Using Pooled Recombinant Plasmids as Control Materials for Diagnostic Real-Time PCR.

BACKGROUND: Despite the potential usefulness of plasmids as reference materials for PCR, there are very few reports documenting the experience with using these materials in quality control (QC) of laboratory developed tests (LDTs), in a clinical diagnostic setting. In this study, an approach to preparing and titering such controls is detailed. The practicality of using plasmids as QC materials was explored and presented. METHODS: Target DNA fragments (n = 11) were amplified from positive samples and cloned using TA-cloning. Identities of the fragments were ascertained using DNA sequencing. Real-time PCR was carried out using TaqMan probes on RGQ or CFX-96 thermal-cyclers. RESULTS: All the 11 targeted DNA fragments were successfully cloned into E. coli vectors. Plasmid pools could be repeatedly reconstituted to generate stable and usable positive controls for multiplex PCR assays in a simple workflow. Importantly, plasmid controls generated meaningful run data that could be used in QC processes for monitoring routine runs. Further, plasmid material could be spiked into clinical specimens for quality assurance (QA) purposes, avoiding the culture of live infectious organisms, showing another routine usefulness of plasmids. CONCLUSIONS: Plasmid pools are useful and inexhaustible sources of reference materials for various routine QC uses in the diagnostic laboratory.

Atypical caseinolytic protease homolog from Plasmodium falciparum possesses unusual substrate preference and a functional nuclear localization signal.

Although ATP-dependent caseinolytic protease (Clp) complexes are important for regulating the pathogenicity, survival, and development of many pathogens, their physiological roles in the pathogenicity of malarial parasites remain unknown. This study reports the cloning, authentication, and characterization of a putative Clp protease subunit from Plasmodium falciparum (PfClpP). Heterologous expression studies showed that signal peptide hindered the soluble expression of the full-length PfClpP. Biochemical analyses of the recombinant PfClpP showed that it did not cleave the known ClpP substrate, succinyl-leucine-tyrosine-7-amido-4-methylcoumarin hydrochloride (AMC). Instead, PfClpP readily hydrolyzed a different substrate, glycine-arginine-AMC. The distinctive substrate preference of PfClpP suggests structural uniqueness in its substrate-binding sites that might be exploitable in anti-malarial drug development. Whether PfClpP resembles most eukaryotic ClpPs in being localized to the mitochondria and chloroplasts was also investigated using a mammalian surrogate host system. The results observed showed that green-fluorescence protein tagged PfClpP proteins were localized to the nucleus. PfClpP may have a unique and specialized role in the plasmodial nucleus. Taken together, this study has shown that PfClpP has a unique peptide cleavage function that is localized at the plasmodial nucleus, probably positioned to elicit a regulatory role in the parasite's pathogenicity.

Comparison of the Biofire FilmArray Respiratory Panel, Seegene AnyplexII RV16, and Argene for the detection of respiratory viruses.

BACKGROUND: Respiratory infections are common reasons for hospital admission, and are associated with enormous economic burden due to significant morbidity and mortality. The wide spectrum of microbial agents underlying the pathology renders the diagnosis of respiratory infections challenging. Molecular diagnostics offer an advantage to the current serological and culture-based methods in terms of sensitivity, coverage, hands-on time, and time to results. OBJECTIVES: This study aimed to compare the clinical performance of three commercial kits for respiratory viral detection. STUDY DESIGN: The performance of FilmArray Respiratory Panel, AnyplexII RV16, and Argene was compared using clinical respiratory samples (n = 224, comprising 189 nasopharyngeal swabs in Universal Transport Medium (UTM) and 35 endotracheal aspirates), based on common overlapping targets across the platforms. Influenza A "equivocal" and "no-subtype" samples by FilmArray were further compared to a laboratory-developed Influenza A/B test. RESULTS AND CONCLUSIONS: The overall performance of all three platforms appeared to be comparable with regards to sensitivities (95.8-97.9%) and specificities (96.1-98.0%), detection of coinfections, and distinguishment of influenza from non-influenza cases. "Equivocal" and "no-subtype" samples by FilmArray mostly represented weak Influenza A by laboratory-developed test. Lower respiratory tract samples had comparable final-run success-rates and discordant-rates as compared to UTM. Coronavirus HKU1, which was not targeted by AnyplexII RV16, were detected as OC43. The expected test volume would be the main determinant for the selection of platform. Among the platforms, the FilmArray is the most automated but is of the lowest-throughput and has the highest reagent cost.

