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.