic pneumonia and sepsis, and lung and cardiac dysfunction in mouse model of pneumococcal infection, in the presence or absence of fluoroquinolone treatment. Completion of the proposal will chart new path to treat pneumococcal infection by targeting the competence regulon.