Development of a real-time assay to determine the frequency of qac genes in methicillin resistant Staphylococcus aureus.

PURPOSE: The emergence of antiseptic resistance and/or antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) may result in failure of decolonization treatments. Plasmid-encoded efflux pump genes qacA/B and qacC (smr) confer tolerance to chlorhexidine and quaternary ammonium compounds. The objective of this study was to develop and validate a multiplex real time-PCR assay for detection of antiseptic-resistance genes, apply the assay on 200 MRSA isolates and explore if carriage of these genes was associated with resistance to topical antibiotics. METHODOLOGY: A SYBR-Green based multiplex real time-PCR assay was developed to detect qacA/B, qacC, and mecA (internal control) simultaneously. The multiplex assay was compared against conventional single-plex PCR followed by agarose gel electrophoresis, using DNA from the first 73 MRSA isolates, followed by multiplex testing of the remaining 127 MRSA isolates. All 200 MRSA isolates were tested for susceptibility to mupirocin, retapamulin, neomycin, bacitracin and octenidine. The genetic diversity of the isolates was investigated by spa-typing. RESULTS: The concordance between multiplex and conventional PCR, in assignments of qacA/B and qacC status were 99%(72/73) and 100%(73/73) respectively. Among 200 MRSA isolates, 48(24%) and 44(23%) were found to harbour qacA/B and qacC genes, respectively. These isolates remained susceptible to many common decolonization agents, except mupirocin. The predominant spa-types were t020 and t1081 (41 and 32 isolates respectively). CONCLUSION: The real-time assay performed acceptably for the detection of qac genes. A high prevalence of antiseptic-resistance genes were detected in the MRSA isolates in our population and appeared to be associated with spa-type t1081.

Plasmodium falciparum pyruvate kinase as a novel target for antimalarial drug-screening.

BACKGROUND: Global travellers are increasingly at risk of contracting malaria. The increasing occurrence of drug-resistance in many endemic areas emphasizes the need for novel drug targets for antimalarial-screening. In this study, the use of pyruvate kinase as a drug-target is evaluated. The functional validation of a gene encoding pyruvate kinase (designated PK1) has previously been reported. However, alternative copies of this enzyme encoded by Plasmodium falciparum could also circumvent the role of PK1. A survey of genome data revealed a putative ORF seemingly coding for another pyruvate kinase (designated PK2). METHODS: The expression of PK1 and PK2 in in vitro cultures were investigated by RT-PCR. Biocomputational analysis was carried out to identify structural differences between the P. falciparum pyruvate kinases and the corresponding enzymes from its human host. RESULTS: Both PK1 and PK2 were indeed actively transcribed during the intraerythrocytic stages, suggesting the involvement of both enzymes during infection. A comparison of amino acid residues at the effector binding sites of PK1 and PK2, to those of the human pyruvate kinases revealed some significant differences that could serve as targets for selective inhibitors to be designed against parasitic pyruvate kinases. CONCLUSION: Experimental evidence for the expression of both PK1 and PK2 during the blood stages of malaria infection was provided. Interestingly, phylogenetic analysis revealed that the "PK2" type of enzyme appears to be confined to Apicomplexans, an important observation with respect to the assessment of PK2 as a drug-target.

Pfnek3: an atypical activator of a MAP kinase in Plasmodium falciparum.

The canonical mitogen-activated protein kinase (MAPK) signal cascade was previously suggested to be atypical in the malaria parasite. This raises queries on the existence of alternative mediators of plasmodial MAPK pathways. This study describes, Pfnek3, a malarial protein kinase belonging to the NIMA (Never in Mitosis, Aspergillus) family. Endogenous Pfnek3 is expressed during late asexual to gametocyte stages and lacks some classical protein kinase sequence motifs. Moreover, Pfnek3 is phylogenetically distant from mammalian NIMA-kinases. Recombinant Pfnek3 was able to phosphorylate and stimulate a malarial MAPK (Pfmap2). Contrastingly, this was not observed with two other kinases, Pfmap1 and human MAPK1, suggesting that the Pfnek3-Pfmap2 interaction may be specific for Pfmap2 regulation. In summary, our data reveal a malarial NIMA-kinase with the potential to regulate a MAPK. Possessing biochemical properties divergent from classical mammalian NIMA-kinases, Pfnek3 could potentially be an attractive target for parasite-selective anti-malarials.

