Plasmodium Iron-Sulfur [Fe-S] cluster assembly protein Dre2 as a plausible target of Artemisinin: Mechanistic insights derived in a prokaryotic heterologous system.

Iron-sulfur (Fe-S) cluster containing proteins have been assigned roles in various essential cellular processes, such as regulation of gene expression, electron transfer, sensing of oxygen and balancing free radical chemistry. However, their role as the drug target remains sparse. Recently the screening of protein alkylation targets for artemisinin in Plasmodium falciparum led to identification of Dre2, a protein involved in redox mechanism for the cytoplasmic Fe-S cluster assembly in different organisms. In the present study, to further explore the interaction between artemisinin and Dre2, we have expressed the Dre2 protein of both P. falciparum and P. vivax in E. coli. The opaque brown colour of the IPTG induced recombinant Plasmodium Dre2 bacterial pellet, suggested iron accumulation as confirmed by the ICP-OES analysis. In addition, overexpression of rPvDre2 in E. coli reduced its viability, growth and increased the ROS levels of bacterial cells, which in turn led to an increase in expression of stress response genes of E. coli such as recA, soxS, mazF. Moreover, the overexpression of rDre2 induced cell death could be rescued by treatment with Artemisinin derivatives suggesting their interaction. The interaction between DHA and PfDre2 was later demonstrated by CETSA and microscale thermophoresis. Overall, this study suggests that Dre2 is the probable target of Artemisinin and the antimalarial activity of DHA/Artemether could also be due to yet unidentified molecular mechanism altering the Dre2 activity in addition to inducing DNA and protein damage.

Delineating the Role of Aedes aegypti ABC Transporter Gene Family during Mosquito Development and Arboviral Infection via Transcriptome Analyses.

Aedes aegypti acts as a vector for several arboviral diseases that impose a major socio-economic burden. Moreover, the absence of a vaccine against these diseases and drug resistance in mosquitoes necessitates the development of new control strategies for vector-borne diseases. ABC transporters that play a vital role in immunity and other cellular processes in different organisms may act as non-canonical immune molecules against arboviruses, however, their role in mosquito immunity remains unexplored. This study comprehensively analyzed various genetic features of putative ABC transporters and classified them into A-H subfamilies based on their evolutionary relationships. Existing RNA-sequencing data analysis indicated higher expression of cytosolic ABC transporter genes (E & F Subfamily) throughout the mosquito development, while members of other subfamilies exhibited tissue and time-specific expression. Furthermore, comparative gene expression analysis from the microarray dataset of mosquito infected with dengue, yellow fever and West Nile viruses revealed 31 commonly expressed ABC transporters suggesting a potentially conserved transcriptomic signature of arboviral infection. Among these, only a few transporters of ABCA, ABCC and ABCF subfamily were upregulated, while most were downregulated. This indicates the possible involvement of ABC transporters in mosquito immunity.

Functional analysis of iron-sulfur cluster biogenesis (SUF pathway) from Plasmodium vivax clinical isolates.

Iron-sulfur (Fe-S) clusters are critical metallo-cofactors required for cell function. Assembly of these cofactors is a carefully controlled process in cells to avoid toxicity from free iron and sulfide. In Plasmodium, two pathways for these Fe-S cluster biogenesis have been reported; ISC pathway in the mitochondria and SUF pathway functional in the apicoplast. Amongst these, SUF pathway is reported essential for the apicoplast maintenance and parasite survival. Many of its components have been studied from P. falciparum and P. berghei in recent years, still few queries remain to be addressed; one of them being the assembly and transfer of Fe-S clusters. In this study, using P. vivax clinical isolates, we have shown the in vitro interaction of SUF pathway proteins SufS and SufE responsible for sulfur mobilization in the apicoplast. The sulfur mobilized by the SufSE complex assembles on the scaffold protein PvSufA along with iron provided by the external source. Here, we demonstrate in vitro transfer of these labile Fe-S clusters from the scaffold protein on to an apo-protein, PvIspG (a protein involved in penultimate step of Isoprenoids biosynthesis pathway) in order to provide an insight into the interaction of different components for the biosynthesis and transfer of Fe-S clusters. Our analysis indicate that inspite of the presence of variations in pathway proteins, the overall pathway remains well conserved in the clinical isolates when compared to that reported in lab strains.

Unraveling the Plasmodium vivax sporozoite transcriptional journey from mosquito vector to human host.

