Stability, denaturation and refolding of Mycobacterium tuberculosis MfpA, a DNA mimicking protein that confers antibiotic resistance

MfpA from Mycobacterium tuberculosis is a founding member of the pentapeptide repeat class of proteins (PRP) that is believed to confer bacterial resistance to the drug fluoroquinolone by mimicking the size, shape and surface charge of duplex DNA. We show that phenylalanine side chain stacking stabilizes the N-terminus of MfpA's pentapeptide thus extending the DNA mimicry analogy. The Lumry-Eyring model was applied to multiple spectral measures of MfpA denaturation revealing that the MfpA dimer dissociates to monomers which undergo a structural transition that leads to aggregation. MfpA retains high secondary and tertiary structure content under denaturing conditions. Dimerization stabilizes MfpA's pentapeptide repeat fold. The high Arrhenius activation energy of the barrier to aggregate formation rationalizes its stability. The mechanism of MfpA denaturation and refolding is a ‘double funnel’ energy landscape where the ‘native’ and ‘aggregate’ funnels are separated by the high barrier that is not overcome during in vitro refolding.     

结核分枝杆菌耐氟喹诺酮类药物的分子机制研究进展

结核病是由结核分枝杆菌(Mycobacterium tuberculosis)通过空气传播引起人类感染的慢性传染病,耐药结核分枝杆菌的流行是目前结核病防治的世界难题。氟喹诺酮类药物是人工合成药物,应用于耐药结核的临床治疗中,在治疗中起着核心的作用。但近年来,氟喹诺酮类药物的抗性菌株不断出现,愈发增加了结核病治疗的困难与治疗失败风险。在临床中氟喹诺酮药物的靶点比较清楚,是结核分枝杆菌的DNA旋转酶。目前发现结核分枝杆菌耐氟喹诺酮类药物的机制主要包括药物靶点DNA旋转酶的关键氨基酸改变、药物外排泵系统、细菌细胞壁厚度的增加以及喹诺酮抗性蛋白MfpA介导的DNA旋转酶活性调控。其中在氟喹诺酮靶标DNA旋转酶功能活性改变的耐药机制方面,编码DNA旋转酶基因突变一直是研究的热点,但近年来发现DNA旋转酶的调控蛋白MfpA以及DNA旋转酶的修饰在细菌耐药性中起着重要的作用,相关机制还亟待发现。本文综述了当前结核分枝杆菌耐氟喹诺酮类药物的作用机制,旨在为研发精准诊断技术和药物发掘提供科学理论基础和参考。     

Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.     

Resistance of M. leprae to Quinolones: A Question of Relativity?

Multidrug resistant leprosy, defined as resistance to rifampin, dapsone and fluoroquinolones (FQ), has been described in Mycobacterium leprae. However, the in vivo impact of fluoroquinolone resistance, mainly mediated by mutations in DNA gyrase (GyrA₂GyrB₂), has not been precisely assessed. Our objective was to measure the impact of a DNA gyrase mutation whose implication in fluoroquinolone resistance has been previously demonstrated through biochemical studies, on the in vivo activity of 3 fluoroquinolones: ofloxacin, moxifloxacin and garenoxacin.

Methodology/Principal Findings

We used the proportional bactericidal method. 210 four-week-old immunodeficient female Nude mice (NMRI-Foxn1^nu/Foxn1^nu) were inoculated in the left hind footpad with 0.03 ml of bacterial suspension containing 5×10³, 5×10², 5×10¹, and 5×10⁰ M. leprae AFB organisms of strain Hoshizuka-4 which is a multidrug resistant strain harboring a GyrA A91V substitution. An additional subgroup of 10 mice was inoculated with 5×10⁻¹ bacilli in the untreated control group. The day after inoculation, subgroups of mice were treated with a single dose of ofloxacin, moxifloxacin, garenoxacin or clarithromycin at 150 mg/kg dosing. 12 months later mice were sacrificed and M. leprae bacilli were numbered in the footpad. The results from the untreated control group indicated that the infective inoculum contained 23% of viable M. leprae. The results from the moxifloxacin and garenoxacin groups indicated that a single dose of these drugs reduced the percentage of viable M. leprae by 90%, similarly to the reduction observed after a single dose of the positive control drug clarithromycin. Conversely, ofloxacin was less active than clarithromycin.

