Membrane lipid composition directs the cellular selectivity of antimicrobial metallohelices

Two enantiomeric pairs of iron(ii) metallohelices, available as water-soluble, stable, and optically pure bimetallic complexes, differ principally in the length of the central hydrophobic region between two cationic domains, and have distinct activity and cell selectivity profiles against Gram-positive and Gram-negative microbes. The effects of dose concentration and temperature on levels of intracellular accumulation in E. coli and S. aureus, studied via isotopic labelling, indicate that the metallohelices enter the microbial cells via passive diffusion, whereupon (as previously determined) they act on intracellular targets. Whilst the metallohelices with the shorter central hydrophobic regions accumulate less readily than those with the longer hydrophobic bridge in both E. coli and S. aureus cells when incubated at the same concentration, an order of magnitude less is actually required per cell to inhibit growth in E. coli, hence they are more active. Furthermore, these more Gram-negative active compounds (with the shorter central hydrophobic region) are less toxic towards human APRE-19 mammalian cells and equine red blood cells. We hypothesise that these cell selectivities originate from the membrane composition. Dynamic light scattering and zeta potential measurements demonstrate that the more lipophilic metallohelices interact more strongly with the membrane-mimetic vesicles, notably in the charge-neutral mammalian model; thus the selectivity is not simply a result of electrostatic effects. For the less lipophilic metallohelices we observe that the binding affinity with the E. coli model vesicles is greater than with S. aureus vesicles, despite the lower negative surface charge, and this corresponds with the cellular accumulation data and the measured MICs. Specifically, the presence of membrane phosphatidylethanolamine (POPE) significantly increases the binding affinity of these metallohelices, and we postulate that a high proportion of such conical, non-lamellar phospholipids is important for metallohelix transport across the membrane. The metallohelices with the shorter hydrophobic bridge studied have a balance of charge and lipophilicity which allows selective cell entry in E. coli over mammalian cells, while the more lipophilic metallohelices are membrane promiscuous and unselective.