49 results on '"Agam P, Singh"'
Search Results
2. Dissecting The role of Plasmodium metacaspase-2 in malaria gametogenesis and sporogony
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Vandana Kumari, Kona Madhavinadha Prasad, Inderjeet Kalia, Gagandeep Sindhu, Rajnikant Dixit, Diwan S. Rawat, O. P. Singh, Agam P. Singh, and Kailash C. Pandey
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Malaria ,metacaspase ,specific inhibitor ,Plasmodium transmission ,sporogony ,An. stephensi ,Infectious and parasitic diseases ,RC109-216 ,Microbiology ,QR1-502 - Abstract
The family of apicomplexan specific proteins contains caspases–like proteins called “metacaspases”. These enzymes are present in the malaria parasite but absent in human; therefore, these can be explored as potential drug targets. We deleted the MCA-2 gene from Plasmodium berghei genome using a gene knockout strategy to decipher its precise function. This study has identified that MCA-2 plays an important role in parasite transmission since it is critical for the formation of gametocytes and for maintaining an appropriate number of infectious sporozoites required for sporogony. It is noticeable that a significant reduction in gametocyte, oocysts, ookinete and sporozoites load along with a delay in hepatocytes invasion were observed in the MCA-2 knockout parasite. Furthermore, a study found the two MCA-2 inhibitory molecules known as C-532 and C-533, which remarkably inhibited the MCA-2 activity, abolished the in vitro parasite growth, and also impaired the transmission cycle of P. falciparum and P. berghei in An. stephensi. Our findings indicate that the deletion of MCA-2 hampers the Plasmodium development during erythrocytic and exo-erythrocytic stages, and its inhibition by C-532 and C-533 critically affects the malaria transmission biology. more...
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- 2022
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Catalog
3. Essential role of a Plasmodium berghei heat shock protein (PBANKA_0938300) in gametocyte development
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Mohammad Kashif, Afshana Quadiri, and Agam Prasad Singh
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Medicine ,Science - Abstract
Abstract The continued existence of Plasmodium parasites in physiologically distinct environments during their transmission in mosquitoes and vertebrate hosts requires effector proteins encoded by parasite genes to provide adaptability. Parasites utilize their robust stress response system involving heat shock proteins for their survival. Molecular chaperones are involved in maintaining protein homeostasis within a cell during stress, protein biogenesis and the formation of protein complexes. Due to their critical role in parasite virulence, they are considered targets for therapeutic interventions. Our results identified a putative P. berghei heat shock protein (HSP) belonging to the HSP40 family (HspJ62), which is abundantly induced upon heat stress and expressed during all parasite stages. To determine the role HspJ62, a gene-disrupted P. berghei transgenic line was developed (ΔHspJ62), which resulted in disruption of gametocyte formation. Such parasites were unable to form subsequent sexual stages because of disrupted gametogenesis, indicating the essential role of HspJ62 in gametocyte formation. Transcriptomic analysis of the transgenic line showed downregulation of a number of genes, most of which were specific to male or female gametocytes. The transcription factor ApiAP2 was also downregulated in ΔHspJ62 parasites. Our findings suggest that the downregulation of ApiAP2 likely disrupts the transcriptional regulation of sexual stage genes, leading to impaired gametogenesis. This finding also highlights the critical role that HspJ62 indirectly plays in the development of P. berghei sexual stages and in facilitating the conversion from the asexual blood stage to the sexual stage. This study characterizes the HspJ62 protein as a fertility factor because parasites lacking it are unable to transmit to mosquitoes. This study adds an important contribution to ongoing research aimed at understanding gametocyte differentiation and formation in parasites. The molecule adds to the list of potential drug targets that can be targeted to inhibit parasite sexual development and consequently parasite transmission. more...
