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

ABSTRACT

The Staphylococcus aureus 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 2, 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, F₄₁₁P₄₁₂G_413, in external loop L7 situated between TMS 13 and 14 led to a decrease of fourfold in S. aureus internalization by A549 cells and a partial effect on tetracycline resistance (4-fold reduction). A three-amino acid deletion, D₃₈D₃₉L₄₀, in external loop L1 situated between TMS 1 and 2, had a similar partial effect on tetracycline resistance but did not affect cell internalization. Using a 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 R₁₀₆ and G₁₅₁ (TMSs 4 and 5) are particularly important for tetracycline resistance without affecting internalization.