Phosphoethanolamine addition to the Heptose I of the Lipopolysaccharide modifies the inner core structure and has an impact on the binding of Polymyxin B to the Escherichia coli outer membrane

Phosphoethanolamine (pEtN) decoration of E. coli Lipopolysaccharide (LPS) provides resistance to the antimicrobial Polymyxin B (PolB). While EptA and EptB enzymes catalyze the addition of pEtN to the Lipid A and Kdo (pEtN-Kdo-Lipid A), EptC catalyzes the pEtN addition to the Heptose I (pEtN-Hept_I). In this study, we investigated the contribution of pEtN-Hept_I to PolB resistance using eptA/eptB and eptC deficient E. coli K12 and its wild-type parent strains. These mutations were shown to decrease the antimicrobial activity of PolB on cells grown under pEtN-addition inducing conditions. Furthermore, the 1-N-phenylnapthylamine uptake assay revealed that in vivo PolB has a reduced OM-permeabilizing activity on the ΔeptA/eptB strain compared with the ΔeptC strain. In vitro, the changes in size and zeta potential of LPS-vesicles indicate that pEtN-Hept_I reduce the PolB binding, but in a minor extent than pEtN-Kdo-Lipid A. Molecular dynamics analysis revealed the structural basis of the PolB resistance promoted by pEtN-Hept_I, which generate a new hydrogen-bonding networks and a denser inner core region. Altogether, the experimental and theoretical assays shown herein indicate that pEtN-Hept_I addition promote an LPS conformational rearrangement, that could act as a shield by hindering the accession of PolB to inner LPS-targets moieties.