Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance

Abstract

Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5-to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5-to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.A major human infectious disease, tuberculosis (TB) is estimated to affect approximately one-third of the world's population, and 95% of cases occur in developing countries (15,30,31). Current estimates show that approximately 9.4 million new cases and nearly 1.7 million deaths from TB occur each year, and TB remains a major cause of premature death (36).The increased incidence of multidrug-resistant (MDR) TB (TB resistant to more than two anti-TB drugs, including rifampin and isoniazid [35]) has hampered the treatment and control of TB and is associated with an increase in mortality rates in people with TB (3,37,40). Consequently, the required drug dosage for the treatment of TB has dramatically increased (38), and fluoroquinolones (FQs) are now considered to be important second-line anti-TB agents (13,20

Extending the Definition of the GyrB Quinolone Resistance-Determining Region in Mycobacterium tuberculosis DNA Gyrase for Assessing Fluoroquinolone Resistance in M. tuberculosis

“…Our results also show that N538T was responsible for a lower level of resistance than N538D (3-to 5-fold increases in MICs and/or IC 50 s, versus 12-to 42-fold increases) and confirmed the results published very recently regarding substitution E540V (Table 4). The data published by Kim et al are close to the results we obtained, suggesting that DNA gyrase assay is a reliable tool in demonstrating resistance and allows comparisons between studies (15).…”

“…The previous study showing that GyrB substitutions could be not implicated in FQ resistance despite being found in resistant M. tuberculosis strains (D473N, P478A, R485H, S486F, A506G, A547V, G551R, and G559A) (20) underlines the need for additional studies of the effect of GyrB substitutions on FQ resistance in M. tuberculosis. So far, only two GyrB substitutions have been unequivocally demonstrated to be involved in FQ resistance: N538D (also called N510D in some publications depending on the numbering system used) and, very recently, E540V (2,15). The present study demonstrates that several GyrB substitutions (D500A, N538T, T539P, and E540V) are responsible for FQ resistance and underlines the presence of a 3-amino-acid hot spot region for substitutions responsible for FQ resistance in M. tuberculosis.…”

“…The present study demonstrates that several GyrB substitutions (D500A, N538T, T539P, and E540V) are responsible for FQ resistance and underlines the presence of a 3-amino-acid hot spot region for substitutions responsible for FQ resistance in M. tuberculosis. Since amino acids 539 and 540 are not included in the M. tuberculosis GyrB QRDR, these results also help us to refine the QRDR, whose limit can therefore be extended from amino acid 500 to amino acid 540, as suggested by previous studies (15,22).…”

“…Substitutions in GyrB in M. tuberculosis clinical strains are being increasingly reported (2, 4, 6, 8, 9, 16, 19, 21, 25-27, 29, 32). However, the implication of gyrB mutations in FQ resistance remains largely unclear, and among the 21 GyrB substitutions described in the literature, only two have been demonstrated to be implicated in FQ resistance so far (N538D and E540V) (2,15,27), whereas eight were reported not to be implicated in FQ resistance despite being found in FQ-resistant M. tuberculosis clinical strains (D473N, P478A, R485H, S486F, A506G, A547V, G551R, and G559A) (20). Consequently, it is scientifically and medically important to clarify the exact role of new GyrB substitutions in FQ resistance in M. tuberculosis.…”

Quinolone Resistance in Tuberculosis Treatment: A Structural Overview

“…1 and 11). Almost all these residues have been shown to be directly involved in the level of resistance to fluoroquinolone when they are modified (Aubry et al, 2004;Matrat et al, 2006;Mokrousov et al, 2008;Von Groll et al, 2009;Kim et al, 2011). The model shows that the geometry of the QBP is crucial for the recognition and the stability of fluoroquinolone in the pocket.…”