Identification and characterisation of two functional antibiotic MATE efflux pumps in the archaeon Halorubrum amylolyticum

Abstract

Multidrug efflux pumps have been found to play a crucial role in drug resistance in bacteria and eukaryotes. In this study, we investigated the presence of functional multidrug and toxic compound extrusion (MATE) efflux pumps, inferred from whole genome sequencing, in the halophilic archaeon Halorubrum amylolyticum CSM52 using Hoechst 33342 dye accumulation and antimicrobial sensitivity tests in the presence and absence of efflux pump inhibitors (EPIs). The whole genome sequence of H. amylolyticum CSM52 contained two putative MATE-type efflux pump genes, which may contribute to the inherent resistance to conventional antimicrobial agents reported in archaea. Antimicrobial susceptibility of the wild-type H. amylolyticum CSM52 testing revealed a lack of sensitivity to a wide range of antimicrobials, including glycopeptides, aminoglycosides, macrolides, fluoroquinolones, tetracycline, and chloramphenicol. However, the presence of EPIs, such as thioridazine, fluoxetine, and chlorpromazine, significantly increased the susceptibility of H. amylolyticum CSM52 to a number of these antimicrobials, indicating the potential involvement of efflux pumps in the observed resistance. A molecular modelling study with EPIs and substrate antimicrobials provided important insights into the molecular interactions with the putative transporter. It suggests that the occupancy of the transporter channel by EPIs has the potential to impact the efflux of antimicrobials. Phylogenetic analysis of the amino acid sequences of both MATE pumps showed low similarity with bacterial representatives, suggesting the presence of novel and distinct MATE efflux pumps in archaea. Our findings provide the first experimental evidence of active antibiotic efflux mechanisms in archaea and their potential roles in antimicrobial resistance, broadening our understanding of mechanisms of archaeal antimicrobial resistance, an overlooked aspect of AMR research.