Discovery and functional characterization of archaeal efflux transporters

Multidrug efflux transporters are critical contributors to antimicrobial resistance. This chapter details methodologies for identifying and functionally characterizing efflux pumps in halophilic archaea, expanding upon recent work that provided the first experimental evidence of active antibiotic efflux in an archaeon. Using Halorubrum amylolyticum CSM52 as a model, we describe genomic identification of putative efflux pump genes via whole-genome sequencing and RAST annotation, followed by phylogenetic analyses and comparative genomics to establish evolutionary context. We then outline functional assays in the native archaeal strain, including a fluorescence-based Hoechst 33342 accumulation assay to detect active efflux and antibiotic susceptibility testing in the presence of efflux pump inhibitors (EPIs) to reveal efflux-mediated resistance. To overcome challenges of manipulating extremophiles, we detail cloning of archaeal MATE (Multidrug and Toxin Extrusion) transporter genes into Escherichia coli and heterologous expression under inducible conditions, enabling characterization of pump activity in a model bacterial system. Fluorometric efflux assays in these E. coli clones confirmed transporter function and inhibitor specificity. We also describe integrative structural approaches: homology modeling of the archaeal MATE pumps (using YASARA and AlphaFold3) and molecular docking of substrates and inhibitors to elucidate mechanistic interactions. The relevance of archaeal efflux pumps to antimicrobial resistance (AMR) is discussed, including their potential to harbor and disseminate novel resistance determinants. Finally, we address the interpretation and limitations of using heterologous systems. This comprehensive methodological framework provides a roadmap for exploring efflux-mediated drug resistance in archaea, an emerging and important aspect of AMR research.