Examination of intrinsic sulfonamide resistance in Bacillus anthracis: a novel assay for dihydropteroate synthase

Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPPi to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/alpha-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.     

A rapid assay for dihydropteroate synthase activity suitable for identification of inhibitors

The enzymes 6-hydroxymethylpterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) catalyze sequential steps in folate biosynthesis. They are present in microorganisms but absent in mammals and therefore are especially suitable targets for antimicrobials. Sulfa drugs (sulfonamides and sulfones) currently are used as antimicrobials targeting DHPS, although resistance to these drugs is increasing. The most widely used assay that measures activity of these enzymes, to assess new inhibitors in vitro, is not amenable to automation. This article describes a simple, coupled, enzymatic spectrophotometric assay where the product of the DHPS reaction, dihydropteroate, is reduced to tetrahydropteroate by excess dihydrofolate reductase (DHFR) using the cofactor NADPH. The oxidation of NADPH is monitored at 340 nm. The activity of both HPPK and DHPS can be measured in this assay, and it has been used to measure kinetic parameters of wild-type and sulfa drug-resistant DHPS enzymes to demonstrate the utility of the assay. It is a sensitive and reproducible assay that can be readily automated and used in multiwell plates. This NADPH-coupled microplate photometric assay could be used for rapid screening of chemical libraries for novel inhibitors of folate biosynthesis as the first step in developing new antimicrobial drugs targeting the folate biosynthetic pathway.     

Design,synthesis, antibacterial evaluation and molecular docking studies of some new quinoxaline derivatives targeting dihyropteroate synthase enzyme

Development of new antimicrobial agents is a good solution to overcome drug-resistance problems. From this perspective, new quinoxaline derivatives bearing various bioactive heterocyclic moieties (thiadiazoles, oxadiazoles, pyrazoles and thiazoles) were designed and synthesized. The newly synthesized compounds were evaluated for their in vitro antibacterial activity against nine bacterial human pathogenic strains using the disc diffusion assay. In general, most of the synthesized compounds exhibited good antibacterial activities. The thiazolyl 11c displayed significant antibacterial activities against P. aeruginosa (MIC, 12.5 µg/mL vs levofloxacin 12.5 µg/mL). Molecular docking studies indicated that the synthesized compounds could occupy both p-amino benzoic acid (PABA) and pterin binding pockets of the dihydropteroate synthase (DHPS), suggesting that the target compounds could act by the inhibition of bacterial DHPS enzyme. The results provide important information for the future design of more potent antibacterial agents.     

Crystal structures of <Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis> dihydropteroate synthase in the apo-form and complexed with the product 7,8-dihydropteroate

Background  

The enzyme dihydropteroate synthase (DHPS) participates in the de novo synthesis of folate cofactors by catalyzing the formation of 7,8-dihydropteroate from condensation of p-aminobenzoic acid with 6-hydroxymethyl-7,8-dihydropteroate pyrophosphate. DHPS is absent from humans, who acquire folates from diet, and has been validated as an antimicrobial therapeutic target by chemical and genetic means. The bacterium Burkholderia cenocepacia is an opportunistic pathogen and an infective agent of cystic fibrosis patients. The organism is highly resistant to antibiotics and there is a recognized need for the identification of new drugs against Burkholderia and related Gram-negative pathogens. Our characterization of the DHPS active site and interactions with the enzyme product are designed to underpin early stage drug discovery.     

Characterization of mutationally altered dihydropteroate synthase and its ability to form a sulfonamide-containing dihydrofolate analog.

Among spontaneous mutants of Escherichia coli selected for resistance against sulfonamides, thermosensitive strains were found. These were shown to possess a changed dihydropteroate synthase (EC 2.5.1.15), which had a substantially higher Km value for its normal substrate, p-aminobenzoic acid, and an about 150-fold higher Km for sulfonamides. The mutationally changed dihydropteroate synthase was found to be thermosensitive by in vitro assays. The thermosensitivity was used as an enzyme marker to demonstrate the complex formation between 2-amino-4-hydroxy-6-pyrophosphorylmethyl pteridine and sulfonamides by partially purified dihydropteroate synthase. The formation of folate from 2-amino-4-hydroxy-6-pyrophosphorylmethyl pteridine and p-aminobenzoylglutamic acid by dihydropteroate synthase was found to be very sensitive to inhibition by sulfonamides and very inefficient with the mutationally changed enzyme.     

Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine-pyrimethamine in Mali

Sulfadoxine-pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.     

Insights into the drug resistance induced by the BaDHPS mutations: molecular dynamic simulations and MM/GBSA studies

Dihydropteroate synthase (DHPS) is essential for the folic acid biosynthetic pathway in prokaryotes; the mutation forms for DHPS are found to be relative to the urgent drug resistance problems. In our study, the Bacillus anthracis DHPS (BaDHPS) was selected for molecular dynamics and binding free energy studies to investigate the biochemistry behaviors of the wild-type and mutation form BaDHPS proteins (D184N and K220Q). It is found that the conformational change of the ligand dihydropteroate sulfathiazole binding site in mutation D184N and K220Q systems is mainly attributed from the Loop 1, Loop 2, and Loop 7 regions, and the binding free energy of these mutation systems is lower than that of the wild-type system. Additionally, some important hydrogen bonds of the mutation systems are disrupted during the simulations. But the shortening of the distance between residue Thr67 and the ligand would cause significant change of the binding pose in the K220Q system. These studies of DHPS family will be helpful for further drug resistance investigations.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:24     

Rational design of biodegradable sulphonamide candidates treating septicaemia by synergistic dual inhibition of COX-2/PGE2 axis and DHPS enzyme

A new series of co-drugs was designed based on hybridising the dihydropteroate synthase (DHPS) inhibitor sulphonamide scaffold with the COX-2 inhibitor salicylamide pharmacophore through biodegradable linkage to achieve compounds with synergistic dual inhibition of COX-2/PGE2 axis and DHPS enzyme to enhance antibacterial activity for treatment of septicaemia. Compounds 5 b, 5j, 5n and 5o demonstrated potent in vitro COX-2 inhibitory activity comparable to celecoxib. 5j and 5o exhibited ED₅₀ lower than celecoxib in carrageenan-induced paw edoema test with % PGE2 inhibition higher than celecoxib. Furthermore, 5 b, 5j and 5n showed gastric safety profile like celecoxib. Moreover, in vivo antibacterial screening revealed that, 5j showed activity against S.aureus and E.coli higher than sulfasalazine. While, 5o revealed activity against E.coli higher than sulfasalazine and against S.aureus comparable to sulfasalazine. Compound 5j achieved the target goal as potent inhibitor of COX-2/PGE2 axis and in vivo broad-spectrum antibacterial activity against induced septicaemia in mice.     

Surface Plasmon Resonance Biosensor for Detection of Bacillus anthracis, the Causative Agent of Anthrax from Soil Samples Targeting Protective Antigen

Bacillus anthracis, the causative agent of anthrax is one of the most important biological warfare agents. In this study, surface plasmon resonance (SPR) technology was used for indirect detection of B. anthracis by detecting protective antigen (PA), a common toxin produced by all live B. anthracis bacteria. For development of biosensor, a monoclonal antibody raised against B. anthracis PA was immobilized on carboxymethyldextran modified gold chip and its interaction with PA was characterized in situ by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, K_D (equilibrium constant) and B_max (maximum binding capacity of analyte) were found to be 20 fM and 18.74, respectively. The change in Gibb’s free energy (∆G = −78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 12 fM purified PA. When anthrax spores spiked soil samples were enriched, PA produced in the sample containing even a single spore of B. anthracis could be detected by SPR. PA being produced only by the vegetative cells of B. anthracis, confirms indirectly the presence of B. anthracis in the samples. The proposed method can be a very useful tool for screening and confirmation of anthrax suspected environmental samples during a bio-warfare like situation.     

Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group

Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.     

Diaminodiphenylsulfone resistance of Mycobacterium leprae due to mutations in the dihydropteroate synthase gene

The nucleotide sequence analysis of the dihydropteroate synthase (DHPS) gene of six diaminodiphenylsulfone-resistant Mycobacterium leprae strains revealed that the mutation was limited at highly conserved amino acid residues 53 or 55. Though the mutation at amino acid residue 55 or its homologous site has been reported in other bacteria, the mutation at residue 53 is the first case in bacteria. This is the first paper which links the mutations in DHPS and sulfonamide resistance in M. leprae. This finding is medically and socially relevant, since leprosy is still a big problem in certain regions.     