Conclusion/Significance

DNA gyrase mutation is not always synonymous of lack of in vivo fluoroquinolone activity in M. leprae. As for M. tuberculosis, in vivo studies allow to measure residual antibiotic activity in case of target mutations in M. leprae.     

Fluoroquinolone-Gyrase-DNA Complexes: TWO MODES OF DRUG BINDING*

DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys⁴⁶⁶ gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly⁸¹ and GyrB-Glu⁴⁶⁶ residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.     

Substitutions of amino acids in α-helix-4 of gyrase A confer fluoroquinolone resistance on <Emphasis Type="Italic">Clostridium perfringens</Emphasis>

DNA gyrase, an essential enzyme that regulates DNA topology in bacteria, is the target of fluoroquinolones. Three fluoroquinolone-resistant mutants derived from one strain of Clostridium perfringens had amino acid substitutions of glycine 81 to cysteine, aspartic acid 87 to tyrosine, or both, in α-helix-4 of gyrase A. The gyrase mutations affected the growth kinetics of mutants differently when the mutants were exposed to increasing concentrations of gatifloxacin and ciprofloxacin. Fluoroquinolone concentration-dependent effects observed during growth in the exponential and stationary phases depended on the presence of particular gyrA mutations. Introduction of a wild-type gyrA gene into the mutants enhanced their susceptibility to fluoroquinolones and decreased their growth rates proportional to increases in fluoroquinolone concentrations. Amino acid substitutions in α-helix-4 of gyrase A protected C. perfringens from fluoroquinolones, and a strain with two substitutions was the most resistant.     

Solution structure and refolding of the Mycobacterium tuberculosis pentapeptide repeat protein MfpA

The pentapeptide repeat is a recently discovered protein fold. Mycobacterium tuberculosis MfpA is a founding member of the pentapeptide repeat protein (PRP) family that confers resistance to the antibiotic fluoroquinolone by binding to DNA gyrase and inhibiting its activity. The size, shape, and surface potential of MfpA mimics duplex DNA. As an initial step in a comprehensive biophysical analysis of the role of PRPs in the regulation of cellular topoisomerase activity and conferring antibiotic resistance, we have explored the solution structure and refolding of MfpA by fluorescence spectroscopy, CD, and analytical centrifugation. A unique CD spectrum for the pentapeptide repeat fold is described. This spectrum reveals a native structure whose beta-strands and turns within the right-handed quadrilateral beta-helix that define the PRP fold differ from canonical secondary structure types. MfpA refolded from urea or guanidium by dialysis or dilution forms stable aggregates of monomers whose secondary and tertiary structure are not native. In contrast, MfpA refolded using a novel "time-dependent renaturation" protocol yields protein with native secondary, tertiary, and quaternary structure. The generality of "time-dependent renaturation" to other proteins and denaturation methods is discussed.     

A novel locus conferring fluoroquinolone resistance in Staphylococcus aureus.

Fluoroquinolones such as ciprofloxacin and ofloxacin are potent antimicrobial agents that antagonize the A subunit of DNA gyrase. We selected and mapped a novel fluoroquinolone resistance gene on the Staphylococcus aureus chromosome. Resistant mutants were selected with ciprofloxacin or ofloxacin and were uniformly localized to the A fragment of chromosomal DNA digested with SmaI and arrayed by pulsed-field gel electrophoresis. Several mutants (cfxB, ofxC) were genetically mapped between the thr and trp loci in the A fragment. A majority of A fragment fluoroquinolone resistance mutations were associated with reduced susceptibility to novobiocin, an antagonist of the B subunit of DNA gyrase. Two genes previously associated with fluoroquinolone resistance, the gyrA gene of DNA gyrase and the norA gene (associated with decreased drug accumulation), were localized to the G and D fragments, respectively. Thus, the fluoroquinolone resistance mutations in the A fragment are distinct from previously identified fluoroquinolone resistance mutations in gyrA and norA. Whether mutations in the A fragment after a second topoisomerase or another gene controlling supercoiling or affect drug permeation is unknown.     

