Regulation of mprF by antisense RNA restores daptomycin susceptibility to daptomycin-resistant isolates of Staphylococcus aureus

Abstract

Mutations in mprF have been shown to result in reduced susceptibility to daptomycin and other cationic antibacterials. An mprF antisense-inducible plasmid was constructed and used to demonstrate that depletion of mprF can reestablish susceptibility to daptomycin. Inducing antisense to mprF also resulted in increased susceptibility to vancomycin and gentamicin but, paradoxically, decreased susceptibility to oxacillin. These results suggest that mprF mutations that reduce susceptibility to cationic antibacterials result in a gain-of-function phenotype.

FIG. 1.

Northern blot for mprF in constructs containing candidate plasmids in the absence and presence of 2% xylose. Eleven candidate plasmids (antisense [AS] clones 2D2, 1E3, 1E5, 1A2, 1E4, 2F2, 1H7, 2C9, 1C3, 1B5, and 1B8) containing various mprF antisense inserts in pSAX-1E were assessed as described in the text. mRNA was isolated from strains grown in the absence (−) or presence (+) of 2% xylose. The oligonucleotide probe was specific to mprF, with the arrow depicting the full-length transcript. pSAX-1EmprF2F2 (2F2) was chosen for further study based on the minimal impact the addition of xylose had on growth (optical density [OD] ratio with or without xylose).

FIG. 2.

Impact of MprF antisense expression on daptomycin time-kill kinetics and the ability of cells to repel cationic cytochrome c. (A) Daptomycin kill kinetics and cells expressing mprF antisense were compared to cells harboring the vector-only control. Strain designations indicate the MprF allele (WT, T345I, or L826F [the same strains as used for MIC analyses]) and the presence of either the pSAX-1EmprF2F2 plasmid expressing mprF antisense (+as) or the pSAX-1E vector-only control (+vc). Drug-free growth controls for strain MW2 expressing antisense (WT+as, GC) or containing the vector only (WT+vc, GC) are representative of growth controls for the other strains (data not shown). The DAP concentration was held constant for each pair, and xylose was used at 20 mM. (B) Binding of cationic cytochrome c relative to cell-free controls was compared for cells expressing mprF antisense or containing the vector-only control; the MW2 and MW2ΔmprF strains were included as controls. Values shown are averages of at least three independent values; error bars indicate standard deviation.