Malaria parasites transmitted by mosquito bite are remarkably efficient in establishing human infections. The infection process requires roughly 30 minutes and is highly complex as quiescent sporozoites injected with mosquito saliva must be rapidly activated in the skin, migrate through the body, and infect the liver. This process is poorly understood for Plasmodium vivax due to low infectivity in the in vitro models. To study this skin-to-liver-stage of malaria, we used quantitative bioassays coupled with transcriptomics to evaluate parasite changes linked with mammalian microenvironmental factors. Our in vitro phenotyping and RNA-seq analyses revealed key microenvironmental relationships with distinct biological functions. Most notable, preservation of sporozoite quiescence by exposure to insect-like factors coupled with strategic activation limits untimely activation of invasion-associated genes to dramatically increase hepatocyte invasion rates. We also report the first transcriptomic analysis of the P. vivax sporozoite interaction in salivary glands identifying 118 infection-related differentially-regulated Anopheles dirus genes. These results provide important new insights in malaria parasite biology and identify priority targets for antimalarial therapeutic interventions to block P. vivax infection.

Recent Advances in the [Fe-S] Cluster Biogenesis (SUF) Pathway Functional in the Apicoplast of Plasmodium.

Iron-sulfur [Fe-S] clusters are one of the most ancient, ubiquitous, structurally and functionally versatile natural biosynthetic prosthetic groups required by various proteins involved in important metabolic processes. Genome mining and localization studies in Plasmodium have shown two evolutionarily distinct biogenesis pathways: the ISC pathway in mitochondria and the SUF pathway in the apicoplast. In recent years, the myriad efforts made to elucidate the SUF pathway have deciphered the role of various proteins involved in the pathway and their importance for the parasite life cycle in both asexual and sexual stages. This review aims to discuss recent research in the apicoplast [Fe-S] biogenesis pathway from Plasmodium to enhance our current understanding of parasite biology with an overall aim to identify gaps to strengthen our fight against malaria.

Deciphering the role of IspD (2­C­methyl­D­erythritol 4­phosphate cytidyltransferase) enzyme as a potential therapeutic drug target against Plasmodium vivax.

Biosynthesis of isoprenoids (MEP Pathway) in apicoplast has an important role during the erythrocytic stages of Plasmodium, as it is the sole pathway to provide the major isoprene units required as metabolic precursor for various housekeeping activities. With the intensifying need to identify a novel therapeutic drug target against Plasmodium, the MEP pathway and its components are considered as potential therapeutic targets, due to the difference in the isoprenoid synthesis route (MVA) functional in the host cells. While few major components have already been studied from this pathway for their potential as a drug target, IspD (2-C-methyl-D-erythritol-4-phosphate cytidyltransferase) enzyme, the enzyme catalyzing the third step of the pathway has only been tested against a synthetic compound from Malaria box called MMV008138, which also has not shown adequate inhibitory activity against P. vivax IspD. In the present study, to validate the potential of PvIspD as a drug target, various antimicrobial agents were screened for their inhibition possibilities, using in-vitro High Throughput Screening (HTS) technique. Shortlisted antimicrobial drug molecules like Cefepime, Tunicamycin and Rifampicin were further validated by in-vitro biochemical enzyme inhibition assays where they showed activity at nanomolar concentrations suggesting them or their derivatives as prospective future antimalarials. This study also confirmed the in-vivo expression of PvIspD protein during asexual stages by sub-cellular localization in apicoplast and explores the importance of the IspD enzyme in the development of new therapeutics.

Pacing Induced Ventricular Dysfunction in a Child: Improvement with Reduction in Paced Rate.

Right ventricular (RV) pacing can be associated with impairment of left ventricular (LV) function due to electrical dyssynchrony and myocardial remodeling (Janousek et al. in J Cardiovasc Electrophysiol 15:470-474, 2004). RV-pacing induced ventricular dysfunction is reversible by techniques such as biventricular pacing and LV apical or LV free wall pacing or turning the pacemaker off which have all been shown to restore synchrony and improve left ventricular function (Janousek et al. in J Cardiovasc Electrophysiol 15:470-474, 2004; Geldorp et al. in Heart Fail Rev 16:305-314, 2011). We describe an infant with RV-pacing induced cardiomyopathy who improved when the pacing rate was reduced thus demonstrating the relationship between pacing rate and development of LV dysfunction.

Characterization of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG) from Plasmodium vivax and it's potential as an antimalarial drug target.

