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Antimicrobial Agents and Chemotherapy, November 1998, p. 3044-3046, Vol. 42, No. 11
New Product Research Laboratories I, Daiichi
Pharmaceutical Co. Ltd., Edogawa-ku, Tokyo 134-8630, Japan
Received 9 June 1998/Returned for modification 4 August
1998/Accepted 1 September 1998
Two altered GrlB proteins (one with an Asp-432 Fluoroquinolones have potent and
broad antibacterial activities against gram-positive and -negative
bacteria. However, the increase in clinical use of quinolones has led
to the widespread emergence of resistance, especially in
Staphylococcus aureus (13). The three known
resistance mechanisms to quinolones in S. aureus are
alteration of two target enzymes and overproduction of quinolone efflux
protein (8). The two target enzymes of quinolones are topoisomerase IV and DNA gyrase, and the alteration of subunit A
proteins (GrlA and GyrA) is reported to be responsible for quinolone resistance on the basis of genetic and enzymological evidence (1,
3-7, 9, 10, 12, 14-20, 22). We have reported that the
inhibitory activities of quinolones against topoisomerase IV
reconstituted with GrlA with an alteration at position 80 and/or 84 and
wild-type GrlB were weaker than those against wild-type enzyme
(17). While there are many reports of quinolone resistance involving GrlA and GyrA, association of the subunit B proteins (GrlB
and GyrB) with quinolone resistance has been investigated only
genetically for S. aureus (6, 9, 15, 17). There has been a report on altered GyrB proteins (with Asp-426 All quinolones used were synthesized at the New Product Research
Laboratories I, Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan. Novobiocin was purchased from Sigma-Aldrich Japan (Tokyo, Japan). The
bacterial strains used in this study were S. aureus FDA
209-P and E. coli MC1061 (17). GrlA and GrlB
proteins of topoisomerase IV of S. aureus FDA 209-P were
purified separately as fusion proteins with maltose-binding protein
(MBP) from overproducing strains of E. coli by using a
protein fusion and purification system (New England Biolabs, Beverly,
Mass.). Mutated grlA and grlB genes prepared by
site-directed mutagenesis with Mutan-K (Takara Syuzo, Shiga, Japan)
were also used for construction of expression vectors. Details of the
purification procedures and the method for determination of inhibitory
activities of quinolones were described previously (17). The
substrate of the topoisomerase IV for determination of decatenation
activity was kinetoplast DNA (Nippon Gene, Toyama, Japan), and the
inhibitory activity of each drug was measured three times. The 50%
inhibitory concentrations (IC50s) were calculated as the
drug concentrations that reduced the decatenation observed with
drug-free controls by 50%.
The altered GrlB proteins with a substitution of Asn (AAT) for Asp-432
(GAT) or of Asp (GAT) for Asn-470, the altered GrlA (Ser-80
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Quinolone Resistance Mutations in the GrlB Protein
of Staphylococcus aureus
ABSTRACT
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Asn alteration and
one with an Asn-470Asp alteration) of Staphylococcus
aureus were purified as fusion proteins to maltose-binding
protein. The 50% inhibitory concentrations of levofloxacin were 14 and
3.4 µg/ml against topoisomerase IV containing GrlB proteins with
alterations at positions 432 and 470, respectively. These results
suggest that the alteration of Asp to Asn at position 432 may be
responsible for quinolone resistance.
TEXT
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Asn and Lys-447Glu alterations) in Escherichia coli
(24). In our previous work (17), a mutation in
the grlB gene [GAT(Asp)-432AAT(Asn)] was found in a
clinical isolate that was highly resistant to quinolones and that also
had alterations in GrlA at position 80 and in GyrA at position 84. Recently, Fournier and Hooper (6) reported a GrlB mutation
encoded at codon 470 (AsnAsp), and the strain conferring the altered
GrlB became, in comparison to the parent strain, slightly resistant to
quinolones (two- to eightfold more) and susceptible to novobiocin. In
this study, these two altered GrlB proteins (with Asp-432Asn and
Asn-470Asp alterations) were purified and the inhibitory activities
of quinolones against mutated topoisomerase IV were determined.
