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

Abstract

Introduction: Murepavadin is an antimicrobial peptide (AMP) in clinical development that selectively targets Pseudomonas aeruginosa 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 P. aeruginosa 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 in vitro. 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 ΔlpxL1 and ΔlpxL2 knock-out mutants obtained from a auxotroph PAO1 derivative.

Results: GWAS revealed a missense variant (A→G p.Thr260Ala in the hisJ 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 lpxL1 and lpxL2 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, ΔlpxL1 and ΔlpxL2 mutants exhibited higher murepavadin MICs than parental PAO1 auxotroph strain (2 and 4 vs 0.5 mg/L respectively). Lipopolysaccharide from murepavadin-resistant mutants triggered lower inflammatory responses in human monocytes. Those with lpxL 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 lpxL1 and lpxL2 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.

Keywords: antibiotic resistance; colistin; host-derived antimicrobial peptides; innate immune system; monocytes; murepavadin.

Figure 1

Similarity tree iTOL construction with the whole genome of the 496 P. aeruginosa isolates included in bioinformatic analysis. Legend differentiates the country of origin and their phenotypic susceptibility to murepavadin and colistin. MUR^R, murepavadin resistant (MIC >0.25 mg/L) and COL^R, colistin resistant (MIC >4mg/L).

Figure 2

Number of P. aeruginosa isolates harboring wild type or A→G p.Thr₂₆₀Ala variant according to their susceptibility to murepavadin. OR, odds ratio; *p-value < 0.05 in Chi-square (χ2) test.

Figure 3

Lipid A profiles in the murepavadin resistant mutants (MUR^R) and murepavadin susceptible parental strains (MUR^S) of P7 (A), P14 (B), P28 (C), P40 (D) and PAO (E) strain pairs. Mass spectrum expressed in relative intensity against maximum intensity peaks of the 10 P. aeruginosa isolates. Peaks corresponding to tetra-acylated lipid A are in yellow, penta-acylated lipid A in orange, hexa-acylated lipid A in red and L-Ara4N modified lipid A in blue. n.i., not identified.

Figure 4

The lipopolysaccharide (LPS) from murepavadin-resistant strains (MUR^R) exhibit lower immunogenicity than their parental murepavadin susceptible strains (MUR^S). Human monocytes (from n=8 different healthy volunteers) were stimulated with 10 mg/L of purified LPS from MUR^R mutant and MUR^S parental strains strains for 16 h. Supernatant from unstimulated cells (Unst) and stimulated with 10 mg/L commercial E. coli O111:B4 LPS were used as negative and positive controls respectively. Levels of inflammatory cytokines TNFα, IL-1β, IL-6 and IFN-γ are shown. *p-value < 0.05; **p-value < 0.01; ns, no significant in Wilcoxon paired t-test.

Figure 5

Murepavadin-resistant strains (MUR^R) strains are more susceptible to host-AMPs than the murepavadin susceptible strains (MUR^S). PA strains were grown in presence and absence of cathelicidin and indolicidin peptides (128 mg/L) and monitored by OD at 600 nm. (A-E) Growth curves of P7 (A), P14 (B), P28 (C), P40 (D), PAO1 (E) strains are shown. ***p-value < 0.001 in Two-way ANOVA and Tukey comparison test between murepavadin resistant mutants (MUR^R) and murepavadin susceptible parental strains (MUR^S) with the same treatment. Data expressed as median+SD from three independent replicates.