The prokaryotic type Methyl Erythritol phosphate (MEP) pathway functional in the apicoplast of Plasmodium is indispensable for the erythrocytic stages of the parasite. It is the sole process of isoprenoids biosynthesis in the parasite and is different from that in humans. Among the seven enzymes known to be functional in the MEP pathway in prokaryotes, most enzymes from Plasmodium are yet uncharacterized. The penultimate enzyme of this pathway 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG), has been shown to act as a key target molecule in prokaryotes, where its deletion results in impairment of many housekeeping functions. The present study is the first detailed report of IspG enzyme from any Plasmodium sp. We report here that the protein is highly conserved across apicomplexans and prokaryotes and it localizes to the apicoplast as evident from the immune-localization studies performed on P. vivax infected blood smears made from clinical patients. The biochemical reconstitution and in silico docking of [4Fe-4S] clusters on the protein indicate their importance for the activity of enzyme. In-silico screening of different drug entities suggested the inhibitory role of alkyne diphosphate analogues and fosmidomycin against the IspG enzyme, suggesting the potential role of this enzyme as an antimalarial target.

Subtalar Joint Instability and Calcaneal Spurs Associated with the Configuration of the Articular Facets of Adult Human Calcaneum in Indian Population.

INTRODUCTION: Morphological variations of articular facets of calcaneum may predispose people to joint instability, ligamentous laxity and development of arthritic changes in the subtalar joint. Knowledge of such variations is essential for treatment and diagnostic procedures in orthopaedic surgeries. AIM: The aim of this study was to determine patterns of articular facets of calcanei and to establish its correlation with calcaneal spurs. MATERIALS AND METHODS: The study was conducted on 580 adult calcanei of Indian origin at Maulana Azad Medical College and pattern of articular facets were observed and classified according to five patterns described in literature. A digital vernier calliper was used to measure separation between anterior and middle facet. Degree of intersecting angle between anterior and medial facets was calculated using UTHSCSA Image Tool software. The calcaneal spurs were observed by visual inspection. RESULTS: Out of 580 calcanei, 66.55% had fused anterior and middle facets (Pattern I), 27.59% had all three facets separate (Pattern II), 5.52% had absence of anterior facet (Pattern III), 0.17% had all three facets fused (Pattern IV) and 0.17% had fused middle and posterior facets (Pattern V). A significant side variation was present in Pattern III with predominance on left side. Mean angle of intersection was 147.700 in Pattern I and 133.340 in Pattern II calcaneum. Calcaneal spurs were found in 61.38% out of which it was associated with Pattern I in 43.62%, Pattern II in 14.66% and Pattern III in 2.76%. CONCLUSION: Individuals with Pattern I and III calcaneum were found to be at a greater risk of subtalar joint instability than individuals with Pattern II. Angle of intersection was obtuse in Pattern I which resulted in ligament laxity and unstable joint. Pattern I was more common in Indian population and this fact necessitates modifications of the western surgical techniques to suit the Indian scenario. An association between the presence of spur and facet configuration was found to be significant.

New Insight into Isoprenoids Biosynthesis Process and Future Prospects for Drug Designing in Plasmodium.

The MEP (Methyl Erythritol Phosphate) isoprenoids biosynthesis pathway is an attractive drug target to combat malaria, due to its uniqueness and indispensability for the parasite. It is functional in the apicoplast of Plasmodium and its products get transported to the cytoplasm, where they participate in glycoprotein synthesis, electron transport chain, tRNA modification and several other biological processes. Several compounds have been tested against the enzymes involved in this pathway and amongst them Fosmidomycin, targeted against IspC (DXP reductoisomerase) enzyme and MMV008138 targeted against IspD enzyme have shown good anti-malarial activity in parasite cultures. Fosmidomycin is now-a-days prescribed clinically, however, less absorption, shorter half-life, and toxicity at higher doses, limits its use as an anti-malarial. The potential of other enzymes of the pathway as candidate drug targets has also been determined. This review details the various drug molecules tested against these targets with special emphasis to Plasmodium. We corroborate that MEP pathway functional within the apicoplast of Plasmodium is a major drug target, especially during erythrocytic stages. However, the major bottlenecks, bioavailability and toxicity of the new molecules needs to be addressed, before considering any new molecule as a potent antimalarial.

Structural and functional characterization of an iron-sulfur cluster assembly scaffold protein-SufA from Plasmodium vivax.