Phe), and
wild-type GrlA and GrlB proteins were purified as fusion proteins with
MBP. When the purified GrlB proteins were reconstituted with GrlA,
decatenation activity was observed. The relative decatenation activity
of the altered enzymes with one substitution in the GrlB sequence were
almost the same as that of the wild-type enzyme; however, the activity
of topoisomerase IV with alterations at position 80 in the GrlA
sequence and at position 432 in the GrlB sequence was half that of the
wild-type enzyme. Thus, the amount of the enzyme with altered GrlA and
GrlB proteins producing 1 U of decatenation activity was twice that of
the wild-type enzyme with GrlA and GrlB. The inhibitory activities of
the quinolones and novobiocin on the decatenation activity of
topoisomerase IV with or without alteration are shown in Table 1. The IC50s of quinolones
against the altered enzyme in which Asn (AAT) was substituted for Asp
(GAT) at position 432 of the GrlB sequence were 5 to 16 times higher
than those against wild-type topoisomerase IV, which suggests that the
change at position 432 in GrlB resulted in low-level quinolone
resistance. In contrast, the IC50s of quinolones against
the enzyme with an alteration in GrlB at position 470 were within
twofold of those against wild-type topoisomerase IV, while the
IC50 of novobiocin was one-fourth of that against the
strain FDA 209-P enzyme. These results reflect the MIC change reported
by Fournier and Hooper (6). Among the quinolones tested,
sitafloxacin (DU-6859a) showed the highest inhibitory activity against
both enzymes.
TABLE 1.
Inhibitory activities of quinolones and novobiocin
against topoisomerase IV
The inhibitory activities of the drugs against topoisomerase IV
reconstituted with altered GrlA (Ser-80Phe) and GrlB (Asp-432Asn) are also shown in Table 1. The IC50 of sitafloxacin against
the double-mutated topoisomerase IV was also the lowest among those of
the quinolones tested. The IC50s of levofloxacin and
ciprofloxacin against topoisomerase IV with a double amino acid change
were 117- and 72-fold, 1.3- and 2.4-fold, and 19- and 4.5-fold higher than those against the wild-type enzyme and the enzymes with GrlA or
GrlB alterations, respectively. These results confirm the association of a mutation in GrlB (Asp-432Asn) with quinolone resistance.
The Asp-432Asn change in the GrlB sequence is considered to
correspond with the Asp-437Asn change in GyrB that is a part of the
quinolone resistance-determining region (QRDR). The QRDR in GyrA was
considered to be a part of the quinolone-binding pocket that is the
binding site of quinolones to the DNA-DNA gyrase complex (23). The crystal structure of the 59-kDa protein of
E. coli GyrA showed that the QRDR was at the periphery of
GyrA and in close proximity to Tyr-122, which is a DNA-binding site
(2). Ito et al. (9) also discussed that the QRDR
around the Ser-84-to-Glu-88 region in GyrA is in close proximity to
Asp-437 and Arg-458 in GyrB in S. aureus. The QRDRs of DNA
gyrase and topoisomerase IV are well conserved, and the mutations
conferring quinolone resistance are similar. In the case of
topoisomerase IV, the region around Ser-80 to Glu-84 in GrlA may be in
close proximity to Asp-432; however, Asn-470 is slightly outside of the
highly conserved QRDR. When the structure of the
topoisomerase-DNA-quinolone complex is revealed, the exact mode of
binding of quinolones to the enzyme-DNA complex will be clarified.
Novobiocin, a coumarin antibacterial agent, acts by inhibiting ATP hydrolysis by the GyrB protein. Lewis et al. reported the crystal structures of a complex between DNA gyrase B protein and novobiocin (11). From their results, the binding sites for ATP and novobiocin overlapped to some degree and mutations which confer resistance to novobiocin were localized at the periphery of the ATP-binding site (6, 11, 21). It was also reported that hypersusceptibility to novobiocin might occur if the altered topoisomerase IV (GrlB altered at position 470) had reduced affinity for ATP. The IC50 of novobiocin against the altered enzyme containing Asn-432 in GrlB and Phe-80 in GrlA was slightly higher than those against topoisomerase IV with other alterations. We speculate that the tertiary structure of the enzyme with the double substitution might be changed.
In our previous studies (16-18), we found that the MICs of levofloxacin and ciprofloxacin against strain 891185, which possessed a grlB mutation at codon 432 and alterations at Phe-80 in GrlA and at Leu-84 in GyrA, were 100 and >800 µg/ml, respectively. Furthermore, the MICs of levofloxacin and ciprofloxacin for S. aureus 90-37 with the same alterations in GrlA and GyrA were 12.5 and 50 µg/ml, respectively. For 75 clinical isolates that possessed the same alterations in GrlA and GyrA but unknown alterations in GrlB and GyrB, the MICs of levofloxacin varied between 3.13 and 50 µg/ml and those of ciprofloxacin varied between 12.5 and 800 µg/ml. From these data and the possibility of the third mechanism of quinolone resistance (efflux), the association of a mutation in GrlB position 432 with quinolone resistance was within 16 times for levofloxacin and 128 times for ciprofloxacin at the MIC level.
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FOOTNOTES |
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* Corresponding author. Mailing address: New Product Research Laboratories I, Daiichi Pharmaceutical Co. Ltd., 16-13 Kitakasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan. Phone: 81-3-3680-0151, ext. 5810. Fax: 81-3-5696-8344. E-mail: tanakpmj{at}daiichipharm.co.jp.
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Antimicrobial Agents and Chemotherapy, November 1998, p. 3044-3046, Vol. 42, No. 11
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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