Uncovering the mechanism of virulence factor maturation in Haemophilus influenzae - Project Summary/Abstract Non-typeable Haemophilus influenzae (NTHi) is a bacterial pathogen that infects the lower airways and is the main pathogen responsible for exacerbations of chronic inflammatory pulmonary disease (COPD). COPD affects 6.4% of American adults and is the fourth leading cause of death worldwide. Due to the persistent nature of NTHi, antibiotic resistance is increasing and new therapeutic strategies are needed. Recently, a new virulence factor termed NTHI1441 was discovered through genome mining efforts. This protein is expressed as part of the NTHI1440-1444 operon that contains the putative biosynthetic genes NTHI1443 and NTHI1444, which show homology to DUF692- and DUF2063-family proteins, respectively. Homologous operons, such as the methanobactin biosynthesis operon identified in methane-oxidizing bacteria, encode a ribosomally synthesized post-translationally modified peptide (RiPP) natural product precursor and biosynthetic DUF692- and DUF2063-like proteins that form a complex to modify the RiPP precursor peptide. Thus, I hypothesize that the NTHI1441 gene encodes a RiPP precursor peptide that is post-translationally modified by NTHI1443 and NTHI1444 to generate the mature virulence factor. The goal of this research proposal is to uncover the molecular details of the maturation, structure, and function of the virulence factor using in vivo and in vitro approaches. In Aim 1, the biochemical properties of the biosynthetic proteins will be examined using spectroscopic, structural, and biophysical techniques. Aim 2 involves a mass spectrometry-based proteomics approach to characterize the post-translational modifications (PTMs) conferred to the peptide substrate as produced by combinatorial heterologous expression, in vitro reconstitution, and isolation from the native organism NTHi to provide a detailed understanding of the molecular structure and biosynthesis of the mature virulence factor. Finally, in Aim 3, genetic knockouts of the biosynthetic proteins in NTHi will be constructed and used for invasion assays to assess whether the PTMs are essential for NTHi virulence. The outcomes of this project will provide insight into the highly conserved yet underexplored DUF692-family of biosynthetic proteins and will also illuminate the structure and function of a key virulence factor, which will establish the foundation for the development of targeted therapeutics to treat NTHi infections.