Biophysical Society Thematic Meeting | Stockholm 2022

Physical and Quantitative Approaches to Overcome Antibiotic Resistance

Monday Speaker Abstracts

STRUCTURAL BASIS OF LIPID A MODIFICATION BY THE AMINOARABINOSE TRANSFERASE ARNT LINKED TO POLYMYXIN RESISTANCE Vasileios I Petrou 1,2 ; Khuram U Ashraf 1,2 ; Filippo Mancia 3 ; 1 Rutgers New Jersey Medical School, Department of Microbiology, Biochemistry and Molecular Genetics, Newark, NJ, USA 2 Rutgers New Jersey Medical School, Center for Immunity and Inflammation, Newark, NJ, USA 3 Columbia University Irving Medical Center, Department of Physiology and Cellular Biophysics, New York, NY, USA Lipid A, the major lipidic component of the lipopolysaccharide (LPS) decorating the outer membrane of Gram-negative (GN) bacteria, can be modified by addition of diverse chemical moieties. Such modifications lead to altered host recognition, evasion of host defenses, and resistance to antimicrobial agents. Modification of the phosphates of Lipid A with the aminoarabinose moiety 4-amino-4-deoxy-L-arabinose (L-Ara4N) leads to charge modification of the outer membrane and is responsible for bestowing resistance against natural cationic antimicrobial peptides (CAMPs) and polymyxin-class antibiotics to GN bacteria. Polymyxins are cationic peptides that associate with the outer bacterial membrane through electrostatic interactions with the phosphate groups of Lipid A. They are currently used as last resort antibiotics, either as monotherapies or in combination with other antibiotics, against multidrug resistant (MDR) GN bacteria. The enzymatic transfer of L-Ara4N to Lipid A to “cap” its phosphate groups is catalyzed by ArnT, an inner membrane lipid-to-lipid glycosyltransferase, and is the major contributor for development of polymyxin resistance in Escherichia coli and Salmonella enterica. Using single particle cryo-electron microscopy (cryoEM) we have determined the structure of ArnT from S. enterica in two states: i) bound to both the acceptor ligand Lipid A and the donor undecaprenyl phosphate (UndP), and ii) only bound to Lipid A, after mutating one of the coordinating residues for UndP. These structures are the first to capture the Lipid A-bound state of ArnT. They allow us to fully characterize substrate binding in the glycosyltransferase ArnT, and to accurately localize the active site of the enzyme. By comparing these structures to existing structures of ArnT from C. metallidurans, we provide further insights towards understanding the structural basis of catalysis and the substrate binding cycle of the glycosyltransferase ArnT.

ENGINEERING RESPONSIVE NANOMATERIALS AGAINST INFECTIONS Georgios Sotiriou Karolinska Institute, Sweden No Abstract

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