Breakage-reunion domain of Streptococcus pneumoniae topoisomerase IV: crystal structure of a gram-positive quinolone target

The 2.7 A crystal structure of the 55-kDa N-terminal breakage-reunion domain of topoisomerase (topo) IV subunit A (ParC) from Streptococcus pneumoniae, the first for the quinolone targets from a gram-positive bacterium, has been solved and reveals a 'closed' dimer similar in fold to Escherichia coli DNA gyrase subunit A (GyrA), but distinct from the 'open' gate structure of Escherichia coli ParC. Unlike GyrA whose DNA binding groove is largely positively charged, the DNA binding site of ParC exhibits a distinct pattern of alternating positively and negatively charged regions coincident with the predicted positions of the grooves and phosphate backbone of DNA. Based on the ParC structure, a new induced-fit model for sequence-specific recognition of the gate (G) segment by ParC has been proposed. These features may account for the unique DNA recognition and quinolone targeting properties of pneumococcal type II topoisomerases compared to their gram-negative counterparts.     

New alkaloid antibiotics that target the DNA topoisomerase I of Streptococcus pneumoniae

Streptococcus pneumoniae has two type II DNA-topoisomerases (DNA-gyrase and DNA topoisomerase IV) and a single type I enzyme (DNA-topoisomerase I, TopA), as demonstrated here. Although fluoroquinolones target type II enzymes, antibiotics efficiently targeting TopA have not yet been reported. Eighteen alkaloids (seven aporphine and 11 phenanthrenes) were semisynthesized from boldine and used to test inhibition both of TopA activity and of cell growth. Two phenanthrenes (seconeolitsine and N-methyl-seconeolitsine) effectively inhibited both TopA activity and cell growth at equivalent concentrations (～17 μM). Evidence for in vivo TopA targeting by seconeolitsine was provided by the protection of growth inhibition in a S. pneumoniae culture in which the enzyme was overproduced. Additionally, hypernegative supercoiling was observed in an internal plasmid after drug treatment. Furthermore, a model of pneumococcal TopA was made based on the crystal structure of Escherichia coli TopA. Docking calculations indicated strong interactions of the alkaloids with the nucleotide-binding site in the closed protein conformation, which correlated with their inhibitory effect. Finally, although seconeolitsine and N-methyl-seconeolitsine inhibited TopA and bacterial growth, they did not affect human cell viability. Therefore, these new alkaloids can be envisaged as new therapeutic candidates for the treatment of S. pneumoniae infections resistant to other antibiotics.     

Action of quinolones against Staphylococcus aureus topoisomerase IV: basis for DNA cleavage enhancement

Topoisomerase IV is the primary cellular target for most quinolones in Gram-positive bacteria; however, its interaction with these agents is poorly understood. Therefore, the effects of four clinically relevant antibacterial quinolones (ciprofloxacin, and three new generation quinolones: trovafloxacin, levofloxacin, and sparfloxacin) on the DNA cleavage/religation reaction of Staphylococcus aureus topoisomerase IV were characterized. These quinolones stimulated enzyme-mediated DNA scission to a similar extent, but their potencies varied significantly. Drug order in the absence of ATP was trovafloxacin > ciprofloxacin > levofloxacin > sparfloxacin. Potency was enhanced by ATP, but to a different extent for each drug. Under all conditions examined, trovafloxacin was the most potent quinolone and sparfloxacin was the least. The enhanced potency of trovafloxacin correlated with several properties. Trovafloxacin induced topoisomerase IV-mediated DNA scission more rapidly than other quinolones and generated more cleavage at some sites. The most striking correlation, however, was between quinolone potency and inhibition of enzyme-mediated DNA religation: the greater the potency, the stronger the inhibition. Dose-response experiments with two topoisomerase IV mutants that confer clinical resistance to quinolones (GrlA(Ser80Phe) and GrlA(Glu84Lys)) indicate that resistance is caused by a decrease in both drug affinity and efficacy. Trovafloxacin is more active against these enzymes than ciprofloxacin because it partially overcomes the effect on affinity. Finally, comparative studies on DNA cleavage and decatenation suggest that the antibacterial properties of trovafloxacin result from increased S. aureus topoisomerase IV-mediated DNA cleavage rather than inhibition of enzyme catalysis.     