Reverse Line Blot Macroarray for Simultaneous Detection and Characterization of Four Biological Warfare Agents

The need for a rapid detection and characterization of biowarfare (BW) agents cannot be over emphasized. With diverse array of potential BW pathogen available presently, rapid identification of the pathogen is crucial, so that specific therapy and control measures can be initiated. We have developed a multiplex polymerase chain reaction based reverse line blot macroarray to simultaneously detect four pathogens of BW importance viz. Bacillus anthracis, Yersinia pestis, Brucella melitensis and Burkholderia pseudomallei. The multiplex PCR utilizes 14 pairs of primers targeting 18 specific markers. These markers include genes which are genus specific, species-specific chromosomal sequences and virulence markers of plasmid origin. The assay was evaluated on various human, environment and animal isolates. The assay w successful in simultaneous detection and characterization of isolates of the four pathogens on as a single platform with sensitivity ranging from 0.3 pg to 0.3 ng of genomic DNA. The assay was able to detect 5 × 10² cfu/ml for B. anthracis, 8 × 10² cfu/ml for Yersinia sp., 1.4 × 10² cfu/ml for B. melitensis and 4 × 10² cfu/ml for B. pseudomallei.     

A Case of Pneumonia Caused by Pneumocystis jirovecii Resistant to Trimethoprim-Sulfamethoxazole

A 50-year-old male visited the outpatient clinic and complained of fever, poor oral intake, and weight loss. A chest X-ray demonstrated streaky and fibrotic lesions in both lungs, and chest CT revealed multifocal peribronchial patchy ground-glass opacities with septated cystic lesions in both lungs. Cell counts in the bronchoalveolar lavage fluid revealed lymphocyte-dominant leukocytosis, and further analysis of lymphocyte subsets showed a predominance of cytotoxic T cells and few T helper cells. Video-assisted wedge resection of the left upper lobe was performed, and the histologic examination was indicative of a Pneumocystis jirovecii infection. Trimethoprim-sulfamethoxazole (TMP-SMX) was orally administered for 3 weeks; however, the patient complained of cough, and the pneumonia was aggravated in the follow-up chest X-ray and chest CT. Molecular studies demonstrated mutations at codons 55 and 57 of the dihydropteroate synthase (DHPS) gene, which is associated with the resistance to TMP-SMX. Clindamycin-primaquine was subsequently administered for 3 weeks replacing the TMP-SMX. A follow-up chest X-ray showed that the pneumonia was resolving, and the cough was also alleviated. A positive result of HIV immunoassay and elevated titer of HCV RNA indicated HIV infection as an underlying condition. This case highlights the importance of careful monitoring of patients with P. jirovecii pneumonia (PCP) during the course of treatment, and the molecular study of DHPS mutations. Additionally, altering the anti-PCP drug utilized as treatment must be considered when infection with drug-resistant P. jirovecii is suspected. To the best of our knowledge, this is the first case of TMP-SMX-resistant PCP described in Korea.     

Sulfonamide resistance gene for plant transformation

The sulfonamide resistance gene from plasmid R46 encodes for a mutated dihydropteroate synthase insensitive to inhibition by sulfonamides. Its coding sequence was fused to the pea ribulose bisphosphate carboxylase/oxygenase transit peptide sequence. Incubation of isolated chloroplasts with the fusion protein synthesised in vitro, showed that the bacterial enzyme was transported to the chloroplast stroma and processed into a mature form. Expression of the gene fusion in transgenic plants resulted in a high level of resistance to sulfonamides. Direct selection of transformed shoots on leaf explants was efficient using sulfonamides as sole selective agents. Transformed shoots rooted normally on sulfonamides at concentrations toxic for untransformed ones. Sulfonamide resistance was transmitted to the progeny of transformed plants as a single Mendelian dominant character. These results demonstrate that this chimeric gene can be used as an efficient and versatile selectable marker for plant transformation.     

