Abstract
The polymixin colistin is a “last line” antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.
Highlights
Antibiotic resistance, in Gram-negative bacteria (GNB) is a serious and growing global threat
Bacterial lipooligosaccharide PEA-transferases such as MCR-1 are organised into two domains: an N-terminal inner membrane-bound domain predicted to contain 5 transmembrane α-helices and a soluble, periplasmic domain containing the putative catalytic centre[8,13,14,15]
Size exclusion chromatography experiments show MCR1ΔTM is monomeric in solution (Supplementary Figure S2), leading us to conclude that this dimeric form is unlikely to be physiologically relevant
Summary
Antibiotic resistance, in Gram-negative bacteria (GNB) is a serious and growing global threat. Widespread dissemination of plasmids containing multiple resistance determinants has eroded treatment options leaving few reliable antibiotics for empiric therapy, a situation exacerbated by the continuing shortage of new antibacterials effective against GNB5. Recently a plasmid-encoded colistin resistance determinant, MCR-1, was identified in an animal-associated E. coli strain, and subsequently found on multi-resistance plasmids from animal, retail meat and human E. coli and K. pneumoniae[8]. MCR-1 confers resistance by modifying the colistin target, catalyzing transfer of phosphoethanolamine (PEA) onto the glucosamine saccharide of lipid A in the bacterial outer membrane (Fig. 1)[8]. This reduces the net negative charge of the lipid A head group and, colistin binding[12]. Colistin susceptibility assays support the proposed importance of both zinc and disulphide bond formation to MCR-1 activity, while density functional theory models of key states on the proposed reaction pathway indicate that both mono- and di-zinc forms of the enzyme may be able to support PEA transfer
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