Lipid A phosphoethanolamine transferase-mediated site-selective modifications show association with colistin resistance phenotypes and fitness

Abstract

ABSTRACTGenes encoding lipid A modifying phosphoethanolamine transferases (PETs) are genetically diverse and can confer resistance to colistin and antimicrobial peptides. To better understand the functional diversity of PETs, we characterized three canonical mobile colistin resistance (mcr) alleles (mcr-1,-3,-9), one intrinsicpet(eptA), and twomcr-like genes (petB,petC). Using an isogenic expression system, we show thatmcr-1andmcr-3are phenotypically similar by conferring colistin resistance with lower fitness costs.mcr-9, which is phylogenetically closely related tomcr-3, andeptAonly provide fitness advantages in the presence of sub-inhibitory concentrations of colistin and significantly reduce fitness in media only. PET-B and PET-C were phenotypically distinct from bonafide PETs; neither conferred colistin resistance or caused considerable fitness cost inEscherichia coli. Strikingly, we found for the first time that different PETs selectively modify different phosphates of lipid A - MCR-1, MCR-3, and PET-C selectively modify the 4’-phosphate, while MCR-9 and EptA modify the 1-phosphate. 4’-phosphate modifications facilitated by MCR-1 and -3 are associated with high levels of colistin resistance and low toxicity. Our results suggest that PETs have a wide phenotypic diversity and that high level colistin resistance is associated with specific lipid A modification patterns that has been largely unexplored thus far.IMPORTANCERising levels of resistance to increasing numbers of antimicrobials has led to the revival of last resort antibiotic colistin. Unfortunately, resistance to colistin is also spreading in the form ofmcrgenes, making it essential to (i) improve identification of resistant bacteria to allow clinicians to prescribe effective drug regimens and (ii) develop new combination therapies effective at targeting resistant bacteria. Our results demonstrate that PETs, including MCR variants, are site-selective inE. coli, with site-selectivity correlating with the level of resistance and fitness costs conferred by certain PETs. Site-selectivity associated with a given PET may not only help predict colistin resistance phenotypes, but may also provide an avenue to (i) improved drug regimens and (ii) development of new combination therapies to better combat colistin resistant bacteria.