Project Summary (Abstract) Helicobacter pylori persistently colonizes the stomach in about 50% of the human population resulting in gastric inflammation and an increased risk of developing gastric diseases including cancer. The World Health Organization (WHO) lists gastric cancer as the third leading cause of cancer-related death worldwide and classifies H. pylori as a type I carcinogen. Increasing incidence of clarithromycin resistance also has led WHO to declare H. pylori a priority target for new antimicrobial development. Bacterial lipoproteins are modified by acylation to help anchor lipoproteins to the inner or outer membrane in Gram-negative bacteria. These proteins are an emerging target of antimicrobial development as inhibitors of bacterial lipoprotein synthesis or localization have been identified. H. pylori lipoproteins contribute to pathogenesis in numerous ways, including a role in delivering an oncoprotein (CagA) to mammalian cells. We hypothesize that lipoprotein sorting and modifications are fundamentally important for H. pylori immunopathogenesis. In previous studies, we showed that two of the three enzymes responsible for acylation of H. pylori lipoproteins and two proteins required for localization of lipoproteins to the inner vs outer membrane are essential for growth. In the current proposal, we will identify additional elements of the H. pylori lipoprotein localization system and determine the impact of H. pylori lipoproteins on immunopathogenesis. In Aim 1, we will characterize lipoprotein localization in H. pylori by identifying localization signals and previously unidentified proteins of the localization system. Further, lipoproteins are recognized by innate receptors of the host, leading to pro- or anti-inflammatory responses depending on the number and variety of acyl chain modifications on lipoproteins. Therefore, in Aim 2, we will determine how H. pylori lipoproteins influence immune cell activation and inflammation by characterizing the acyl chains present on H. pylori lipoproteins, defining how the number and variety of acyl chains affect innate signaling, and analyzing inflammation and disease in mice infected with H. pylori strains expressing tri- vs diacylated lipoproteins. The proposed studies will increase our understanding of lipoprotein biology and its impact on gastric disease thereby potentiating our ability to treat H. pylori infection and gastric cancer. Results will lay the foundation for future studies aimed at determining structure-activity relationships of novel lipoprotein localization components, antimicrobial development, and defining the role of lipoproteins and innate signaling in immune tolerance in H. pylori pathogenesis.