A monoclonal antibody that inhibits mycobacterial DNA gyrase by a novel mechanism

Abstract

DNA gyrase is a DNA topoisomerase indispensable for cellular functions in bacteria. We describe a novel, hitherto unknown, mechanism of specific inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis DNA gyrase by a monoclonal antibody (mAb). Binding of the mAb did not affect either GyrA-GyrB or gyrase-DNA interactions. More importantly, the ternary complex of gyrase-DNA-mAb retained the ATPase activity of the enzyme and was competent to catalyse DNA cleavage-religation reactions, implying a new mode of action different from other classes of gyrase inhibitors. DNA gyrase purified from fluoroquinolone-resistant strains of M.tuberculosis and M.smegmatis were inhibited by the mAb. The absence of cross-resistance of the drug-resistant enzymes from two different sources to the antibody-mediated inhibition corroborates the new mechanism of inhibition. We suggest that binding of the mAb in the proximity of the primary dimer interface region of GyrA in the heterotetrameric enzyme appears to block the release of the transported segment after strand passage, leading to enzyme inhibition. The specific inhibition of mycobacterial DNA gyrase with the mAb opens up new avenues for designing novel lead molecules for drug discovery and for probing gyrase mechanism.

Figure 1

Inhibition of different enzymatic activities of DNA gyrase by mAb:C3. (A) Supercoiling: one unit of M.smegmatis DNA gyrase was pre-incubated with antibody (IgG or Fab as indicated), as described in Materials and Methods, and supercoiling assays were carried out using relaxed pUC18 as substrate. Samples were analysed on 1.2% agarose gels; R and S are relaxed and negatively supercoiled pUC18 DNA, respectively. (B) ATP-independent DNA relaxation: mAb was pre-incubated with gyrase and relaxation assays were carried out using supercoiled pUC18 DNA as substrate. Samples were analysed as described for the supercoiling assay. (C) Decatenation activity: reactions were carried out in the presence of kDNA (lane 1) with M.smegmatis enzyme (lane 2) along with ciprofloxacin (Q, lane 3), novobiocin (N, lane 4), mAb:C3 and E9 (C3 and E9, lanes 5 and 6, respectively). kN, kDNA network; BN, broken network.

Figure 2

Effect of mAb:C3 on DNA gyrase subunit interaction. (A) Immunoprecipitation assay: Gyrase was immunoprecipitated with mAb:C3 or mAb:E9 or control mouse IgG. Immunocomplexes were recovered using protein G agarose. Both pellet (P) and supernatant (S) protein fractions were analysed by SDS–PAGE followed by western blotting with GyrA- or GyrB-specific polyclonal antibodies. (B) Surface plasmon resonance: a sensogram showing the interaction of gyrase subunits on rabbit α-mouse (RAM) Fc immobilized CM5 surface through mAb:C3. The various steps of interaction are shown schematically along with the bound response units. I: binding of C3 to α-mouse Fc; II: binding of GyrA to C3; III: binding of GyrB to GyrA; IV: dissociation of GyrB after salt wash.

Figure 3

mAb binding to the gyrase–DNA complex. (A) EMSA with 240 bp labelled DNA fragment (lane 1) was carried out with gyrase (lane 2) incubated with control mouse immunoglobulin (NM Ig, lane 3) or gyrase with mAb:C3 (lane 4). The samples were electrophoresed on a 3.5% native polyacrylamide gel and autoradiographed. (B) Immunoprecipitation of covalently closed circular DNA. The enzyme was incubated with various concentrations of mAb (mAb:C3 or mAb:E9), followed by the addition of radiolabelled relaxed pUC18 DNA and immunoprecipitation was carried out. The DNA present in the GyrA–antibody complex (pellet) and the unbound (supernatant) were measured using liquid scintillation counter.

Figure 4

Effect of mAb on quinolone-stabilized cleavage and religation reactions. Cleavage reactions using a 240 bp labelled fragment of DNA (A and E) or negatively supercoiled pBR322 (C) as substrate in the presence of varying amounts of enzyme and different mAbs as indicated. (B) Quantitative representation of cleaved products of (A). (D) EMSA using 240 bp labelled fragment of DNA in the presence of 5× or 10× competitor unlabelled DNA and ciprofloxacin. (E) EDTA-induced religation reaction. A gyrase–CFX–DNA cleavage reaction (lane 2) was further incubated without (lanes 3 and 4) or with EDTA treatment (lanes 5 and 6). In lanes 4 and 6, the enzyme was pre-incubated with MsGyrA:C3.

Figure 5

Effect of mAb on ATP hydrolysis. ATPase assays were carried out with 75 nM of M.smegmatis DNA gyrase in the presence of supercoiled pBR322 DNA. The enzyme (E) was pre-incubated in the presence of mAb:C3 (C3 IgG), control mouse IgG (NM IgG) or novobiocin, and ATPase reactions were carried out as described previously. The DNA-dependent ATPase rate is shown.

Figure 6

Effect of mAb on the strand-passage reaction. (A) The indicated amounts of enzyme (1 U = 25 fmol) were pre-incubated with 24 pmol of the mAb:C3 or mAb:E9 at 4°C for 15 min. An aliquot of 250 fmol of the relaxed DNA were added to the enzyme–antibody complex in supercoiling buffer and then incubated for 2 h at 37°C. SDS (0.16%) and proteinase K (90 μg/ml) were than added and the incubation continued for 30 min at 37°C. Samples were analysed on a 1.2% agarose gel electrophoresed at 25 V for 30 h. The slow migrating band in lanes 5 and 6 is thought to be a catenated species, as indicated. (B) Single round supercoiling. The assays are carried out as above with 15 U of enzyme (lanes 2–5). Lane 1, no enzyme; lane 5, 0.5 mM ADPNP is present along with mAb:C3.

Figure 7

Inhibition of DNA gyrase from quinolone-resistant mycobacteria. Supercoiling reactions were carried out in the presence of varying concentrations of ciprofloxacin (CFX) (A) or mAb:C3 (B), with CFX-sensitive or CFX-resistant enzymes, isolated from wild-type and drug-resistant M.smegmatis, as indicated. R and S are relaxed and negatively supercoiled pUC18 DNA, respectively. (C) Supercoiling reactions carried out with DNA gyrase isolated from an ofloxacin-resistant M.tuberculosis strain, in the presence of CFX and mAb:C3, as indicated.

Figure 8

Model for the interaction of mAb:C3 with DNA gyrase. The different steps of the supercoiling reaction are depicted, based on the proposed model for DNA gyrase (39,40). The GyrA N-terminal domain is shown in blue, and the GyrB N- and C-terminal domains are shown in green and yellow, respectively. The C-terminal domain of GyrA is not shown for clarity. The solid bar (grey) represents double-stranded DNA with the segment of DNA that is cleaved (G segment) in red and the segment of DNA that is transported through the enzyme (T segment) in purple. Gyrase transiently cleaves the G segment and transports the T segment through this break before its religation. mAb:C3 (shown as red ‘Y’-shaped molecule) is proposed to interact with GyrA near the exit gate such as to prevent the release of the T segment (step III).