A relevant in vitro eukaryotic live-cell system for the evaluation of plasmodial protein localization.

Understanding the functional genomics and proteomics of plasmodia underpins the development of new approaches to antimalarial chemotherapy. Although genome databanks (e.g. PlasmoDB) and biocomputing tools (e.g. PlasMit, PlasmoAP, PATS) are useful in providing a global albeit predictive view of the myriad of about 5000 genes, only 40% are annotated, with few cases of endorsed subcellular localizations of the corresponding proteins in animal models. Progress in plasmodial protein trafficking has been hampered by the lack of a simple yet reliable method for studying subcellular localization of plasmodial proteins. In this study, we have used a combination of fluorescent markers, organelle-specific probes, phase contrast microscopy, and confocal microscopy to locate a selection of signal peptides from 10 plasmodial proteins in CHO-K1 cells. These eukaryotic cells serve as an in vitro living system for studying the cellular destinations of four mitochondrial-targeted TCA cycle proteins (citrate synthase, CS; isocitrate dehydrogenase, ICDH; branched chain alpha-keto-acid dehydrogenase E1alpha subunit, BCKDH; succinate dehydrogenase flavoprotein-subunit, SDH), two nuclear-targeted proteins (histone deacetylase, HDAC; RNA polymerase, RPOL), two apicoplast-targeted proteins (pyruvate kinase 2, PK2; glutamate dehydrogenase, GDH), and two cytoplasmic resident proteins (malate dehydrogenase, MDH; glycerol kinase, GK). The respective localizations of these malarial proteins have complied with the selected molecular targets, viz. mitochondrial, nuclear and cytoplasmic. Interestingly, MDH that is widely known to be resident in eukaryotic mitochondria was found to be cytoplasmic, probably due to the absence of molecular target sequences. Since the localization of plasmodial proteins is central to the authentication of their pathophysiological roles, this experimental system will serve as a useful a priori approach.

Novel cost-effective quality control approach for the Cepheid Xpert CT/NG assay for the detection of Chlamydia Trachomatis and Neisseria Gonorrhoeae.

The Xpert CT/NG is a rapid assay for detection of Neisseria gonorrhoeae and Chlamydia trachomatis. QC materials must be formulated to emulate human specimens, and are prohibitively expensive. A creative, cost-effective QC approach is proposed. The acceptable sample types for the Xpert CT/NG assay were extended to include eye swabs.

A Novel System Control for Quality Control of Diagnostic Tests Based on Next-Generation Sequencing.

BACKGROUND: We describe a novel system control (SC) implemented in an automated AmpliSeq™-based next-generation sequencing (NGS)2 run that simultaneously acts as (a) an external positive/sensitivity control, (b) a spike-in QC for DNA extraction, and (c) a nontemplate control to detect exogenous DNA contamination. METHODS: Plasmids carrying wild-type tobacco mosaic virus sequence and a sequence with three designed mutations were synthesized and mixed, such that the mutations are present at 5% variant frequency in the mixture designated as SC. SC was used as a stand-alone sample and spiked into each sample in each run. A cell line-derived reference material, in both a formalin-fixed paraffin-embedded (FFPE) sample and genomic DNA (gDNA), was sequenced in the same runs. RESULTS: By interpolation, 100 fg SC spiked in FFPE sample produced sequencing coverage equivalent to approximately 3 fg in the gDNA. In the SC-only sample, all three designed mutations were recovered around 5% as expected, while no significant reads of human genome were present. In samples with a common PCR inhibitor, coverage for both SC and target amplicons were eliminated. An inverse relationship between the coverage of SC and DNA input was observed. In clinical samples, the ratio of SC to the median coverage of sample can be used to indicate insufficient DNA input. CONCLUSIONS: The SC is an elegant and comprehensive QC concept for NGS-based diagnostic tests.

microRNAs in breast cancer: regulatory roles governing the hallmarks of cancer.