Pentapeptide repeat proteins

The pentapeptide repeat protein (PRP) family has more than 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F][S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable similarity in size, shape, and electrostatics to DNA.     

GyrI: a counter-defensive strategy against proteinaceous inhibitors of DNA gyrase

DNA gyrase is the target of two plasmid-encoded toxins CcdB and microcin B17, which ensure plasmid maintenance. These proteins stabilize gyrase–DNA covalent complexes leading to double-strand breaks in the genome. In contrast, the physiological role of chromosomally encoded inhibitor of DNA gyrase (GyrI) in Escherichia coli is unclear and its mechanism of inhibition has not been established. We demonstrate that the mode of inhibition of GyrI is distinct from all other gyrase inhibitors. It inhibits DNA gyrase prior to, or at the step of, binding of DNA by the enzyme. GyrI reduces intrinsic as well as toxin-stabilized gyrase–DNA covalent complexes. Furthermore, GyrI reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin, substantiating the in vitro results. Thus, GyrI is an antidote to DNA gyrase-specific proteinaceous poisons encoded by plasmid addiction systems.     

Rapid assay for detecting gyrA and parC mutations associated with fluoroquinolone resistance in Enterobacteriaceae

We developed a PCR-RFLP assay to detect mutations in the quinolone-resistance determining regions of gyrA and parC associated with fluoroquinolone resistance in Enterobacteriaceae. The assay detected mutations associated with reduced susceptibility to fluoroquinolones and may therefore serve as a specific, rapid, inexpensive, and simple testing alternative.     

Structure Based In Silico Analysis of Quinolone Resistance in Clinical Isolates of Salmonella Typhi from India

Enteric fever is a major cause of morbidity in several parts of the Indian subcontinent. The treatment for typhoid fever majorly includes the fluoroquinolone group of antibiotics. Excessive and indiscriminate use of these antibiotics has led to development of acquired resistance in the causative organism Salmonella Typhi. The resistance towards fluoroquinolones is associated with mutations in the target gene of DNA Gyrase. We have estimated the Minimum Inhibitory Concentration (MIC) of commonly used fluoroquinolone representatives from three generations, ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin, for 100 clinical isolates of Salmonella Typhi from patients in the Indian subcontinent. The MICs have been found to be in the range of 0.032 to 8 μg/ml. The gene encoding DNA Gyrase was subsequently sequenced and point mutations were observed in DNA Gyrase in the quinolone resistance determining region comprising Ser83Phe/Tyr and Asp87Tyr/Gly. The binding ability of these four fluoroquinolones in the quinolone binding pocket of wild type as well as mutant DNA Gyrase was computationally analyzed by molecular docking to assess their differential binding behaviour. This study has revealed that mutations in DNA Gyrase alter the characteristics of the binding pocket resulting in the loss of crucial molecular interactions and consequently decrease the binding affinity of fluoroquinolones with the target protein. The present study assists in understanding the underlying molecular and structural mechanism for decreased fluoroquinolone susceptibility in clinical isolates as a consequence of mutations in DNA Gyrase.     

Identification of an ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate as a catalytic inhibitor of DNA gyrase

Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections and target bacterial type-II topoisomerases (DNA gyrase and topoisomerase IV). Fluoroquinolones, however potent, are susceptible to bacterial resistance with prolonged use, which limits their use in the clinic. Quinazoline-2,4-diones also target bacterial type-II topoisomerases and are not susceptible to bacterial resistance similar to fluoroquinolones, however, their potency pales in comparison to fluoroquinolones. To meet the increasing demand for antibacterial development, nine modified quinazoline-2,4-diones were developed to probe quinazoline-2,4-dione structure modification for possible new binding contacts with the bacterial type-II topoisomerase, DNA gyrase. Evaluation of compounds for inhibition of the supercoiling activity of DNA gyrase revealed a novel ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative as a modest inhibitor of DNA gyrase, having an IC₅₀ of 3.5 μM. However, this ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate does not trap the catalytic intermediate like fluoroquinolones or typical quinazoline-2,4-diones do. Thus, the ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative discovered in this work acts as a catalytic inhibitor of DNA gyrase and therefore represents a new structural type of catalytic inhibitor of DNA gyrase.     

