Unravelling the mechanisms causing murepavadin resistance in : lipopolysaccharide alterations and its consequences.

Introduction: Murepavadin is an antimicrobial peptide (AMP) in clinical development that selectively targets LptD and whose resistance profile remains unknown. We aimed to explore genomic modifications and consequences underlying murepavadin and/or colistin susceptibility.

Methods: To define genomic mechanisms underlying resistance, we performed two approaches: 1) a genome-wide association study (GWAS) in a clinical collection (n=496), considering >0.25 mg/L as tentative cut-off of murepavadin acquired resistance; 2) a paired genomic comparison in a subset of 5 isolates and their isogenic murepavadin-resistant mutants obtained . Lipid-A composition, immunogenicity and cathelicidin and indolicidin effects on bacterial growth were also tested in this last subset of isolates. Murepavadin MICs were determined in Δ and Δ knock-out mutants obtained from a auxotroph PAO1 derivative.

Results: GWAS revealed a missense variant (A→G p.Thr260Ala in the gene) associated with murepavadin resistance although both resistant and susceptible strains harbored it (21% and 12% respectively, OR=1.92, p=0.012 in χ² test). Among the isolate subset, murepavadin-resistant mutants with deletions in and genes showed lower abundance of hexa-acylated lipid-A (m/z 1616, 1632). 4-aminoarabinose addition was found only in colistin-resistant isolates but not in the other ones, irrespective of murepavadin susceptibility. Accordingly, Δ and Δ mutants exhibited higher murepavadin MICs than parental PAO1 auxotroph strain (2 and 4 0.5 mg/L respectively). Lipopolysaccharide from murepavadin-resistant mutants triggered lower inflammatory responses in human monocytes. Those with mutations and hexa-acylated lipid-A loss also exhibited greater growth reduction when exposed to host-derived AMPs cathelicidin and indolicidin.

Discussion: High murepavadin-resistance seems to be linked to and mutations and lower hexa-acylated lipid-A, corresponding to lower inflammatory induction and higher susceptibility to host-derived AMPs. Although GWAS identified one variant associated with the murepavadin-resistant phenotype, data revealed that there was no unique single genetic event underlying this phenotype. Our study provides insight into the mechanisms underlying murepavadin susceptibility.