Staphylococcus aureus Tet38 Efflux Pump Structural Modeling and Roles of Essential Residues in Drug Efflux and Host Cell Internalization

The Tet38 membrane protein has distinct functions, including drug efflux and host cell attachment and internalization mediated by interaction with host cell CD36. Using structural modeling and site-directed mutagenesis, we identified key amino acids involved in different functions. Tet38, a member of the major facilitator superfamily, is predicted to have 14 transmembrane segments (TMS), 6 cytoplasmic loops, and 7 external loops. Cysteine substitutions of arginine 106 situated at the junction of TMS 4 and external loop L2, and glycine 151 of motif C on TMS 5, resulted in complete or near-complete (8- to 16-fold) reductions in Tet38-mediated resistance to tetracycline, with minimal to no effect on A549 host cell internalization. In contrast, a three-amino-acid deletion, F P G , in external loop L7 situated between TMS 13 and 14 led to a decrease of 4-fold in internalization by A549 cells and a partial effect on tetracycline resistance (4-fold reduction). A three-amino-acid deletion, D D L , in external loop L1 situated between TMS-1 and TMS-2, had a similar partial effect on tetracycline resistance but did not affect cell internalization. Using an Ni column retention assay, we showed further that the L7, but not the L1, deletion impaired binding to CD36. Thus, the L7 domain of Tet38 is key for interaction with CD36 and host cell internalization, and amino acids R and G (TMSs 4 and 5) are particularly important for tetracycline resistance without affecting internalization.