Enzymology of a three gene pathway required for cationic antimicrobial peptide resistance by Vibrio cholerae
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Produced by all domains of life, cationic antimicrobial peptides (CAMPs) are the most ubiquitous antibacterial compound in nature. In higher order eukaryotes CAMPs are critical components of innate immunity. Unicellular eukaryotes and prokaryotes use CAMPs for competitive exclusion in ecological niches. Some classes of CAMPs, such as polymyxins, are used as last-resort antibiotics in the treatment of bacterial infections. However, many bacteria have evolved resistance mechanisms to the toxic effects of CAMPs. For example, the current pandemic strain of Vibrio cholerae (O1 El Tor) is resistant to polymyxins, whereas the previous pandemic strain (O1 classical) is polymyxin sensitive. For decades how El Tor V. cholerae evolved polymyxin resistance was unknown, until the recent identification of a three-gene operon responsible for >100-fold improvement in resistance to polymyxin B. Classical strains lack a functional version of this operon. Renamed almEFG, this operon was shown to be necessary for the esterification of one, sometimes two, glycine residues to the major Gram-negative surface molecule lipopolysaccharide (LPS). Covalent attachment of glycine to LPS likely prevents CAMP binding through minimization of the negatively charged bacterial surface, since the amine terminus of glycine or diglycine remains freely exposed. Here, a mechanistic description of the almEFG operon is provided. Guided by predictive models, AlmF is shown to be a genuine aminoacyl carrier protein. Also identified is the V. cholerae enzyme required for post-translational activation of AlmF to a functional carrier protein. A combination of biochemical approaches reveal that AlmE specifically adds glycine to activated AlmF. Ultimately, AlmG transfers glycine from glycyl-AlmF to the lipid A membrane anchor of surface-displayed LPS in vivo, a process that evidence indicates can be done sequentially for diglycine LPS modification. The trio of proteins in the AlmEFG system forms a chemical pathway that resembles the division of labor observed in non-ribosomal peptide synthetases. They also bear striking resemblance to a related Gram-positive cell-wall remodeling strategy that promotes CAMP resistance. This biochemical study has the power to inform therapeutics against V. cholerae infection and provides a highly detailed mechanism of Gram-negative CAMP resistance.