The pentapeptide repeat proteins MfpAMt and QnrB4 exhibit opposite effects on DNA gyrase catalytic reactions and on the ternary gyrase-DNA-quinolone complex

Abstract

MfpA(Mt) and QnrB4 are two newly characterized pentapeptide repeat proteins (PRPs) that interact with DNA gyrase. The mfpA(Mt) gene is chromosome borne in Mycobacterium tuberculosis, while qnrB4 is plasmid borne in enterobacteria. We expressed and purified the two PRPs and compared their effects on DNA gyrase, taking into account host specificity, i.e., the effect of MfpA(Mt) on M. tuberculosis gyrase and the effect of QnrB4 on Escherichia coli gyrase. Whereas QnrB4 inhibited E. coli gyrase activity only at concentrations higher than 30 microM, MfpA(Mt) inhibited all catalytic reactions of the M. tuberculosis gyrase described for this enzyme (supercoiling, cleavage, relaxation, and decatenation) with a 50% inhibitory concentration of 2 microM. We showed that the D87 residue in GyrA has a major role in the MfpA(Mt)-gyrase interaction, as D87H and D87G substitutions abolished MfpA(Mt) inhibition of M. tuberculosis gyrase catalytic reactions, while A83S modification did not. Since MfpA(Mt) and QnrB4 have been involved in resistance to fluoroquinolones, we measured the inhibition of the quinolone effect in the presence of each PRP. QnrB4 reversed quinolone inhibition of E. coli gyrase at 0.1 microM as described for other Qnr proteins, but MfpA(Mt) did not modify M. tuberculosis gyrase inhibition by fluoroquinolones. Crossover experiments showed that MfpA(Mt) also inhibited E. coli gyrase function, while QnrB4 did not reverse quinolone inhibition of M. tuberculosis gyrase. In conclusion, our in vitro experiments showed that MfpA(Mt) and QnrB4 exhibit opposite effects on DNA gyrase and that these effects are protein and species specific.

FIG. 1.

MfpA_Mt inhibits the catalytic activity of M. tuberculosis DNA gyrase in topoisomerase assays. (A) Concentration-dependent inhibitory effect of MfpA_Mt on supercoiling activity of M. tuberculosis DNA gyrase. Supercoiling assays were performed using relaxed pBR322 as the substrate (TR) and final concentrations of MfpA_Mt (μM) as indicated. R, relaxed pBR322; S, supercoiled pBR322. (B) Concentration-dependent inhibitory effect of MfpA_Mt on ATP-independent relaxation of supercoiled DNA. Supercoiled pBR322 (TS) was the substrate. (C) Concentration-dependent inhibitory effect of MfpA_Mt on decatenation activity of M. tuberculosis DNA gyrase. Interlinked kDNA (kDNA) was used as the substrate (TI). Decatenated minicircles were visualized in three forms: relaxed (R), linearized (L), and supercoiled (S).

FIG. 2.

The direct effect of QnrB4 on E. coli DNA gyrase (A) and M. tuberculosis gyrase (B) supercoiling is minimal and is observed only at high concentrations. Supercoiling assays were carried out with 400 ng of relaxed pBR322 as the substrate (pBR) and 1 U of E. coli DNA gyrase or 1 U of M. tuberculosis DNA gyrase with increasing concentrations of QnrB4-His₆ (μM) as indicated. R, relaxed pBR322; S, supercoiled pBR322. QC, QnrB4 with pBR322 without gyrase.

FIG. 3.

MfpA_Mt inhibits E. coli DNA gyrase supercoiling activity with an IC₅₀ of 3 μM. Supercoiling assays were performed with 400 ng of relaxed pBR322 as the substrate (pBR) and 1 U of E. coli DNA gyrase with increasing concentrations of MfpA_Mt (μM) as indicated. R, Relaxed pBR322; S, supercoiled pBR322.

FIG. 4.