Iron-sulfur (Fe-S) clusters are utilized as prosthetic groups in all living organisms for diverse range of cellular processes including electron transport in respiration and photosynthesis, sensing of ambient conditions, regulation of gene expression and catalysis. In Plasmodium, two Fe-S cluster biogenesis pathways are reported, of which the Suf pathway in the apicoplast has been shown essential for the erythrocytic stages of the parasite. While the initial components of this pathway detailing the sulfur mobilization have been elucidated, the components required for the assembly and transfer of Fe-S clusters are not reported from the parasite. In Escherichia coli, SufB acts as a scaffold protein and SufA traffics the assembled Fe-S cluster from SufB to target apo-proteins. However, in Plasmodium, the homologs of these proteins are yet to be characterized for their function. Here, we report a putative SufA protein from Plasmodium vivax with signature motifs of A-type scaffold proteins, which is evolutionarily conserved. The presence of the [Fe4S4](3+) cluster under reduced conditions was confirmed by UV-visible and EPR spectroscopy and the interaction of these clusters with the conserved cysteine residues of chains A and B of PvSufA, validates its existence as a dimer, similar to that in E. coli. The H-bond interactions at the PvSufA-SufB interface demonstrate SufA as a scaffold protein in conjunction with SufB for the pre-assembly of Fe-S clusters and their transfer to the target proteins. Co-localization of the protein to the apicoplast further provides an experimental evidence of a functional scaffold protein SufA for the biogenesis of Fe-S clusters in apicoplast of Plasmodium.

A prospective study on adult patients of severe malaria caused by Plasmodium falciparum, Plasmodium vivax and mixed infection from Bikaner, northwest India.

BACKGROUND & OBJECTIVES: Description of severe vivax malaria and mixed species infection requires good clinical study. The present study was undertaken to evalute the characteristics of severe malaria patients in Bikaner, northwest India. METHODS: This prospective study included 539 admitted adult patients of severe malaria (Plasmodium falciparum 274, P. vivax 221, and mixed infection of Pv + Pf 44). The diagnosis was confirmed by polymerase chain reaction. The categorization of severe malaria was done strictly as per WHO criteria. RESULTS: The distribution of severe manifestation was similar in severe vivax, falciparum and mixed infections except more cases of thrombocytopenia in P. vivax (p=0.030) and in mixed infection (p=0.004). The risk of developing severe malaria was greatest in patients of mixed infection [53.01% (44/83)] in comparison to Plasmodium falciparum malaria [49.37% (274/555), RR= 1.135; p=0.616] and P. vivax malaria [45.38% (221/ 487), RR = 1.299, p=0.243]. Hepatic dysfunction was the commonest pernicious syndrome [P. falciparum 50% (137/274), P. vivax 43.89% (97/221), and mixed infections 54.55% (24/44)]. Multiorgan dysfunction was present in 40.26% (217/539) patients, the risk was greatest in mixed infection [90.90% (40/44)] in comparison to P. falciparum monoinfection [37.59% (103/274), RR = 12.238; p=0.0001] or P. vivax monoinfection [33.48% (74/ 221), RR = 13.25; p=0.0001]. The risk of mortality in severe malaria was 6.31% (34/539) in which mixed infection had greater risk [9.09% (4/44)] in comparison to P. falciparum [7.30% (20/274); OR = 1.270 (CI 0.347-4.217); p=0.757] or P. vivax [4.52% (10/221); 0R 2.110 (CI 0.527-7.826); p=0.260]. INTERPRETATION & CONCLUSION: Severe vivax or falciparum malaria had almost similar features and prognosis including mortality. Risk of developing severe malaria, multiorgan dysfunction and mortality was more in patients of mixed infection in comparison to P. falciparum or P. vivax monoinfection. A multicentric study on larger number of patients requires further confirmation.

Natural antisense transcripts in Plasmodium falciparum isolates from patients with complicated malaria.

Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how the parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which are best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. Using a custom designed strand specific whole genome microarray, a total of 797 NATs targeted against annotated loci have been detected. Out of these, 545 NATs are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. However, 96 genes showed a change in sense/antisense ratio on comparison between uncomplicated and complicated disease conditions. The antisense transcripts map to a broad range of biochemical/metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action of NATs, which would further help in understanding the in vivo pathological adaptations of these parasites.

Dataset of natural antisense transcripts in P. vivax clinical isolates derived using custom designed strand-specific microarray.

