An allelic variant of the PmrB sensor kinase responsible for colistin resistance in an Escherichia coli strain of clinical origin

Abstract

We investigated the colistin resistance mechanism in an Escherichia coli strain (LC711/14) isolated in Italy in 2014, from an urinary tract infection, which was previously shown to express a colistin resistance mechanism different from mcr-1. LC711/14 was found to carry a novel mutation in the pmrB gene, resulting in a leucine to proline amino acid substitution at position 10 of the PmrB sensor kinase component of the PmrAB signal transduction system. The role of this substitution in colistin resistance was documented by expression of the wild-type and mutated alleles in a pmrB deletion derivative of the E. coli reference strain MG1655, in which expression of the mutated allele conferred colistin resistance and upregulation of the endogenous pmrHFIJKLM lipid A modification system. Complementation of LC711/14 with the wild-type pmrB allele restored colistin susceptibility and decreased expression of pmrHFIJKLM, confirming the role of this PmrB mutation. Substitution of leucine at position 10 of PmrB with other amino acids (glycine and glutamine) resulted in loss of function, underscoring a key role of this residue which is located in the cytoplasmic secretion domain of the protein. This work demonstrated that mutation in this domain of the PmrB sensor kinase can be responsible for acquired colistin resistance in E. coli strains of clinical origin.

Figure 1

Protein sequence alignment of PmrB of E. coli MG1655 (accession no. NC_000913.3), ECOR35 (accession no. JN032071.1), LC711/14 (this work), and ZTA11/01748 (Col-R strain isolate from swine faeces, carrying a PmrB mutation putatively associated with colistin resistance. Asterisks indicate conserved amino acid residues. Amino acids polymorphisms not associated with colistin resistance are underlined. The L10P substitution found in LC711/14 and demonstrated to be involved in colistin resistance in this work, and the V161G substitution described in a veterinary E. coli strain and putatively associated with colistin resistance are boldfaced. Provean (Protein Variation Effect Analyzer: http://provean.jcvi.org/seq_submit.php) analysis was carried out for the Leu₁₀Pro (−2,67) and Val₁₆₁Gly (−5.66) mutations, and indicated that both mutations had an impact on the PmrB topology. Structural and functional domains of the protein by Phobius prediction (http://phobius.sbc.su.se/) are also indicated: TM1 and TM2: transmembrane 1 and transmembrane 2 domains, are shaded in dark grey, while the periplasmic domain is shaded in light grey. The sub-domains (DHp: histidine phosphotransfer; CA: catalytic and ATP-binding) of the cytoplasmic portion of PmrB are overlined by black bars. The number of residues of each domain are indicated in brackets.

Figure 2

Secondary structure prediction of PmrB of MG1655 E. coli and its mutant derivatives. Analysis of the region encompassing the first 60 aa of the protein. In white are highlighted the coil regions. The helix regions are shaded in dark grey while the strand regions are boxed. The mutated aa in position 10 are boldfaced.