Exploration of fluoroquinolone resistance in Streptococcus pyogenes: comparative structure analysis of wild‐type and mutant DNA gyrase

Quinolone resistance‐determining region is known to be the druggability site of the target protein that undergoes frequent mutation and thus renders quinolone resistance. In the present study, ligands were tested for their inhibitory activity against DNA gyrase of Streptococcus pyogenes involved in DNA replication. In silico mutational analysis on modelled gyrase A revealed that GLU85 had the most possible interactions with all the ligands used for the study. The amino acid residue GLU85 had also been predicted with an essential role of maintaining the three‐dimensional structure of the protein. When introduced with a mutation (GLU 85 LYS) on this particular residue, it had readily denatured the whole α‐helix (from 80 to 90 amino acids). This was confirmed through the molecular dynamics simulation and revealed that this single mutation can cause many functional and structural changes. Furthermore, LYS85 mutation has altered the original secondary structure of the protein, which in turn led to the steric hindrance during the ligand–receptor interaction. The results based on the G‐score revealed that ligands have reduced interaction with the mutant protein. The semisynthetic fluoroquinolone 6d, which is an exception, forms a strong interaction with the mutant protein and was experimentally verified using the antimicrobial test. Hence, the present study unravels the fact that mutation at the drug binding site is the major cause for different level of resistance by the S. pyogenes when exposed against the varying concentrations of the fluoroquinolones. Furthermore, a comparative assessment of quinolone derivative with the older generation fluoroquinolones will be of great impact for S. pyogenes–related infections. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparison of the performance of two mycobacteriophage D29-based protocols for fluoroquinolone susceptibility testing in Mycobacterium tuberculosis

We tested a mycobacteriophage D29-based method for fluoroquinolone susceptibility assessment in clinical isolates of Mycobacterium tuberculosis. The method was incapable of identifying susceptible strains as such, although a slightly different published protocol successfully identified resistant and susceptible strains. Thus, caution is necessary when choosing an “in-house” D29-based protocol for testing of drug resistance.     

Structural characterization of the fusion of two pentapeptide repeat proteins, Np275 and Np276, from Nostoc punctiforme: resurrection of an ancestral protein

The Nostoc punctiforme genes Np275 and Np276 are two adjacently encoded proteins of 98 and 75 amino acids in length and exhibit sequences composed of tandem pentapeptide repeats. The structures of Np275 and a fusion of Np275 and Np276 were determined to 2.1 and 1.5 A, respectively. The two Nostoc proteins fold as highly symmetric right-handed quadrilateral beta-helices similar to the mycobacterial protein MfpA implicated in fluoroquinolone resistance and DNA gyrase inhibition. The sequence composition of the intervening coding region and the ability to express a fused protein by removing the stop codon for Np275 suggests Np275 and Np276 were recently part of a larger ancestral pentapeptide repeat protein.     

Inhibition of translesion DNA polymerase by archaeal reverse gyrase

Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.     

Functional characterisation of mycobacterial DNA gyrase: an efficient decatenase

A rapid single step immunoaffinity purification procedure is described for Mycobacterium smegmatis DNA gyrase. The mycobacterial enzyme is a 340 kDa heterotetrameric protein comprising two subunits each of GyrA and GyrB, exhibiting subtle differences and similarities to the well-characterised Escherichia coli gyrase. In contrast to E.coli gyrase, the M.smegmatis enzyme exhibits strong decatenase activity at physiological Mg²⁺ concentrations. Further, the enzymes exhibited marked differences in ATPase activity, DNA binding characteristics and susceptibility to fluoroquinolones. The holoenzyme showed very low intrinsic ATPase activity and was stimulated 20-fold in the presence of DNA. The DNA-stimulated ATPase kinetics revealed apparent K_0.5 and k_cat of 0.68 mM and 0.39 s^–1, respectively. The dissociation constant for DNA was found to be 9.2 nM, which is 20 times weaker than that of E.coli DNA gyrase. The differences between the enzymes were further substantiated as they exhibited varied sensitivity to moxifloxacin and ciprofloxacin. In spite of these differences, mycobacterial DNA gyrase is a functionally and mechanistically conserved enzyme and the variations in activity seem to reflect functional optimisation for its physiological role during mycobacterial genome replication.