Molecular investigation of drug efflux pumps by deep mutational scanning

Bacteria protect themselves from the noxious action of antibiotics and other toxic compounds by the expression of drug efflux pumps, which are highly efficient in extruding them out of the cell. Recent advances in structure determination using cryo-EM and structure predictions via AlphaFold have provided detailed insights into the architecture and conformational space of drug efflux machineries. In contrast, comprehensive functional data of high quality are scarce, although they are crucial to understand the enigmatic phenomenon of drug efflux at the molecular level. Recently, we have applied the well-established method of deep mutational scanning (DMS) to drug efflux pumps and thereby shed light on the molecular mechanism of drug transport mediated by the heterodimeric ABC exporter EfrCD of the opportunistic pathogen Enterococcus faecalis. In DMS, a single site-saturation library is generated by substituting a subset or all residues to the other 19 amino acids. The resulting library is subjected to a functional screen, where in the case of drug efflux pumps, variant activity in the presence of a transport substrate is reflected in the growth phenotype of the expressing cells. NGS is then used to quantify enrichment or depletion of variants relative to the wild-type transporter. Such experiments allow for the systematic assessment of thousands of variants in parallel, under identical conditions and across multiple substrates, thereby delivering comprehensive mutational landscapes of drug transport. In this chapter, we outline the technical details how DMS projects on drug efflux pumps are planned and executed.