Defining ICR-Mo, an intrinsic colistin resistance determinant from <i>Moraxella osloensis</i>

<div><p>Polymyxin is the last line of defense against severe infections caused by carbapenem-resistant gram-negative pathogens. The emergence of transferable MCR-1/2 polymyxin resistance greatly challenges the renewed interest in colistin (polymyxin E) for clinical treatments. Recent studies have suggested that <i>Moraxella</i> species are a putative reservoir for MCR-1/2 genetic determinants. Here, we report the functional definition of ICR-Mo from <i>M</i>. <i>osloensis</i>, a chromosomally encoded determinant of colistin resistance, in close relation to current MCR-1/2 family. ICR-Mo transmembrane protein was prepared and purified to homogeneity. Taken along with an <i>in vitro</i> enzymatic detection, MALDI-TOF mass spectrometry of bacterial lipid A pools determined that the ICR-Mo enzyme might exploit a possible “ping-pong” mechanism to accept the <u>p</u>hospho<u>e</u>thanol<u>a</u>mine (PEA) moiety from its donor <u>p</u>hosphatidyl<u>e</u>thanolamine (PE) and then transfer it to the 1(or 4’)-phosphate position of lipid A via an ICR-Mo-bound PEA adduct. Structural decoration of LPS-lipid A by ICR-Mo renders the recipient strain of <i>E</i>. <i>coli</i> resistant to polymyxin. Domain swapping assays indicate that the two domains of ICR-Mo cannot be functionally-exchanged with its counterparts in MCR-1/2 and EptA, validating its phylogenetic position in a distinct set of MCR-like genes. Structure-guided functional mapping of ICR-Mo reveals a PE lipid substrate recognizing cavity having a role in enzymatic catalysis and the resultant conference of antibiotic resistance. Expression of <i>icr-Mo</i> in <i>E</i>. <i>coli</i> significantly prevents the formation of reactive oxygen species (ROS) induced by colistin. Taken together, our results define a member of a group of intrinsic colistin resistance genes phylogenetically close to the MCR-1/2 family, highlighting the evolution of transferable colistin resistance.</p>