The three-dimensional structure of the bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/dihydropteroate synthase of Saccharomyces cerevisiae

In Saccharomyces cerevisiae and other fungi, the enzymes dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) are encoded by a polycistronic gene that is translated into a single polypeptide having all three functions. These enzymatic functions are essential to both prokaryotes and lower eukaryotes, and catalyse sequential reactions in folate biosynthesis. Deletion or disruption of either function leads to cell death. These enzymes are absent from mammals and thus make ideal antimicrobial targets. DHPS is currently the target of antifolate therapy for a number of infectious diseases, and its activity is inhibited by sulfonamides and sulfones. These drugs are typically used as part of a synergistic cocktail with the 2,4-diaminopyrimidines that inhibit dihydrofolate reductase. A gene encoding the S.cerevisiae HPPK and DHPS enzymes has been cloned and expressed in Escherichia coli. A complex of the purified bifunctional polypeptide with a pterin monophosphate substrate analogue has been crystallized, and its structure solved by molecular replacement and refined to 2.3A resolution. The polypeptide consists of two structural domains, each of which closely resembles its respective monofunctional bacterial HPPK and DHPS counterpart. The mode of ligand binding is similar to that observed in the bacterial enzymes. The association between the domains within the polypeptide as well as the quaternary association of the polypeptide via its constituent DHPS domains provide insight into the assembly of the trifunctional enzyme in S.cerevisiae and probably other fungal species.     

Cloning, sequencing, and enhanced expression of the dihydropteroate synthase gene of Escherichia coli MC4100.

The Escherichia coli gene coding for dihydropteroate synthase (DHPS) has been cloned and sequenced. The protein has 282 amino acids and a compositional molecular mass of 30,314 daltons. Increased expression of the enzyme was realized by using a T7 expression system. The enzyme was purified and crystallized. A temperature-sensitive mutant was isolated and found to express a DHPS with a lower specific activity and lower affinities for para-aminobenzoic acid and sulfathiazole. The allele had a point mutation that changed a phenylalanine codon to a leucine codon, and the mutation was in a codon that is conserved among published DHPS sequences.     

A folate independent role for cytosolic HPPK/DHPS upon stress in Arabidopsis thaliana

Cytosolic HPPK/DHPS (cytHPPK/DHPS) in Arabidopsis is a functional enzyme with activity similar to its mitochondrial isoform. Genomic complementation of the cytHPPK/DHPS knockout mutant with the wild type gene led to a complete rescue of the stress sensitive mutant phenotype in seed germination tests under abiotic stress conditions. Moreover, over-expression of the gene resulted in higher germination rate under stress as compared to the wild-type, confirming its role in stress resistance. Analysis of folates in seedlings, inflorescence and dry seeds showed unchanged levels in the wild-type, mutant and over-expressor line, upon stress and normal conditions, suggesting a role for cytHPPK/DHPS distinct from folate biosynthesis and a folate-independent stress resistance mechanism. This apparently folate-independent mechanism of stress resistance points towards a possible role of pterins, since the product of HPPK/DHPS is dihydropteroate.     

Use of high-resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination

Single nucleotide polymorphisms (SNPs) are important diagnostic markers for the detection and differentiation of Bacillus anthracis. High-Resolution Melting (HRM) and Melting Temperature (Tm)-shift methods are two approaches that enable SNP detection without the need for expensive labeled probes. We evaluated the potential diagnostic capability of those methods to discriminate B. anthracis from the other members of the B. cereus group. Two assays targeting B. anthracis-specific SNPs in the plcR and gyrA genes were designed for each method and used to genotype a panel of 155 Bacilli strains. All B. anthracis isolates (n = 65) were correctly and unambiguously identified. Assays also proved to be appropriate for the direct genotyping of biological samples. They could reliably detect B. anthracis in contaminated organs containing as little as 10³ CFU/ml, corresponding to a few genome equivalents per reaction. The HRM and Tm-shift applications described here represent valuable tools for specific identification of B. anthracis at reduced cost.     

Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph

Summary Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA ^–auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35854, predicted from the deduced amino acid sequence, was similar to the 38 000 M_r determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.