Natural antisense transcripts (NATs) have been detected in many organisms and shown to regulate gene expression. Similarly, NATs have also been observed in malaria parasites with most studies focused on Plasmodium falciparum. There were no reports on the presence of NATs in Plasmodium vivax, which has also been shown to cause severe malaria like P. falciparum, until a recent study published by us. To identify in vivo prevalence of antisense transcripts in P. vivax clinical isolates, we performed whole genome expression profiling using a custom designed strand-specific microarray that contains probes for both sense and antisense strands. Here we describe the experimental methods and analysis of the microarray data available in Gene Expression Omnibus (GEO) under GSE45165. Our data provides a resource for exploring the presence of antisense transcripts in P. vivax isolated from patients showing varying clinical symptoms. Related information about the description and interpretation of the data can be found in a recent publication by Boopathi and colleagues in Infection, Genetics and Evolution 2013.

An in vivo transcriptome data set of natural antisense transcripts from Plasmodium falciparum clinical isolates.

Antisense transcription is pervasive among biological systems and one of the products of antisense transcription is natural antisense transcripts (NATs). Emerging evidences suggest that they are key regulators of gene expression. With the discovery of NATs in Plasmodium falciparum, it has been suggested that these might also be playing regulatory roles in this parasite. However, all the reports describing the diversity of NATs have come from parasites in culture condition except for a recent study published by us. In order to explore the in vivo diversity of NATs in P. falciparum clinical isolates, we performed a whole genome expression profiling using a strand-specific 244 K microarray that contains probes for both sense and antisense transcripts. In this report, we describe the experimental procedure and analysis thereof of the microarray data published recently in Gene Expression Omnibus (GEO) under accession number GSE44921. This published data provide a wealth of information about the prevalence of NATs in P. falciparum clinical isolates from patients with diverse malaria related disease conditions. Supplementary information about the description and interpretation of the data can be found in a recent publication by Subudhi et al. in Experimental Parasitology (2014).

Revealing natural antisense transcripts from Plasmodium vivax isolates: evidence of genome regulation in complicated malaria.

Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130-435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is less studied and poorly understood, in spite of these facts. Emerging evidence of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating the parasite directly from infected patients is the best way to study its biology and pathogenic mechanisms. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. The mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of Natural Antisense Transcripts (NATs) in Plasmodium falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed microarray with strand specific probes. Majority of NATs identified from this study shows positive correlation with the expression pattern of the sense (S) transcript. Our data also shows condition specific expression patterns of varying S and antisense (AS) transcript levels. Genes with AS transcripts enrich to various biological processes. To our knowledge this is the first report on the presence of NATs from P. vivax obtained from infected patients with different disease complications. The data suggests differential regulation of gene expression in diverse clinical conditions, as shown by differing sense/antisense ratios and would lead to future detailed investigations of gene regulation.

Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax.

The 28S rRNA gene was amplified and sequenced from P. falciparum and P. vivax isolates collected from northwest India. Based upon the sequence diversity of the Plasmodium 28SrRNA gene in comparison with its human counterpart, various nested polymerase chain reaction (PCR) primers were designed from the 3R region of the 28SrRNA gene and evaluated on field isolates. This is the first report demonstrating the utility of this gene for species-specific diagnosis of malaria for these two species, prevalent in India. The initial evaluation on 363 clinical isolates indicated that, in comparison with microscopy, which showed sensitivity and specificity of 85·39% and 100% respectively, the sensitivity and specificity of the nested PCR assay was found to be 99·08% and 100% respectively. This assay was also successful in detecting mixed infections that are undetected by microscopy. Our results demonstrate the utility of the 28S rRNA gene as a diagnostic target for the detection of the major plasmodial species infecting humans.

Comparative evaluation of microscopy, OptiMAL(®) and 18S rRNA gene based multiplex PCR for detection of Plasmodium falciparum & Plasmodium vivax from field isolates of Bikaner, India.

OBJECTIVE: To evaluate microscopy, OptiMAL(®) and multiplex PCR for the identification of Plasmodium falciparumm (P. falciparum) and Plasmodium vivax (P. vivax) from the field isolates of Bikaner, Rajasthan (Northwest India). METHODS: In this study, a multiplex PCR (P. falciparum and P. vivax) was further developed with the incorporation of Plasmodium malariae (P. malariae) specific primer and also a positive control. The performance of microscopy, plasmodium lactate dehydrogenase (pLDH) based malaria rapid diagnostic test OptiMAL(®) and 18S rRNA gene based multiplex PCR for the diagnosis of P. falciparum and P. vivax was compared. RESULTS: The three species multiplex PCR (P. falciparum, P. vivax and P. malariae) with an inbuilt positive control was developed and evaluated. In comparison with multiplex PCR, which showed the sensitivity and specificity of 99.36% (95%CI, 98.11%-100.00%) and 100.00% (95%CI, 100.00%-100.00%), the sensitivity and specificity of microscopy was 90.44% (95%CI, 88.84%-95.04%) and 99.22% (95%CI, 97.71%-100.00%), and OptiMAL(®) was 93.58% (95%CI, 89.75%-97.42%) and 97.69% (95%CI, 95.10%-100.00%). The efficiencies were 99.65%, 95.10% and 95.45% for multiplex PCR, microscopy and OptiMAL(®), respectively. CONCLUSIONS: Our results raise concerns over the overall sensitivities of microscopy and OptiMAL(®), when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites. This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.