The MfpA_Mt effect on DNA gyrase catalysis is abolished with M. tuberculosis DNA gyrase harboring the D87G but not the A83S substitution of GyrA. (A) Supercoiling assays were performed with wild-type M. tuberculosis gyrase and relaxed pBR322 as a substrate. R, relaxed pBR322; S, supercoiled pBR322. Reactions were carried out without MfpA_Mt (lane 0) and with increasing concentrations of MfpA_Mt (final concentrations in μM are indicated above the gel). MfpA_Mt inhibited gyrase supercoiling in a concentration-dependent manner. (B) Same experiment with an altered M. tuberculosis DNA gyrase composed of GyrA A83S and wild-type GyrB subunit, which efficiently supercoiled pBR322 (lane 0). MfpA_Mt inhibited altered gyrase supercoiling in a concentration-dependent manner, as for the wild-type gyrase. (C) Same experiment with an altered M. tuberculosis DNA gyrase composed of GyrA D87G and the wild-type GyrB subunit, which efficiently supercoiled pBR322 (lane 0). No modification of this supercoiling activity in the presence of MfpA_Mt (from 0.5 to 5 μM) was observed.

FIG. 5.

MfpA_Mt does not modify ciprofloxacin inhibition of M. tuberculosis gyrase. (A) Supercoiling assays were performed with relaxed pBR322 as a substrate (pBR). In the absence of fluoroquinolone, M. tuberculosis DNA gyrase efficiently supercoiled pBR322 (SC). In the presence of 50 μM ciprofloxacin, supercoiling activity was inhibited (lane 0). Increasing concentrations of MfpA_Mt (final concentrations in μM are indicated above the gel) were added to reaction mixtures containing M. tuberculosis DNA gyrase and ciprofloxacin. (B) Determination of the ciprofloxacin IC₅₀ in the absence and presence of MfpA_Mt. The IC₅₀ was measured by comparing the intensities of the bands corresponding to supercoiled pBR322.

FIG. 6.

QnrB4 has a protective effect on fluoroquinolone inhibition of E. coli DNA gyrase supercoiling activity but not toward M. tuberculosis gyrase. Supercoiling assays were performed with 400 ng of relaxed pBR322 (pBR) as the substrate and 1 U of E. coli DNA gyrase or 1 U of M. tuberculosis DNA gyrase in the presence of increasing concentrations (μM) of QnrB4. R, relaxed pBR322; S, supercoiled pBR322. (A) In the presence of 6 μM ciprofloxacin, supercoiling activity was inhibited (lane 0); pBR322 remained in a relaxed form (R). When QnrB4 was added (final concentrations in μM are indicated above the gel), the intensity of the band corresponding to the supercoiled form of pBR322 (S) increased (lanes 0.1 to 5), showing that the inhibition of E. coli DNA gyrase supercoiling activity by ciprofloxacin was diminished. (B) Same experiment with QnrB4, M. tuberculosis gyrase, and ciprofloxacin at 50 μM. When QnrB4 was added, the topoisomeres produced by gyrase inhibition by ciprofloxacin remained and no supercoiling form (result of gyrase protection) was observed, even at high concentrations (5 μM) of QnrB4.

FIG. 7.

MfpA_Mt does not protect against E. coli gyrase inhibition by quinolones even in the presence of KGlu. (A) Comparison of inhibition by ciprofloxacin of pBR322 (pBR) supercoiling by E. coli gyrase in the presence of QnrB4 or MfpA_Mt at the same concentration (0.5 μM). R, relaxed pBR322; S, supercoiled pBR322. (B) Similar experiments with 100 mM KGlu in the assay buffer.

FIG. 8.

Comparison of sequences of four PRPs shows major differences in the region including residue 115, which was demonstrated previously to be involved in gyrase protection for quinolones. An alignment of MfpA (29), MfpA_Mt (Rv 3361c), QnrB4 (AAZ04784), and VPA0085 is shown. Conserved residues in the four sequences are in black, and those in three sequences are in light gray. C115 is indicated.