532 Three-dimensional structure of a microbial nanomachine and its role in acne vulgaris

Abstract

Treating acne with antibiotics has led to the emergence of multidrug resistant (MDR) bacteria. MDR bacterial infections incur high healthcare costs and result in higher patient mortality. New antibiotics that target specific bacteria to avoid collateral microbiota damage are needed. Antibiotics used to treat acne reduce levels of Cutibacterium acnes. Acne-associated C. acnes genomes include putative, non-ribosomal peptide synthetase (NRPS) genes. Microbes use NRPS proteins to assemble unique metabolites. Some of these metabolites are virulence factors (e.g., Escherichia coli siderophores). Inhibiting bacterial virulence factors has been proposed as a strategy to minimize antibiotic collateral damage. No C. acnes NRPS has yet been characterized. One of the putative, acne-associated NRPS C. acnes genes (PPA-RS12630) is found almost exclusively within the Propionibacterium genus, which includes C. acnes. Based on sequence homology, we hypothesize that PPA-RS12630 is an iterative NRPS with a tri-domain structure consisting of adenylation, peptidyl carrier, and condensation domains. Microbes synthesize the siderophore vicibactin using this type of NRPS. Our group used structure prediction programs to design a PPA-RS12630 E. coli expression construct. Recombinant PPA-RS12630 was expressed, purified, and used in biochemical and structural investigations. Negative-stain electron microscopy of recombinant PPA-RS12630 demonstrated a flexible, tri-domain structure consistent with an iterative NRPS. Cryogenic electron microscopy (cryoEM) data was collected on PPA-RS12630 to determine its three-dimensional structure. The high-resolution structure of this protein will provide a road map to guide the biochemical characterization of PPA-RS12630 and other related C. acnes proteins. These investigations will help us to understand the role of C. acnes in human diseases, develop precision C. acnes therapeutics, and decrease the burden of MDR bacterial infections.