Inhibition of lipoprotein synthesis in Helicobacter pylori

Helicobacter pylori is a Gram-negative bacterium that colonizes the stomach in about 50% of the human population (i.e. over 4 billion people). Without antibiotic therapy, H. pylori can persist in the stomach for decades. Over time, H. pylori can lead some infected individuals to develop severe diseases including peptic ulcers or gastric cancer, the third leading cause of cancer-related death worldwide. This has led the World Health Organization (WHO) to classify H. pylori as a type I carcinogen. The effectiveness of therapeutic approaches is becoming increasingly compromised by the growing incidence of antibiotic resistance, leading WHO to declare H. pylori a priority pathogen for development of new antimicrobials. Bacterial lipoproteins are modified by acylation to anchor them to the inner or outer membrane in Gram-negative bacteria where these proteins carry out a variety of essential functions. The proteins responsible for lipoprotein synthesis and proper subcellular localization are emerging targets of antimicrobial development. In previous studies, we identified the enzymes responsible for lipoprotein synthesis in H. pylori. Although the H. pylori proteins only exhibit 20-40% amino acid identity to their E. coli counterparts, each was able to complement an E. coli mutant strain. In the current proposal, we hypothesize that inhibitors of H. pylori lipoprotein synthesis would interfere with the ability of H. pylori to survive in the stomach. This hypothesis is based on the following. First, we found that five proteins required for lipoprotein synthesis and localization (biogenesis) are essential for H. pylori growth in vitro. Second, we found that a sixth lipoprotein biogenesis protein (not essential in vitro) is essential for the ability of H. pylori to colonize the stomach. The current proposal aims to develop assays suitable for high-throughput screening to identify inhibitors of lipoprotein synthesis in H. pylori. In Aim 1, we will develop complementary assays to monitor both acylation of a synthetic peptide by recombinant H. pylori prolipoprotein diacylglyceryl transferase (Lgt) and a byproduct of the acylation reaction. In Aim 2 we will develop an assay to monitor signal peptide cleavage by recombinant H. pylori lipoprotein signal peptidase (LspA). In Aim 3, we will develop complementary assays to monitor both acylation of a synthetic peptide by recombinant H. pylori apolipoprotein N-acyltransferase (Lnt) and a byproduct of the acylation reaction. The assays developed in this R21 Exploratory/Developmental proposal will provide the foundation for the future identification of inhibitors of H. pylori lipoprotein biogenesis using computationally driven virtual screening, fragment-based lead discovery, and traditional high-throughput screening. We will then optimize the activities of identified compounds, determine whether the inhibitors are specific to H. pylori or are active against other bacteria, and test the effectiveness of inhibitors using established animal models of H. pylori infection. Project Number: 1R21AI187891-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: MARK MCCLAIN | Institution: VANDERBILT UNIVERSITY MEDICAL CENTER, NASHVILLE, TN | Award Amount: $481,250 | Activity Code: R21 | Study Section: Anti-Infective Resistance and Targets Study Section [AIRT] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI18789101