Abstract
Cationic antimicrobial peptides (CAMPs), such as polymyxins, are used as a last-line defense in treatment of many bacterial infections. However, some bacteria have developed resistance mechanisms to survive these compounds. Current pandemic O1 Vibrio cholerae biotype El Tor is resistant to polymyxins, whereas a previous pandemic strain of the biotype Classical is polymyxin-sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to antimicrobial peptides through aminoacylation of lipopolysaccharide (LPS), expected to decrease the negatively charged surface of the V. cholerae outer membrane. This Gram-negative system bears striking resemblance to a related Gram-positive cell-wall remodeling strategy that also promotes CAMP resistance. Mutants defective in AlmEF-dependent LPS modification exhibit reduced fitness in vivo Here, we present investigation of AlmG, the hitherto uncharacterized member of the AlmEFG pathway. Evidence for AlmG glycyl to lipid substrate transferase activity is demonstrated in vivo by heterologous expression of V. cholerae pathway enzymes in a specially engineered Escherichia coli strain. Development of a minimal keto-deoxyoctulosonate (Kdo)-lipid A domain in E. coli was necessary to facilitate chemical structure analysis and to produce a mimetic Kdo-lipid A domain AlmG substrate to that synthesized by V. cholerae. Our biochemical studies support a uniquely nuanced pathway of Gram-negative CAMPs resistance and provide a more detailed description of an enzyme of the pharmacologically relevant lysophosphospholipid acyltransferase (LPLAT) superfamily.
Highlights
Cationic antimicrobial peptides (CAMPs), such as polymyxins, are used as a last-line defense in treatment of many bacterial infections
Research on bacterial lysophosphospholipid acyltransferase (LPLAT) has shown their impact on the chemical composition of membrane lipids, and how variation in composition contributes to organismal fitness, but has demonstrated how specific environmental factors trigger remodeling of bacterial membranes through sensory pathways that coordinate regulation of LPLAT activities [1,2,3]
Biochemical characterization of Lipid A biosynthesis late acyltransferases (LABLAT) members was first performed with source material from Escherichia coli, where secondary acyltransferases esterify acyl groups to primary hydroxylacyl chains of nascently synthesized Kdo2-lipid IVA [4, 5]
Summary
Cationic antimicrobial peptides (CAMPs), such as polymyxins, are used as a last-line defense in treatment of many bacterial infections. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to antimicrobial peptides through aminoacylation of lipopolysaccharide (LPS), expected to decrease the negatively charged surface of the V. cholerae outer membrane. This Gram-negative system bears striking resemblance to a related Gram-positive cell-wall remodeling strategy that promotes CAMP resistance. Evidence for AlmG glycyl to lipid substrate transferase activity is demonstrated in vivo by heterologous expression of V. cholerae pathway enzymes in a specially engineered Escherichia coli strain. Biochemical characterization of LABLAT members was first performed with source material from Escherichia coli, where secondary acyltransferases esterify acyl groups to primary hydroxylacyl chains of nascently synthesized Kdo2-lipid IVA [4, 5]. Inactivating mutants of distantly related LABLAT members from other organisms such as Klebsiella, Escherichia, Salmonella, and Acinetobacter display sensitivity to CAMPs [9, 10, 12, 13] but not to the same degree as LpxN inactivation, which produces an order of magnitude more sensitivity to CAMPS [10, 11]
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