Browsing by Author "Garg, Shilpi"

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Analysis of elongation factor Tu (tuf A) of apicoplast from Indian Plasmodium vivax isolates
(Elsiever, 2007-09) Garg, Shilpi; Saxena, Vishal; Das, Ashis
The Apicomplexan parasite Plasmodium vivax is responsible for causing greater than 50% of human malaria cases in Central and South America, Southeastern Asia and the Indian subcontinent. The rising severity of the disease and the resistance shown by the parasite towards usual therapeutic regimen has put forth a demand for a novel drug target to combat this disease. Apicoplast, an organelle of prokaryotic origin, and its circular genome are being looked upon as a potential drug target. The Apicoplast genome is known to carry various genes of functional importance, including the gene encoding for the protein Elongation factor Tu (tuf A) that participates in the translational process in prokaryotes. The tuf A gene is translationally active within the organelle and is believed to be one of the best functionally conserved protein throughout the species. Till date there are no reports of this gene from another major human malaria parasite P. vivax. This is the first report detailing any complete gene analysis from the Apicoplast genome of the Indian P. vivax isolates. The study predicts and evaluates the complete Apicoplast Elongation factor tuf A gene and EF–Tu protein at primary, secondary and tertiary structure level. In addition, a comparative phylogenetic analysis using this gene is done to understand the evolutionary status of Indian P. vivax isolates. Our study shows that although the Indian P. vivax EF–Tu is not showing any major difference at the structural and predicted functional level, it is diverging way ahead from the P. vivax clade.
Awareness on critical prevention practices to combat the drug resistance in Plasmodium falciparum and Plasmodium vivax malaria from Rajasthan, India
(IJMR, 2020) Garg, Shilpi
The ability of the Plasmodium parasites to develop resistance to commonly used antimalarial is the biggest challenge for malaria elimination. This threat is being accelerated by the systematic misuse or overuse of antimalarial, as well as inadequate infection prevention and control. Most disease control strategies require active community participation, which in turn depends on an individual’s knowledge and alertness to the disease. This study was undertaken to understand the awareness of the local public, predominantly rural, regarding the use of antimalarial, current status of drug resistance and critical prevention practices or alternatives used to combat malaria in and around Jhunjhunu, Rajasthan. A questionnaire-based cross-sectional study was designed and data was collected using an observational checklist. On the whole, our data shows that while nearly three fourth of the respondents were aware of the mosquito bite, only 10% knew about the parasite Plasmodium. About one-tenth of the people were aware of Chloroquine as well as drug resistance. Further, to fill the knowledge gaps of the target audiences, malaria control strategies were personalized as an effort to strengthen existing malaria prevention approaches and proper usage of drug regimens to eliminate malaria.
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
(Elsiever, 2017-03) Garg, Shilpi; Yadav, Sushil Kumar; Saxena, Vishal
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.
Clinical Features of Children Hospitalized with Malaria—A Study from Bikaner, Northwest India
(ASTMH, 2010) Garg, Shilpi; Saxena, Vishal; Das, Ashis
Severe Plasmodium vivax malaria in adults has been reported from Bikaner (northwestern India) but the reports on children are scanty. This prospective study was done on 303 admitted children of malaria. The diagnosis was done by peripheral blood smear and rapid diagnostic test. Further confirmation of severe P. vivax monoinfection was done by polymerase chain reaction (PCR). The proportion of P. falciparum, P. vivax, and mixed (P. falciparum and P. vivax) infection was 61.01%, 33.99%, and 4.95%, respectively. Severe disease was present in 49.5% (150/303) children with malaria, with the risk greatest among P. vivax monoinfection (63.1% [65/103]) compared with P. falciparum, either alone (42.7% [79/185]; odds ratio [OR] = 2.3 [95% confidence interval (CI) = 1.40–3.76], P = 0.001) or mixed infections (40% [6/15]; OR = 2.57 [95% CI = 0.88–7.48]). In children < 5 years of age, the proportion of severe malaria attributable to P. vivax rose to 67.4% (31/46) compared with 30.4% (14/46) of P. falciparum (OR = 4.7 [95% CI = 2.6–8.6], P < 0.0001) and 2.2% (1/46) of mixed infection (OR = 92 [95% CI = 24.6–339.9], P < 0.0001). The proportion of patients having severe manifestations, which included severe anemia, thrombocytopenia, cerebral malaria, acute respiratory distress syndrome, hepatic dysfunction, renal dysfunction, abnormal bleeding was significantly high in association with P. vivax monoinfection in 0–5 year age group, while the same was significantly high in association with P. falciparum monoinfection in 5–10 year age group. Similarly P. vivax monoinfection had greatest propensity to cause multiorgan dysfunction in 0–5 year age group (34.1% [17/41], P < 0.0001) in comparison to P. falciparum monoinfection, which had similar propensity in 5–10 year age group (36.8% [35/95], P = 0.039). Plasmodium vivax monoinfection was almost equally serious to cause significant mortality in comparison to P. falciparum (case fatality rate of severe P. vivax was 3.9% versus 3.2% of severe P. falciparum malaria; P = 1.0). This study reaffirms the evidence of severe P. vivax malaria in children in Bikaner.