A large number of etiological factors and the complexity of breast cancers present challenges for prevention and treatment. Recently, the emergence of microRNAs (miRNAs) as cancer biomarkers has added an extra dimension to the 'molecular signatures' of breast cancer. Bioinformatic analyses indicate that each miRNA can regulate hundreds of target genes and could serve functionally as 'oncogenes' or 'tumour suppressor' genes, and co-ordinate multiple cellular processes relevant to cancer progression. A number of studies have shown that miRNAs play important roles in breast tumorigenesis, metastasis, proliferation and differentiation of breast cancer cells. This review provides a comprehensive overview of miRNAs with established functional relevance in breast cancer, their established target genes and resulting cellular phenotype. The role and application of circulating miRNAs in breast cancer is also discussed. Furthermore, we summarize the role of miRNAs in the hallmarks of breast cancer, as well as the possibility of using miRNAs as potential biomarkers for detection of breast cancer.

Detecting novel genetic variants associated with isoniazid-resistant Mycobacterium tuberculosis.

BACKGROUND: Isoniazid (INH) is a highly effective antibiotic central for the treatment of Mycobacterium tuberculosis (MTB). INH-resistant MTB clinical isolates are frequently mutated in the katG gene and the inhA promoter region, but 10 to 37% of INH-resistant clinical isolates have no detectable alterations in currently known gene targets associated with INH-resistance. We aimed to identify novel genes associated with INH-resistance in these latter isolates. METHODOLOGY/PRINCIPAL FINDINGS: INH-resistant clinical isolates of MTB were pre-screened for mutations in the katG, inhA, kasA and ndh genes and the regulatory regions of inhA and ahpC. Twelve INH-resistant isolates with no mutations, and 17 INH-susceptible MTB isolates were subjected to whole genome sequencing. Phylogenetically related variants and synonymous mutations were excluded and further analysis revealed mutations in 60 genes and 4 intergenic regions associated with INH-resistance. Sanger sequencing verification of 45 genes confirmed that mutations in 40 genes were observed only in INH-resistant isolates and not in INH-susceptible isolates. The ratios of non-synonymous to synonymous mutations (dN/dS ratio) for the INH-resistance associated mutations identified in this study were 1.234 for INH-resistant and 0.654 for INH-susceptible isolates, strongly suggesting that these mutations are indeed associated with INH-resistance. CONCLUSION: The discovery of novel targets associated with INH-resistance described in this study may potentially be important for the development of improved molecular detection strategies.

Identification of circulating microRNA signatures for breast cancer detection.

PURPOSE: There is a quest for novel noninvasive diagnostic markers for the detection of breast cancer. The goal of this study is to identify circulating microRNA (miRNA) signatures using a cohort of Asian Chinese patients with breast cancer, and to compare miRNA profiles between tumor and serum samples. EXPERIMENTAL DESIGN: miRNA from paired breast cancer tumors, normal tissue, and serum samples derived from 32 patients were comprehensively profiled using microarrays or locked nucleic acid real-time PCR panels. Serum samples from healthy individuals (n = 22) were also used as normal controls. Significant serum miRNAs, identified by logistic regression, were validated in an independent set of serum samples from patients (n = 132) and healthy controls (n = 101). RESULTS: The 20 most significant miRNAs differentially expressed in breast cancer tumors included miRNA (miR)-21, miR-10b, and miR-145, previously shown to be dysregulated in breast cancer. Only 7 miRNAs were overexpressed in both tumors and serum, suggesting that miRNAs may be released into the serum selectively. Interestingly, 16 of the 20 most significant miRNAs differentially expressed in serum samples were novel. MiR-1, miR-92a, miR-133a, and miR-133b were identified as the most important diagnostic markers, and were successfully validated; receiver operating characteristic curves derived from combinations of these miRNAs exhibited areas under the curves of 0.90 to 0.91. CONCLUSION: The clinical use of miRNA signatures as a noninvasive diagnostic strategy is promising, but should be further validated for different subtypes of breast cancers.

Improving indel detection specificity of the Ion Torrent PGM benchtop sequencer.