Clerocidin interacts with the cleavage complex of Streptococcus pneumoniae topoisomerase IV to induce selective irreversible DNA damage

Clerocidin (CL), a diterpenoid natural product, alkylates DNA through its epoxide moiety and exhibits both anticancer and antibacterial activities. We have examined CL action in the presence of topoisomerase IV from Streptococcus pneumoniae. CL promoted irreversible enzyme-mediated DNA cleavage leading to single- and double-stranded DNA breaks at specific sites. Reaction required the diterpenoid function: no cleavage was seen using a naphthalene-substituted analogue. Moreover, drug-induced DNA breakage was not observed using a mutant topoisomerase IV (ParC Y118F) unable to form a cleavage complex with DNA. Sequence analysis of 102 single-stranded DNA breaks and 79 double-stranded breaks revealed an overwhelming preference for G at the −1 position, i.e. immediately 5′ of the enzyme DNA scission site. This specificity contrasts with that of topoisomerase IV cleavage with antibacterial quinolones. Indeed, CL stimulated DNA breakage by a quinolone-resistant topoisomerase IV (ParC S79F). Overall, the results indicate that topoisomerase IV facilitates selective irreversible CL attack at guanine and that its cleavage complex differs markedly from that of mammalian topoisomerase II which promotes both irreversible and reversible CL attack at guanine and cytosine, respectively. The unique ability to form exclusively irreversible DNA breaks suggests topoisomerase IV may be a key intracellular target of CL in bacteria.     

The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae

Background

Streptococcus pneumoniae is the major cause of community-acquired pneumonia and is also associated with bronchitis, meningitis, otitis and sinusitis. The emergence and increasing prevalence of resistance to penicillin and other antibiotics has led to interest in other anti-pneumonococcal drugs such as quinolones that target the enzymes DNA gyrase and topoisomerase IV. During crystallization and in the avenues to finding a method to determine phases for the structure of the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae, obstacles were faced at each stage of the process. These problems included: majority of the crystals being twinned, either non-diffracting or exhibiting a high mosaic spread. The crystals, which were grown under conditions that favoured diffraction, were difficult to flash-freeze without loosing diffraction. The initial structure solution by molecular replacement failed and the approach proved to be unviable due to the complexity of the problem. In the end the successful structure solution required an in-depth data analysis and a very detailed molecular replacement search.

Methodology/Principal Findings

Crystal anti-twinning agents have been tested and two different methods of flash freezing have been compared. The fragility of the crystals did not allow the usual method of transferring the crystals into the heavy atom solution. Consequently, it was necessary to co-crystallize in the presence of the heavy atom compound. The multiple isomorphous replacement approach was unsuccessful because the 7 cysteine mutants which were engineered could not be successfully derivatized. Ultimately, molecular replacement was used to solve the structure by sorting through a large number of solutions in space group P1 using CNS.

Conclusions/Significance

The main objective of this paper is to describe the obstacles which were faced and overcome in order to acquire data sets on such difficult crystals and determine phases for successful structure solution.     

Experimental infection of rhesus macaques with Streptococcus pneumoniae: a possible model for vaccine assessment

BACKGROUND: We explored the possibility of using normal adult rhesus macaques for the preclinical assessment of safety, immunogenicity, and efficacy of newly developed vaccines against Streptococcus pneumoniae infection of the lung. METHODS: Our primary objective was to determine whether an intra-bronchial inoculum of at least 10(6)S. pneumoniae colony-forming units, or one as high as 10(8)-10(9) organisms, could detectably survive in rhesus macaques for a period longer than 1-2 weeks. If so, we hypothesized, it would be possible to observe signs of pneumonia commonly observed in humans, and discriminate between vaccinated/protected animals and controls. Infection was detectable in bronchoalveolar lavage fluids 3-5 weeks post-inoculation. RESULTS: The clinical course of disease mimicked aspects of that of human pneumococcal pneumonia. Signs of inflammation typical of the disease in humans, such as elevated concentrations of neutrophils and of pro-inflammatory cytokines in bronchoalveolar lavage fluids were also observed. CONCLUSIONS: These findings underscore the utility of this model to assess the safety, immunogenicity, and efficacy of newly developed S. pneumoniae vaccines.     

Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.

DNA topoisomerase IV mediates chromosome segregation and is a potential target for antibacterial agents including new antipneumococcal fluoroquinolones. We have used hybridization to a Staphylococcus aureus gyrB probe in concert with chromosome walking to isolate the Streptococcus pneumoniae parE-parC locus, lying downstream of a putative new insertion sequence and encoding 647-residue ParE and 823-residue ParC subunits of DNA topoisomerase IV. These proteins exhibited greatest homology respectively to the GrlB (ParE) and GrlA (ParC) subunits of S. aureus DNA topoisomerase IV. When combined, whole-cell extracts of Escherichia coli strains expressing S. pneumoniae ParC or ParE proteins reconstituted a salt-insensitive ATP-dependent decatenase activity characteristic of DNA topoisomerase IV. A second gyrB homolog isolated from S. pneumoniae encoded a 648-residue protein which we identified as GyrB through its close homology both to counterparts in S. aureus and Bacillus subtilis and to the product of the S. pneumoniae nov-1 gene that confers novobiocin resistance. gyrB was not closely linked to gyrA. To examine the role of DNA topoisomerase IV in fluoroquinolone action and resistance in S. pneumoniae, we isolated mutant strains stepwise selected for resistance to increasing concentrations of ciprofloxacin. We analysed four low-level resistant mutants and showed that Ser-79 of ParC, equivalent to resistance hotspots Ser-80 of GrlA and Ser-84 of GyrA in S. aureus, was in each case substituted with Tyr. These results suggest that DNA topoisomerase IV is an important target for fluoroquinolones in S. pneumoniae and establish this organism as a useful gram-positive system for resistance studies.     

The structural basis for T-antigen hydrolysis by Streptococcus pneumoniae: a target for structure-based vaccine design

Streptococcus pneumoniae endo-alpha-N-acetylgalactosaminidase is a cell surface-anchored glycoside hydrolase from family GH101 involved in the breakdown of mucin type O-linked glycans. The 189-kDa mature enzyme specifically hydrolyzes the T-antigen disaccharide from extracellular host glycoproteins and is representative of a broadly important class of virulence factors that have remained structurally uncharacterized due to their large size and highly modular nature. Here we report a 2.9 angstroms resolution crystal structure that remarkably captures the multidomain architecture and characterizes a catalytic center unexpectedly resembling that of alpha-amylases. Our analysis presents a complete model of glycoprotein recognition and provides a basis for the structure-based design of novel Streptococcus vaccines and therapeutics.     

Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site

Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/c)(A/t)A*GNNCt(T/a)N(C/a) and GN4G(G/c)(A/c)G*GNNCtTN(C/a) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between -1 and +1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. +1G/+4C, -4G/+8C, and particularly the novel -2A/+6T, but with no preference at +2/+3 within the staggered 4-bp overhang. Asymmetric elements include -3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique -2A/+6T and other preferences.     

Structure and inhibition of a quorum sensing target from Streptococcus pneumoniae

