Functional Phenotyping of Diverse Small Multidrug Resistance Transporters

The goal of this project is a broad functional screen of small multidrug resistance (SMR) transporters selected from human and animal pathogens. SMR transporters are the smallest known active transporters. They are found in human and livestock pathogens and are shared between food-borne pathogens on mobile genetic elements. The few that have been studied in the lab were characterized as drug efflux pumps and contribute to antibiotic resistance in bacteria. However, several recent discoveries suggest that the SMR family might be much more functionally diverse than previously thought, including members that may not be drug transporters at all. These novel findings emphasize the need for a broader study of SMR transporter substrate profiles, native function, and transport mechanism.Here I propose to screen a wide variety of SMR transporters from diverse bacteria, focusing on those found in human and livestock pathogens. The objectives listed below will (i) Create substrate specificity profiles of these little-known transporters, providing a broader picture of their functional diversity. (ii) Test whether WT SMR transporters have the capability to perform proton-motive-force-driven concentrative uptake of substrates, providing insight into both functional versatility and test the potential of substrates to "reverse" SMR transport and co-opt this drug-resistance mechanism for drug-delivery. (iii) Develop the tools needed for future in vitro experiments to determine the transport mechanism underlying the SMR functional profiles.The long-term goal for this Hatch project is to understand the breadth of native function of SMR transporters so that when they are identified in pathogenic bacteria - in the clinic, in livestock, or in the food supply - we understand what threat they present to human or animal health or food safety and can develop strategies to combat SMR-caused drug resistance or bacterial virulence.Aim 1: Functional phenotyping of drug and metabolite transport by SMR family members. We will test whether SMR pumps confer susceptibility or resistance to a panel of antibiotics, antiseptics, metabolites, and osmolytes. Completion of this aim will significantly expand our knowledge of the diversity of substrates transported by SMR family members and reveal whether any WT SMR transporters confer both resistance and susceptibility to different substrates.Aim 2: Is the "native" function of SMR transporters drug efflux or do they have other biologically significant roles in biofilm formation, osmoregulation, and pH homeostasis?This aim will test whether non-drug-transport function are common to many SMR transporters or are isolated to a few family members. Completion of this aim will show whether the small multidrug resistance name accurately reflects the function of this family of transporters.Aim 3: Identification of SMR transporters suitable for detailed in vitro characterization. To confirm that the observed phenotypes arise through the inferred mechanism - transport of a specific substrate, proton-coupled symport or antiport - requires follow-up with in vitro testing. We will use standard GFP-fusion strategies to assess which SMR homologs are suitable for future more detailed studies of substrate selectivity and mechanism.