Clinical profiles of 13 children with Plasmodium vivax cerebral malaria
(Taylor & Francis, 2011) Garg, Shilpi; Das, Ashis
Bikaner region is endemic for both P. vivax and P. falciparum malaria. Usually, cerebral malaria is caused by P. falciparum but it has been reported recently also in P. vivax mono-infection. Epidemiologic studies and clinical descriptions of P. vivax cerebral malaria in children are rare.
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
(Elsiever, 2013-05-13) Garg, Shilpi; Saxena, Vishal; Das, Ashis
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).
Dataset of natural antisense transcripts in P. vivax clinical isolates derived using custom designed strand-specific microarray
(Elsiever, 2014-12) Garg, Shilpi; Saxena, Vishal; Das, Ashis
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.
Deciphering the molecular targets of Plasmodium and Anopheline interactions for malaria control
(Wiley, 2025-09) Garg, Shilpi
Malaria is a severe disease that is transmitted by female Anopheles mosquitoes and caused by the Plasmodium parasite. Despite a decrease in mortality rate, it continues to pose significant challenges such as resistance to antimalarial drugs and insecticides, which necessitates the need for novel malaria control and elimination strategies. To identify new molecular targets for malaria control, there is a need to understand the molecular interaction between mosquitoes and parasites. Plasmodium ookinetes interact with the mosquito midgut proteins during midgut invasion and sporozoites interact with the mosquito salivary gland (SG) proteins. These interactions are crucial for the parasite's invasion of the mosquito midgut and SG, respectively. This review explores the involvement of various Plasmodium genes in male and female gametogenesis and parasite transmission, their interaction with the mosquito genes that facilitate parasite invasion, and how the mosquito immune system defends itself from the invading parasite. Understanding the biology underlying the interaction between mosquitoes and parasites may lead to a better comprehension of the disease and could help design efficient vector control strategies.
Deciphering the role of IspD (2‑C‑methyl‑D‑erythritol 4‑phosphate cytidyltransferase) enzyme as a potential therapeutic drug target against Plasmodium vivax
(Elsiever, 2018-10-30) Garg, Shilpi; Saxena, Vishal
IspD(2‑C‑methyl‑d-erythritol 4-phosphate cytidyltransferase) PvIspDPlasmodium vivax IspD PfIspDPlasmodium falciparum IspD AtIspDArabidopsis thaliana IspD MEPMethyl erythritol phosphate (Non-mevalonate) HTSHigh Throughput Screening NEATnuclear encoded apicoplast targeted CTPcytidine 5′-triphosphate CDP-ME4-diphosphocytidyl-2C-methyl‑D-erythritol ACDacid citrate dextrose IHECInstitute Human Ethics Committee PCRPolymerase chain reaction MBPMaltose binding protein RIG plasmidPlasmid with arginine (R), Isoleucine (I) and Glycine (G) tRNA genes DOPEDiscrete Optimized Protein Energy pMALMaltose FITCFluorescein isothiocyanate DAPI4′,6-diamidino-2-phenylindole Qdotquantum dot RMSDroot-mean-square deviation
Delineating the Role of Aedes aegypti ABC Transporter Gene Family during Mosquito Development and Arboviral Infection via Transcriptome Analyses
(MDPI, 2021-09-02) Garg, Shilpi; Saxena, Vishal
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.
Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax
(Taylor & Francis, 2013-04-12) Garg, Shilpi; Saxena, Vishal; Das, Ashis
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.
Functional analysis of iron-sulfur cluster biogenesis (SUF pathway) from Plasmodium vivax clinical isolates
(Elsiever, 2019-03) Garg, Shilpi; Saxena, Vishal
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.
A glimpse into the clinical proteome of human malaria parasites Plasmodium falciparum and Plasmodium vivax
(Wiley, 2009-11-09) Das, Ashis Kumar; Garg, Shilpi
Malaria causes a worldwide annual mortality of about a million people. Rapidly evolving drug-resistant species of the parasite have created a pressing need for the identification of new drug targets and vaccine candidates. By developing fractionation protocols to enrich parasites from low-parasitemia patient samples, we have carried out the first ever proteomics analysis of clinical isolates of early stages of Plasmodium falciparum (Pf) and P. vivax. Patient-derived malarial parasites were directly processed and analyzed using shotgun proteomics approach using high-sensitivity MS for protein identification. Our study revealed about 100 parasite-coded gene products that included many known drug targets such as Pf hypoxanthine guanine phosphoribosyl transferase, Pf L-lactate dehydrogenase, and Plasmepsins. In addition, our study reports the expression of several parasite proteins in clinical ring stages that have never been reported in the ring stages of the laboratory-cultivated parasite strain. This proof-of-principle study represents a noteworthy step forward in our understanding of pathways elaborated by the parasite within the malaria patient and will pave the way towards identification of new drug and vaccine targets that can aid malaria therapy.
An in vivo transcriptome data set of natural antisense transcripts from Plasmodium falciparum clinical isolates
(Elsiever, 2014-12) Garg, Shilpi; Saxena, Vishal; Das, Ashis
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).
Midgut immune profiling and functional characterization of Aedes aegypti ABC transporter gene(s) using systemic and local bacterial challenges
(Springer, 2025-01) Garg, Shilpi; Saxena, Vishal
The mosquito midgut is crucial for digestion and immune interactions. It produces several immune factors that protect the organ from invading pathogens and can limit their propagation. Studies on mosquito midgut transcriptome following pathogen exposure have revealed the presence of non-canonical immune genes, such as ABC transporters, whose function in insect immunity remains unexplored. Therefore, this study focuses on identifying and characterising the immune role of ABC transporters in the midgut of Aedes aegypti, a primary arboviral vector.
Natural antisense transcripts in Plasmodium falciparum isolates from patients with complicated malaria
(Elsiever, 2014-06) Das, Ashis Kumar; Saxena, Vishal; Garg, Shilpi
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.
New Insight into Isoprenoids Biosynthesis Process and Future Prospects for Drug Designing in Plasmodium
(Frontiers, 2016-09-13) Garg, Shilpi; Saxena, Vishal
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.
Novel mutations in the antifolate drug resistance marker genes among Plasmodium vivax isolates exhibiting severe manifestations
(Elsiever, 2012-12) Garg, Shilpi; Saxena, Vishal; Chowdhury, Shibasish; Das, Ashis
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.
Novel point mutations in sulfadoxine resistance genes of Plasmodium falciparum from India
(Elsiever, 2009-04) Garg, Shilpi; Saxena, Vishal; Das, Ashis
Point mutations in the dhfr and dhps genes of Plasmodium falciparum are associated with pyrimethamine and sulfadoxine resistance respectively. In this study we have analyzed these genes from Bikaner (situated in North-West region of India), where both uncomplicated and severe manifestations of P. falciparum malaria are seen. A majority of isolates showed double mutant allele for DHFR. In contrast, the only reported mutation present in DHPS was A437G in few isolates. In addition, three novel non-synonymous mutations were observed in the PfDHPS from this region viz., S587F, N666K and C668W. The mutations at the 666 and 668 codon seem to form a bend in the big loop region of the DHPS enzyme and may affect the binding of the drug to the enzyme. Molecular docking of sulfadoxine to this mutated structure indicates reduction in its binding affinity to this enzyme
Piercing Power Is More Than the Human Power
(IJCRT, 2020) Garg, Shilpi