Streptococcus pneumoniae 5'-methylthioadenosine/S-adenosylhomocysteine hydrolase (MTAN) catalyzes the hydrolytic deadenylation of its substrates to form adenine and 5-methylthioribose or S-ribosylhomocysteine (SRH). MTAN is not found in mammals but is involved in bacterial quorum sensing. MTAN gene disruption affects the growth and pathogenicity of bacteria, making it a target for antibiotic design. Kinetic isotope effects and computational studies have established a dissociative S(N)1 transition state for Escherichia coli MTAN, and transition state analogues resembling the transition state are powerful inhibitors of the enzyme [Singh, V., Lee, J. L., Nú?ez, S., Howell, P. L., and Schramm, V. L. (2005) Biochemistry 44, 11647-11659]. The sequence of MTAN from S. pneumoniae is 40% identical to that of E. coli MTAN, but S. pneumoniae MTAN exhibits remarkably distinct kinetic and inhibitory properties. 5'-Methylthio-Immucillin-A (MT-ImmA) is a transition state analogue resembling an early S(N)1 transition state. It is a weak inhibitor of S. pneumoniae MTAN with a K(i) of 1.0 microM. The X-ray structure of S. pneumoniae MTAN with MT-ImmA indicates a dimer with the methylthio group in a flexible hydrophobic pocket. Replacing the methyl group with phenyl (PhT-ImmA), tolyl (p-TolT-ImmA), or ethyl (EtT-ImmA) groups increases the affinity to give K(i) values of 335, 60, and 40 nM, respectively. DADMe-Immucillins are geometric and electrostatic mimics of a fully dissociated transition state and bind more tightly than Immucillins. MT-DADMe-Immucillin-A inhibits with a K(i) value of 24 nM, and replacing the 5'-methyl group with p-Cl-phenyl (p-Cl-PhT-DADMe-ImmA) gave a K(i) value of 0.36 nM. The inhibitory potential of DADMe-Immucillins relative to the Immucillins supports a fully dissociated transition state structure for S. pneumoniae MTAN. Comparison of active site contacts in the X-ray crystal structures of E. coli and S. pneumoniae MTAN with MT-ImmA would predict equal binding, yet most analogues bind 10(3)-10(4)-fold more tightly to the E. coli enzyme. Catalytic site efficiency is primarily responsible for this difference since k(cat)/K(m) for S. pneumoniae MTAN is decreased 845-fold relative to that of E. coli MTAN.     

Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Jn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (Km^R) and carries a 5’truncated lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cls-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. Km^R LacZ⁺ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular Km^R LacZ⁺ mutant, AB1156, does not produce the brown pigment (Pig) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposonlinked mutation into a wild-type background results in a Pig phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and β-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.     

Streptococcus pneumoniae isoprenoid biosynthesis is downregulated by diphosphomevalonate: an antimicrobial target

The toll that Streptococcus pneumoniae exacts on the welfare of humanity is enormous. This organism claims the lives of approximately 3700 people daily, the majority of whom are children below the age of 5, and the situation could worsen due to the increasing incidence of pernicious, multiple-antibiotic-resistant strains. Here we report the discovery and characterization of a new allosteric site, shown to be absent in humans, that can be used to switch off an essential pathway in S. pneumoniae, the mevalonate pathway. Diphosphomevalonate (DPM), an intermediate in the pathway, binds with high affinity (K(d) = 530 nM) to mevalonate kinase, the first enzyme in the pathway, and inactivates it. Steady-state and equilibrium binding measurements reveal that DPM binding is noncompetitive versus substrates. DPM binds at an allosteric site, and inhibition cannot be overcome by an increasing substrate concentration. The DPM-binding site is a promising target for the development of new antimicrobial agents.     

Alteration of Escherichia coli topoisomerase IV conformation upon enzyme binding to positively supercoiled DNA

Escherichia coli topoisomerase IV (topo IV) is an essential enzyme that unlinks the daughter chromosomes for proper segregation at cell division. In vitro, topo IV readily distinguishes between the two possible chiralities of crossing segments in a DNA substrate. The enzyme relaxes positive supercoils and left-handed braids 20 times faster, and with greater processivity, than negative supercoils and right-handed braids. Here, we used chemical cross-linking of topo IV to demonstrate that enzyme bound to positively supercoiled DNA is in a different conformation from that bound to other forms of DNA. Using three different reagents, we observed novel cross-linked species of topo IV when positively supercoiled DNA was in the reaction. We show that the ParE subunits are in close enough proximity to be cross-linked only when the enzyme is bound to positively supercoiled DNA. We suggest that the altered conformation reflects efficient binding by topo IV of the two DNA segments that participate in the strand passage reaction.     

Contribution of the ATP binding site of ParE to susceptibility to novobiocin and quinolones in Streptococcus pneumoniae

In Streptococcus pneumoniae, an H103Y substitution in the ATP binding site of the ParE subunit of topoisomerase IV was shown to confer quinolone resistance and hypersensitivity to novobiocin when associated with an S84F change in the A subunit of DNA gyrase. We reconstituted in vitro the wild-type topoisomerase IV and its ParE mutant. The ParE mutant enzyme showed a decreased activity for decatenation at subsaturating ATP levels and was more sensitive to inhibition by novobiocin but was as sensitive to quinolones. These results show that the ParE alteration H103Y alone is not responsible for quinolone resistance and agree with the assumption that it facilitates the open conformation of the ATP binding site that would lead to novobiocin hypersensitivity and to a higher requirement of ATP.     