Novel mutations in the antifolate drug resistance marker genes among Plasmodium vivax isolates exhibiting severe manifestations.

Plasmodium vivax is the predominant species of the human malaria parasite present in the Indian subcontinent. There have been recent reports on Chloroquine (CQ) resistance and severe manifestations shown by P. vivax from different regions of the world including India. This study focuses on Bikaner, India where during the last few years there have been continuous reports of severe manifestations by both Plasmodium falciparum and P. vivax. This region has a widespread use of Chloroquine and Sulfadoxine-Pyrimethamine for the treatment of malaria, but the resistance profiles of these drugs are not available. We report here the profile of mutations in marker genes associated with Chloroquine and antifolate drug resistance among the P. vivax parasites obtained from patients with severe (n=30) and non-severe (n=48) manifestations from this region. Most isolates showed the wild type alleles for both the Chloroquine and antifolate resistance markers (P<0.0005). Except for one isolate showing Y976F mutation in the Pvmdr-1 gene, no reported mutation was observed in the Pvmdr-1 or Pvcrt gene. This is in accordance with the fact that till date no Chloroquine resistance has been reported from this region. However, the single isolate with a mutation in Pvmdr-1 may suggest the beginning of the trend towards decreased susceptibility to Chloroquine. The frequency of PvDHFR-PvDHPS two locus mutations was higher among the patients showing severe manifestations than the patient group with non-severe (uncomplicated) malaria (P<0.003). None of the parasites from patients with uncomplicated P. vivax malaria showed the mutant PvDHPS genotype. Novel mutations in PvDHFR (S117H) and PvDHPS (F365L, D459A and M601I) were observed only in the parasite population obtained from patients exhibiting severe complications. Preliminary homology modeling and molecular docking studies predicted that these mutations apparently do not have any effect on the binding of the drug molecule to the enzyme. However, the presence of novel mutations in the PvDHPS gene indicate a degree of polymorphism of this molecule which is in contrast to available published information.

Plasmodium vivax apicoplast genome: a comparative analysis of major genes from Indian field isolates.

The apicomplexan parasite Plasmodium vivax is responsible for causing more than 70% of human malaria cases in Central and South America, Southeastern Asia and the Indian subcontinent. The rising severity of the disease and the increasing incidences of resistance shown by this parasite towards usual therapeutic regimens have necessitated investigation of putative novel drug targets to combat this disease. The apicoplast, an organelle of procaryotic origin, and its circular genome carrying genes of possible functional importance, are being looked upon as potential drug targets. The genes on this circular genome are believed to be highly conserved among all Plasmodium species. Till date, the plastid genome of P. falciparum, P. berghei and P. chabaudi have been detailed while partial sequences of some genes from other parasites including P. vivax have been studied for identifying evolutionary positions of these parasites. The functional aspects and significance of most of these genes are still hypothetical. In one of our previous reports, we have detailed the complete sequence, as well as structural and functional characteristics of the Elongation factor encoding tufA gene from the plastid genome of P. vivax. We present here the sequences of large and small subunit rRNA (lsu and ssu rRNA) genes, sufB (ORF470) gene, RNA polymerase (rpo B, C) subunit genes and clpC (casienolytic protease) gene from the plastid genome of P. vivax. A comparative analysis of these genes between P. vivax and P. falciparum reveals approximately 5-16% differences. A codon usage analysis of major plastid genes has shown a high frequency of codons rich in A/T at any or all of the three positions in all the species. TTA, AAT, AAA, TAT, and ATA are the major preferred codons. The sequences, functional domains and structural analysis of respective proteins do not show any variations in the active sites. A comparative analysis of these Indian P. vivax plastid genome encoded genes has also been done to understand the evolutionary position of the Indian parasite in comparison to other Plasmodium species.