Induction of erm(C) expression by noninducing antibiotics

Abstract

Ketolides, which represent the newest macrolide antibiotics, are generally perceived to be noninducers of inducible erm genes. In the study described in this paper we investigated the effects of several macrolide and ketolide compounds on the expression of the inducible erm(C) gene by Escherichia coli cells. Exposure to 14-member-ring macrolide drugs and to azithromycin led to a rapid and pronounced increase in the extent of dimethylation of Erm(C) target residue A2058 in 23S rRNA. When cells were incubated with subinhibitory concentrations of ketolides, the extent of A2058 dimethylation was also increased, albeit to a lower level and with kinetics slower than those observed with macrolides. The induction of erm(C) expression by ketolides was further confirmed by using a reporter construct which allows the colorimetric detection of induction in a disc diffusion assay. Most of the ketolides tested, including the clinically relevant compounds telithromycin and cethromycin, were able to induce the reporter expression, even though the induction occurred within a more narrow range of concentrations compared to the concentration range at which induction was achieved with the inducing macrolide antibiotics. No induction of the reporter expression was observed with 16-member-ring macrolide antibiotics or with a control drug, chloramphenicol. The deletion of three codons of the erm(C) leader peptide eliminated macrolide-dependent induction but left ketolide-dependent induction unchanged. We conclude that ketolides are generally capable of inducing erm genes. The narrow range of ketolide inducing concentrations, coupled with the slow rate of induction and the lower steady-state level of ribosome methylation, may mask this effect in MIC assays.

FIG. 1.

Conformational transition in erm(C) mRNA leading to the induction of erm(C) expression. The amino acid sequence of the leader peptide and the first three amino acids of Erm(C) are indicated. The ribosome binding sites of the leader peptide gene erm(C)L (RBS_L) and erm(C) (RBS_E) are marked. Complementary segments of mRNA that form two hairpins (ovals 1 and 2 and ovals 3 and 4) in the noninduced structure or one hairpin (ovals 2 and 3) in the induced structure are marked. The position of the ribosome in the leader ORF in the hypothetical stalled complex induced by the interaction of the nascent peptide, ribosome, and antibiotic (AB) and leading to the induction of erm(C) expression is shown.

FIG. 2.

Chemical structures of the macrolide and ketolide antibiotics used in this work.

FIG. 3.

Physical maps of the 4,889-bp pERMCT plasmid carrying the inducible erm(C) cassette and the 3,738-bp pERMZα modular reporter plasmid in which the erm(C) cistron was replaced with codons 2 to 60 of the lacZ gene encoding the β-galactosidase α peptide. The β-lactamase gene (bla) and the lac repressor gene (lacI^q) are indicated by gray arrows. The P_tac promoter is indicated by a black arrow; the tryptophan terminator is shown by a black triangle. The relevant restriction sites are marked.

FIG. 4.

Increase in dimethylation of A2058 in 23S rRNA upon exposure of cells to macrolide and ketolide antibiotics. (A) Principle of detection of A2058 dimethylation by primer extension arrest. In the presence of dATP, dGTP, dTTP, and ddCTP, the 18-nucleotide (nt)-long primer is extended by reverse transcriptase by 4 nucleotides when A2058 is unmodified or by only 2 nucleotides when primer extension is impeded by the dimethylation of A2058. (B) Induction of A2058 dimethylation in cells carrying inducible erm(C) by different antibiotics. Lanes V and E, RNA samples prepared from cells transformed with an empty pPOT1AE vector lacking inducible erm(C) and RNA samples prepared from cells transformed with plasmid pERMCT carrying the inducible erm(C) cassette, respectively. Cells were exposed to antibiotics at approximately one-fourth the MIC for 4 h. (C) Kinetics of induction of A2058 dimethylation by the cladinose-containing macrolide erythromycin or the ketolide telithromycin.

FIG. 5.

Induction of expression of the pERMZα reporter in E. coli JM109 cells by various antibiotics. The plates contained ca. 1.5 ×10⁸ cells plated in 0.6% LB agar on top of 1.5% LB agar containing ampicillin, IPTG, and X-Gal. (A) Induction by erythromycin. The Etest strip (AB Biodisk) contains a gradient of erythromycin concentrations from 0.016 to 256 μg/ml. (B) Induction by 14-member-ring macrolides and ketolides. Antibiotic discs were impregnated with the cladinose-containing macrolides erythromycin (Ery; 4.5 mg/ml), RU69874 (874; 10 mg/ml), RU66252 (252; 10 mg/ml), and clarithromycin (Clr; 8 mg/ml) and the cladinose-containing ketolides telithromycin (Tel; 1.5 mg/ml), HMR3004 (004; 1.5 mg/ml), and RU56066 (006; 12 mg/ml). Chloramphenicol (Chl; 0.3 mg/ml) was used as a control. The lack of reporter induction and of a growth inhibition zone around the RU56006 disc is likely explained by the low level of activity of this ketolide against E. coli cells. (C) Sixteen-member-ring macrolides josamycin (Jos), spiramycin (Spi), and tylosin (Tyl), all at 10 mg/ml. Erythromycin (Ery) and telithromycin (Tel) were used as controls. All plates were photographed after 36 to 48 h of incubation.

FIG. 6.

Mutations in the leader ORF affect the spectrum of inducing antibiotics. Plated cells contained the pERMZα reporter with the wild-type leader peptide ORF (A); a mutant lacking codons 2 to 4 of the leader peptide ORF (pERMZΔ2-4) (B); and a third mutant, pERMZ7, with a similar leader truncation and two additional mutations (which changed the nature of amino acid residues 2 and 3) (C). The sequence of the nascent leader peptide in the hypothetical stalled complex is indicated. The discs contained cladinose-containing macrolides (discs 1 to 5) or ketolides (discs 6 to 10) (all at 25 mg/ml): 1, erythromycin; 2, roxithromycin; 3, clarithromycin; 4, azithromycin; 5, RU66252; 6, HMR3004; 7, RU70645; 8, RU3562; 9, cethromycin; 10, telithromycin.