Atp-bound topoisomerase ii as a target for antitumor drugs

Topoisomerase II (TOP2) poisons interfere with the breakage/reunion reaction of TOP2 resulting in DNA cleavage. In the current studies, we show that two different classes (ATP-sensitive and -insensitive) of TOP2 poisons can be identified based on their differential sensitivity to the ATP-bound conformation of TOP2. First, in the presence of 1 mm ATP or the nonhydrolyzable analog adenosine 5'-(beta,gamma-imino)triphosphate, TOP2-mediated DNA cleavage induced by ATP-sensitive TOP2 poisons (e.g. doxorubicin, etoposide, mitoxantrone, and 4'-(9-acridinylamino)methanesulfon-m-anisidide) was 30-100-fold stimulated, whereas DNA cleavage induced by ATP-insensitive TOP2 poisons (e.g. amonafide, batracylin, and menadione) was only slightly (less than 3-fold) affected. In addition, ADP was shown to strongly antagonize TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive TOP2 poisons. Second, C427A mutant human TOP2alpha, which exhibits reduced ATPase activity, was shown to exhibit cross-resistance to all ATP-sensitive but not ATP-insensitive TOP2 poisons. Third, using ciprofloxacin competition assay, TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive poisons was shown to be antagonized by ciprofloxacin. These results suggest that ATP-bound TOP2 may be the specific target of ATP-sensitive TOP2 poisons. Using Lac repressor-operator complexes as roadblocks, we show that ATP-bound TOP2 acts as a circular clamp capable of entering DNA ends and sliding on unobstructed duplex DNA.     

Assembly of transcriptionally active chromatin in vitro: a possible role for topoisomerase II

Some models of in vitro chromatin assembly suggest a biphasic molecular mechanism. The first phase, nucleosome formation, is comprised of the formation of histone-DNA complexes which mature into a canonical nucleosome structure. The second phase represents the process by which these nucleosomes become properly spaced with a regular periodicity on the DNA. In this report, we examine the role of DNA topoisomerases in the latter phase of chromatin assembly. To study this process, we use a Xenopus laevis cell-free extract, which assembles quantitative amounts of chromatin on circular DNA templates, and the type II topoisomerase-specific antitumor drugs VM-26 and endrofloxicin. Our results suggest that nucleosome formation is unaffected by the presence of VM-26 or endrofloxicin. However, periodic spacing of nucleosomes is inhibited significantly by these drugs. In the absence of proper chromatin assembly, circular DNA molecules are processed into nucleoprotein complexes which are transcribed poorly. Taken together, these results indicate that the antitumor drugs VM-26 and endrofloxicin influence gene expression indirectly by blocking the periodic spacing of nucleosomes.     

DNA topoisomerase I from parasitic protozoa: a potential target for chemotherapy

The growing occurrence of drug resistant strains of unicellular prokaryotic parasites, along with insecticide-resistant vectors, are the factors contributing to the increased prevalence of tropical diseases in underdeveloped and developing countries, where they are endemic. Malaria, cryptosporidiosis, African and American trypanosomiasis and leishmaniasis threaten human beings, both for the high mortality rates involved and the economic loss resulting from morbidity. Due to the fact that effective immunoprophylaxis is not available at present; preventive sanitary measures and pharmacological approaches are the only sources to control the undesirable effects of such diseases. Current anti-parasitic chemotherapy is expensive, has undesirable side effects or, in many patients, is only marginally effective. Under this point of view molecular biology techniques and drug discovery must walk together in order to find new targets for chemotherapy intervention. The identification of DNA topoisomerases as a promising drug target is based on the clinical success of camptothecin derivatives as anticancer agents. The recent detection of substantial differences between trypanosome and leishmania DNA topoisomerase IB with respect to their homologues in mammals has provided a new lead in the study of the structural determinants that can be effectively targeted. The present report is an up to date review of the new findings on type IB DNA topoisomerase in unicellular parasites and the role of these enzymes as targets for therapeutic agents.