The emergence of benchtop sequencers has made clinical genetic testing using next-generation sequencing more feasible. Ion Torrent's PGM™ is one such benchtop sequencer that shows clinical promise in detecting single nucleotide variations (SNVs) and microindel variations (indels). However, the large number of false positive indels caused by the high frequency of homopolymer sequencing errors has impeded PGM™'s usage for clinical genetic testing. An extensive analysis of PGM™ data from the sequencing reads of the well-characterized genome of the Escherichia coli DH10B strain and sequences of the BRCA1 and BRCA2 genes from six germline samples was done. Three commonly used variant detection tools, SAMtools, Dindel, and GATK's Unified Genotyper, all had substantial false positive rates for indels. By incorporating filters on two major measures we could dramatically improve false positive rates without sacrificing sensitivity. The two measures were: B-Allele Frequency (BAF) and VARiation of the Width of gaps and inserts (VARW) per indel position. A BAF threshold applied to indels detected by UnifiedGenotyper removed ~99% of the indel errors detected in both the DH10B and BRCA sequences. The optimum BAF threshold for BRCA sequences was determined by requiring 100% detection sensitivity and minimum false discovery rate, using variants detected from Sanger sequencing as reference. This resulted in 15 indel errors remaining, of which 7 indel errors were removed by selecting a VARW threshold of zero. VARW specific errors increased in frequency with higher read depth in the BRCA datasets, suggesting that homopolymer-associated indel errors cannot be reduced by increasing the depth of coverage. Thus, using a VARW threshold is likely to be important in reducing indel errors from data with higher coverage. In conclusion, BAF and VARW thresholds provide simple and effective filtering criteria that can improve the specificity of indel detection in PGM™ data without compromising sensitivity.

Development of a next-generation sequencing method for BRCA mutation screening: a comparison between a high-throughput and a benchtop platform.

In a clinical setting, next-generation sequencing (NGS) approaches for the enrichment and resequencing of DNA targets may have limitations in throughput, cost, or accuracy. We evaluated an NGS workflow for targeted DNA sequencing for mutation detection. Targeted sequence data of the BRCA1 and BRCA2 genes, generated using a PCR-based, multiplexed NGS approach using the SOLiD 4 (n = 24) and Ion Torrent PGM (n = 20) next-generation sequencers, were evaluated against sequence data obtained by Sanger sequencing. The overall sensitivity for SOLiD and PGM were 97.8% (95% CI = 94.7 to 100.0) and 98.9% (95% CI = 96.8 to 100.0) respectively. The specificity for the SOLiD platform was high, at 100.0% (95% CI = 99.3 to 100.0). PGM correctly identified all 3 indels, but 68 false-positive indels were also called. Equimolar normalization of amplicons was not necessary for successful NGS. Both platforms are highly amenable to scale-up, potentially reducing the reagent cost for BRCA testing to

Evaluation of nanofluidics technology for high-throughput SNP genotyping in a clinical setting.

The current need for high-throughput genotyping platforms for targeted validation of disease-associated single nucleotide polymorphisms (SNPs) motivated us to evaluate a novel nanofluidics platform for genotyping DNA extracted from peripheral blood and buccal wash samples. SNP genotyping was performed using a Fluidigm 48.48 Dynamic Array biochip on the BioMark polymerase chain reaction platform and results were compared against standard TaqMan assays and DNA sequencing. Pilot runs using these dynamic arrays on 90 samples against 20 SNP assays had an average call rate of 99.7%, with 100% call rates for 16 of the assays. Manual TaqMan genotyping of these samples against three SNPs demonstrated 100% correlation between the two platforms. To understand the influence of DNA template variability, three sources of blood samples (CH-1, n = 20; CH-2, n = 47; KK, n = 47) and buccal washes (n = 37) were genotyped for 24 SNPs. Although both CH-1 and CH-2 batches showed good base calling (≥98.8%), the KK batch and buccal wash samples exhibited lower call rates (82.1% and 94.0%). Importantly, repurification of the KK and buccal wash samples resulted in significant improvements in their call rates (to ≥97.9%). Scale-up for genotyping 1698 cases and controls for 24 SNPs had overall call rates of 97.6% for KK and 99.2% for CH samples. The Dynamic Array approach demonstrated accuracy similar to that of TaqMan genotyping, while offering significant savings in DNA, effort, time, and costs.

Molecular basis for thermal properties of Streptomyces thermovulgaris fumarase C hinge at hydrophilic amino acids R163, E170 and S347.

