Molecular basis of antibiotic multiresistance transfer in Staphylococcus aureus

Abstract

Multidrug-resistant Staphylococcus aureus infections pose a significant threat to human health. Antibiotic resistance is most commonly propagated by conjugative plasmids like pLW1043, the first vancomycin-resistant S. aureus vector identified in humans. We present the molecular basis for resistance transmission by the nicking enzyme in S. aureus (NES), which is essential for conjugative transfer. NES initiates and terminates the transfer of plasmids that variously confer resistance to a range of drugs, including vancomycin, gentamicin, and mupirocin. The NES N-terminal relaxase-DNA complex crystal structure reveals unique protein-DNA contacts essential in vitro and for conjugation in S. aureus. Using this structural information, we designed a DNA minor groove-targeted polyamide that inhibits NES with low micromolar efficacy. The crystal structure of the 341-residue C-terminal region outlines a unique architecture; in vitro and cell-based studies further establish that it is essential for conjugation and regulates the activity of the N-terminal relaxase. This conclusion is supported by a small-angle X-ray scattering structure of a full-length, 665-residue NES-DNA complex. Together, these data reveal the structural basis for antibiotic multiresistance acquisition by S. aureus and suggest novel strategies for therapeutic intervention.

Fig. 1.

NES structure from S. aureus pLW1043. (A) Crystal structure of the NES N-terminal relaxase (1–195) bound to a 30-nt oriT-containing hairpin (OriTHP30). (B) Crystal structure of the C-terminal domain (254–593) with domains highlighted in different shades of magenta.

Fig. 2.

DNA binding by S. aureus NES. (A) Schematic of the NES relaxase–DNA contacts with bases examined using variant oligos in boldface type. (B) Minor groove-binding hairpin loop 1 of NES. (C) Major groove-binding hairpin loop 2 of NES. (D) Active site region of NES, with the buried Gua26 base highlighted.

Fig. 3.

Functional analysis of NES. (A) NES wild-type (WT) and variant relaxase activities, including that of loop 1 (L1) and loop 2 (L2) deletions. (B) Activity of NES relaxase with variant oligos, including an abasic G26 site. (C) Effect of DNA oligo length on NES relaxase and full-length activity. (D) Upper, SAXS envelope shape (pink) representing averaged and filtered dummy atom model of the NES–DNA complex, with the relatively electron-rich region indicated (yellow) and NES crystal structures docked by SUPCOMB. Lower, a positively charged region of the NES C-terminal domain is in proximity to the DNA-bound active site of the NES relaxase in the docked model.

Fig. 4.

NES in S. aureus and polyamide inhibition. (A) Effect of nes deletion (KO) and complementation with WT and designed NES loop 1 (L1), loop 2 (L2), and C-terminal deletion (ΔC-term) mutants on conjugation in S. aureus. (B) Structures of the Match Polyamide 1 and Mismatch Polyamide 2. (C) Inhibition of full-length NES with Match 1 (solid bars) and Mismatch 2 (shaded bars) polyamides. (D) Eighteen- and 21-μM IC₅₀ values of the Match Polyamide 1 for the full-length and relaxase forms of NES, respectively.