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Phosphoethanolamine modification to lipid A found in five distinct Akkermansia species

ABSTRACT: We investigated how lipid A is remodeled across the Akkermansia genus, focusing on whether structurally distinct species share common modifications and how these changes relate to antimicrobial peptide resistance. Lipid A was extracted from representative isolates spanning five Akkermansia species (A. muciniphila, A. massiliensis, A. biwaensis, A. ignis, and A. durhamii; total N=55) and profiled by negative-ion MALDI mass spectrometry using an on-target FLAT workflow. Across all species, we detected lipid A species bearing a +123 Da mass shift consistent with phosphoethanolamine (pEtN) addition, and confirmed the modification by tandem MS on dominant modified and unmodified lipid A ions. We quantified isolate-to-isolate variation in the relative abundance of pEtN-modified versus unmodified lipid A and tested functional relevance by measuring colistin susceptibility in representative strains. Higher pEtN modification ratios correlated with decreased colistin susceptibility. We further assessed regulation of this remodeling by sampling across growth phases and found that pEtN-modified lipid A increased as cultures entered stationary phase, independent of colistin exposure. Finally, we used label-free proteomics to identify cell-envelope and envelope biogenesis-associated proteins that change with growth stage in strains that do versus do not upregulate pEtN-modified lipid A, providing candidate pathways linked to outer membrane remodeling. Overall, this work reports a conserved pEtN lipid A modification across diverse Akkermansia species and connects its relative abundance to antimicrobial susceptibility and growth-phase-dependent regulation, laying groundwork for future studies of species-specific host–microbe interactions in the gut.

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ORGANISM(S): Akkermansia Muciniphila

SUBMITTER: Silas Porter Crenna

LAB HEAD: Helena Pětrošová

PROVIDER: PXD074583 | Pride | 2026-04-27

REPOSITORIES: Pride

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Project description:Background: The rapid evolution and dissemination of mobilized colistin resistance gene (mcr) family has revealed as a severe threat to the global public health. Nevertheless, dramatic reduction in the prevalence of mcr-1, the major member of mcr family, was observed after the withdrawal of colistin in animal fodder in China since 2017, demonstrating that colistin acts as a selective stress to promote the dissemination of mcr-1. As the second largest lineage, mcr-3 was firstly discovered in 2017 and has been identified from numerous sources. However, whether the spreading of mcr-3 is driven by colistin remains unknown. Methods: To this end, we investigated the global prevalence of mcr-3 from 2005 to 2022 by an up-to-date systematic review, along with a nation-wide epidemiological study to establish the change of mcr-3 prevalence in China before and after 2017. To investigate the fitness cost imposed by MCR-3 upon bacterial host, in vitro and in vivo competitive assays were employed, along with morphological study and fluorescent observation. Moreover, by replacing non-optimal codons with optimal codons, synonymous mutations were introduced into the 5’-coding region of mcr-3 to study mechanisms accounting for the distinct fitness cost conferred by MCR-1 and MCR-3. Furthermore, by combining AlphaFold and molecular dynamics (MD) simulation, we provided a complete characterization on the putative lipid A binding pocket localized at the linker domain of MCR-3. Crucially, inhibitors targeting at the putative binding pocket of MCR-1 or MCR-3 were identified from small molecules library using the pipeline of virtual screening. Findings: The global prevalence of mcr-3 increased continuously from 2005 to 2022. The average prevalence was 0.18% during 2005-2014 and rapidly increased to 3.41% during 2020-2022. The prevalence of mcr-3 in China increased from 0.79% in 2016 to 5.87% in 2019. We found that the fitness of mcr-3-bearing E. coli and empty plasmid control was comparable but higher than that of mcr-1-positive strain. Although the putative lipid A binding pocket of MCR-3 was similar to that of in MCR-1, mcr-3 occupies remarkable codon bias at the 5’-end of coding region that disrupted the stability of mRNA, further reduced its protein expression in E. coli, resulting in the low fitness burden of bacterial host. Moreover, the 5’-end codon usage frequency appeared as a critical factor related with the evolution of mcr family. Furthermore, based on the similar lipid A binding pocket among MCR family protein, we identified three novel MCR inhibitors targeting at such pocket by screening from small-molecule library, which effectively restored the colistin susceptibility of mcr-bearing E. coli. Interpretation: For the first time, we found that the prevalence of mcr-3 increased continuously during 2016-2019 in China, demonstrating that the withdrawal of colistin in husbandry failed to prevent the dissemination of mcr-3. Our study evidenced that the 5’-end codon bias appeared as a crucial regulator upon the fitness cost conferred by horizontally transferred genes. Most importantly, the putative lipid A binding pocket verified from current study was a promising target site for designing inhibitors against mcr-positive strains.

			Action	DRS
	1813_A1_C_1.pepXML	Pepxml
	1813_A1_interact-C_1.pep.xml	Pepxml
	1813_A1_ion.tsv	Tabular
	1813_A1_peptide.tsv	Tabular
	1813_A1_protein.fas	Other

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Phosphoethanolamine modification to lipid A found in five distinct Akkermansia species

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