Industrially, the use of high temperatures (40-60 degrees C) in the L: -malate production process could result in rapid inactivation of the mesophilic fumarases, warranting constant replenishment of the biocatalyst. Thus, a thermostable fumarase C that is active and stable at high temperatures would be ideal. Biochemical studies using recombinant fumarase C from thermophilic Streptomyces thermovulgaris (stFUMC) indicated that it was optimally active at 50 degrees C and highly stable even after 24 h of incubation at 40 degrees C. The same gene from mesophilic Streptomyces coelicolor (scfumC) was also cloned and expressed as soluble proteins for comparison in thermal properties of both enzymes. In contrast to stFUMC, scFUMC exhibited a lower temperature optima of 30 degrees C and was rapidly denatured at 50 degrees C. The specific activity of stFUMC was also higher than that of scFUMC by 20-fold. After primary sequence comparison, three hydrophilic amino acid residues, R163, E170 and S347, were forged into the thermolabile scFUMC either singly or in combination for the investigation of their contributions in the thermal properties of the mutant enzymes. Of the mutants studied, the A347S scFUMC mutant resulted in the highest increase in optimum temperature of 10 degrees C and a fourfold enhancement in specific activity. G163R/G170E and G163R/G170E/A347S scFUMC mutants are more thermostable than wild-type scFUMC. These findings support stFUMC as a highly efficient, thermostable fumarase C with industrial potential and suggest that R163, E170 and S347 are involved in the enhancement of thermal properties in fumarase C.

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum.

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K(m) of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1mM pyridoxal-5'-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K(i) of 0.8mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.

Malate synthase from Streptomyces clavuligerus NRRL3585: cloning, molecular characterization and its control by acetate.

Malate synthase is a key enzyme of the glyoxylate cycle, which is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, encoding malate synthase from Streptomyces clavuligerus NRRL 3585, was cloned using PCR and fully sequenced. The ORF obtained encodes 541 amino acids with a deduced Mr of 60000, consistent with the observed Mr (62000-64000) of most malate synthase enzymes reported so far. The aceB gene has a high G+C content (71.5 mol%), especially in the third codon position. A 50 bp region upstream of the malate synthase ORF was predicted to be a prokaryotic promoter region. The relationship between carbon source, antibiotic (cephalosporin) biosynthesis and malate synthase activity was investigated. Growth of S. clavuligerus on acetate as the major carbon source was delayed, compared to that on glycerol. Furthermore, high levels of malate synthase activity were associated with the presence of acetate in the growth medium. Growth on acetate also resulted in lower levels of cephalosporin production, compared to that on glycerol. The cloned S. clavuligerus aceB gene was expressed in Escherichia coli BL21(DE3). Transformants exhibited an approximately 71-fold increase in malate synthase activity, compared to the control, thereby demonstrating high-level expression of soluble and enzymically active malate synthase in the heterologous host.

Recombinant Plasmodium falciparum NADP-dependent isocitrate dehydrogenase is active and harbours a unique 26 amino acid tail.

During infection, Plasmodium spp. require reducing equivalents such as NADPH to support the function of specific enzymes in overcoming oxidative stress. The catalysis of isocitrate by the NADP-dependent isocitrate dehydrogenase of Plasmodium falciparum (pfICDH) generates NADPH and is thus crucial for the parasite's survival and pathogenecity. In this study, pfICDH was cloned from a clinical isolate of P. falciparum. This was facilitated by designing primers based on the P. falciparum genome sequence resource PlasmoDB. DNA sequence of the cloned gene revealed an ORF that encodes a protein of 468 aa. Furthermore, after expression in Esherichia coli BL21, enzyme assays of cell-free extracts confirmed the overexpression and function of pfICDH. Further, pfICDH purified by affinity chromatography retained its enzyme activity. Substitution of NADP for NAD, or the use of EDTA, in enzyme assays abolished pfICDH activity. ATP and chloroquine, as well as cupric and argentic ions, inhibited pfICDH activity. Phylogenetic analysis revealed high primary structure homology (45-97%) among genes coding for eukaryal NADP-dependent ICDH, and the occurrence of three subfamilies of ICDH genes. Interestingly, there were significant sequence dissimilarities between pfICDH and its mammalian or bacterial homologs, particularly at the N- and C-termini. Confirming the functionality of the cloned pfICDH, and asserting its distance from the human homolog by molecular definitions, are important prerequisites for promoting this gene as a drug target screen.