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An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State.

ABSTRACT: The cyclic antimicrobial lipopeptide daptomycin (DAP) triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence-specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, non-susceptibility to DAP by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaRW73C). We have determined the structure of the activated E. faecium LiaR protein at 3.2Å resolution and, in combination with solution studies, show that the activation of LiaR induces the formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaRW73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to DAP by the upregulation of the LiaFSR regulon. A comparison of the E. faecalis LiaR, E. faecium LiaR, and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with DAP resistance suggests that physicochemical properties such as oligomerization state and DNA specificity, although tuned to the biology of each organism, share some features that could be targeted for new antimicrobials.

SUBMITTER: Davlieva M

PROVIDER: S-EPMC5085866 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State.

Davlieva Milya M Tovar-Yanez Angel A DeBruler Kimberly K Leonard Paul G PG Zianni Michael R MR Arias Cesar A CA Shamoo Yousif Y

Journal of molecular biology 20160923 22

The cyclic antimicrobial lipopeptide daptomycin (DAP) triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence-specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, non-susceptibility to DAP by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaR<sup>W73C</sup>). We have determined ...[more]

PMID: 27670715

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Project description:The emergence of antimicrobial-resistant, livestock-associated Enterococcus faecalis represents a public health concern. Here, we report the isolation, molecular detection of virulence and antimicrobial resistance determinants, in addition to the phylogenetic analyses of 20 Enterococcus species using whole genome sequencing analysis of 15 Enterococcus faecalis strains including six strains of three novel sequence types, three Enterococcus faecium and two Enterococcus durans. All strains were isolated from food chain animals in South Africa. Enterococcus strains were isolated on bile aesculin azide agar, followed by identification using MALDI-TOF MS analysis. Antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method. The genomic DNA of the isolates was extracted and sequencing was performed using the Illumina MiSeq platform. Sequence reads were trimmed and de novo assembled. The assembled contigs were analyzed for antimicrobial resistance genes and chromosomal mutations, extra-chromosomal plasmids, and multi-locus sequence type (MLST). Multidrug antimicrobial resistance genes conferring resistance to aminoglycosides (ant(6)-Ia, aph(3')-IIIa, sat4, and spw), lincosamides (lnu(B), lsa(A), and lsa(E)), macrolides (erm(B)), trimethoprim (dfrG) and tetracyclines (tet(L) and tet(M)) were identified. Plasmid replicons were detected in seven E. faecalis and three E. faecium isolates. The sequence type (ST) of each isolate was determined using the Enterococcus PubMLST database. Ten STs were identified in the collection, three of which (ST1240, ST1241, and ST1242) have not been previously reported and are described in the present study for the first time. To compare the sequenced strains to other previously sequenced E. faecalis strains, assembled sequences of E. faecalis from livestock were downloaded from the PubMLST database. Core genome-based phylogenetic analysis was performed using ParSNP. The detection of multiple drug-resistance in Enterococcus including E. faecalis and E. faecium highlights the significance of genomic surveillance to monitor the spread of antimicrobial resistance in food chain animals. In addition, the genome sequences of Enterococcus strains reported in the present study will serve as a reference point for future molecular epidemiological studies of livestock-associated and antibiotic-resistant E. faecalis in Africa. In addition, this study enables the in-depth analysis of E. faecalis genomic structure, as well as provides valuable information on the phenotypic and genotypic antimicrobial resistance, and the pathogenesis of livestock-associated E. faecalis and E. faecium.

Project description:Monitoring the use of antimicrobials and the emergence of resistance in animals and people is important for the control of antimicrobial resistance, and for establishing sustainable and effective disease management practices. In this study, we used Enterococcus spp. and Escherichia coli as indicator species to investigate antimicrobial susceptibility patterns and how these change over time, on ten Swedish pig farms. Indoor environmental sock sampling was performed once a month during the entire production cycle of one batch of pigs on each farm, resulting in 60 samples collected in total. Selective culture for E. coli and Enterococcus spp. resulted in 122 isolates of E. coli, 74 isolates of E. faecium, but no isolates of E. faecalis. Microdilution was used to determine minimum inhibitory concentrations for twelve antimicrobial substances in E. coli and fifteen substances in E. faecium. The overall prevalence of resistance was low. Among the E. coli isolates, the proportions non-wild type (resistant, NWT) isolates were as follows: azithromycin and amikacin 1% (n = 1), trimethoprim and sulfamethoxazole 2% (n = 3), ampicillin 6% (n = 7) and tetracycline 9% (n = 11). Among the E. faecium isolates, the NWT proportions were: teicoplanin, linezolid and gentamicin 1% (n = 1), daptomycin 3% (n = 2), erythromycin 26% (n = 19), tetracycline 27% (n = 20), quinupristin/dalfopristin 58% (n = 42). The resistance patterns differed between the farms, likely due to different antimicrobial use, biosecurity measures and source of the animals. The NWT prevalence among E. coli decreased over time, whereas no similar trend could be observed in E. faecium. The results of the current study illustrate the complex factors affecting the antimicrobial resistance patterns observed on each farm, indicating that specific practices and risk factors have an impact on the prevalence and type of antimicrobial resistance. Further studies of the farm environments in combination with antimicrobial use and other risk factor data are needed to elucidate the multifaceted drivers of antimicrobial resistance development on livestock farms.

Dataset Information

An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State.

Publications

An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State.

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