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- 2021
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4. Identification and characterization of protective CD8+ T‐epitopes in a malaria vaccine candidate SLTRiP
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Afshana Quadiri, Inderjeet Kalia, Mohammad Kashif, and Agam P. Singh
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liver‐stage malaria ,T‐cell epitopes ,vaccine ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Abstract Introduction Efforts are required at developing an effective vaccine that can inhibit malaria prevalence and transmission. Identifying the critical immunogenic antigens and understanding their interactions with host proteins forms a major focus of subunit vaccine development. Previously, our laboratory showed that SLTRiP conferred protection to the liver stage of Plasmodium growth in rodents. In the follow‐up of earlier research, we demonstrate that SLTRiP‐mediated protection is majorly concentrated in specific regions of protein. Method To identify particular protective regions of protein, we synthesized multiple nonoverlapping fragments from SLTRiP protein. From this, we designed a panel of 8‐20mer synthetic peptides, which were predicted using T‐epitope‐based prediction algorithm. We utilized the IFN‐γ enzyme‐linked immunosorbent spot assay to identify immunodominant peptides. The latter were used to immunize mice, and these mice were challenged to assess protection. Results The protective polypeptide fragment SLTRiP C3 and SLTRiP C4 were identified, by expressing and testing multiple fragments of PbSLTRiP protein. The immune responses generated by these fragments were compared to identify the immunodominant fragment. The T‐epitopes were predicted from SLTRiP protein using computer‐based algorithms. The in vitro immune responses generated by these peptides were compared with each other to identify the immunodominant T‐epitope. Immunization using these peptides showed significant reduction in parasite numbers during liver stage. Conclusion Our findings show that the protective efficacy shown by SLTRiP is localized in particular protein fragments. The peptides designed from such regions showed protective efficacy equivalent to whole protein. The sequence conservation analysis with human Plasmodium species also showed that these peptides were conserved. In conclusion, these peptides or their equivalent from other Plasmodium species could impart protection against malaria in their respective hosts too. Our studies provide a basis for the inclusion of these peptides in clinical vaccine constructs against malaria. more...
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- 2020
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5. Plasmodium berghei-Released Factor, PbTIP, Modulates the Host Innate Immune Responses
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Inderjeet Kalia, Rajesh Anand, Afshana Quadiri, Shreya Bhattacharya, Bijayalaxmi Sahoo, and Agam Prasad Singh
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immunomodulation ,immune evasion ,T cell immunomodulatory protein ,malaria ,macrophages altered phenotype ,M2 macrophages ,Immunologic diseases. Allergy ,RC581-607 - Abstract
The Plasmodium parasite has to cross various immunological barriers for successful infection. Parasites have evolved mechanisms to evade host immune responses, which hugely contributes to the successful infection and transmission by parasites. One way in which a parasite evades immune surveillance is by expressing molecular mimics of the host molecules in order to manipulate the host responses. In this study, we report a Plasmodium berghei hypothetical protein, PbTIP (PbANKA_124360.0), which is a Plasmodium homolog of the human T-cell immunomodulatory protein (TIP). The latter possesses immunomodulatory activities and suppressed the host immune responses in a mouse acute graft-versus-host disease (GvHD) model. The Plasmodium berghei protein, PbTIP, is expressed on the merozoite surface and exported to the host erythrocyte surface upon infection. It is shed in the blood circulation by the activity of an uncharacterized membrane protease(s). The shed PbTIP could be detected in the host serum during infection. Our results demonstrate that the shed PbTIP exhibits binding on the surface of macrophages and reduces their inflammatory cytokine response while upregulating the anti-inflammatory cytokines such as TGF-β and IL-10. Such manipulated immune responses are observed in the later stage of malaria infection. PbTIP induced Th2-type gene transcript changes in macrophages, hinting toward its potential to regulate the host immune responses against the parasite. Therefore, this study highlights the role of a Plasmodium-released protein, PbTIP, in immune evasion using macrophages, which may represent the critical strategy of the parasite to successfully survive and thrive in its host. This study also indicates the human malaria parasite TIP as a potential diagnostic molecule that could be exploited in lateral flow-based immunochromatographic tests for malaria disease diagnosis. more...
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- 2021
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6. The Multistage Antimalarial Compound Calxinin Perturbates P. falciparum Ca2+ Homeostasis by Targeting a Unique Ion Channel
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Yash Gupta, Neha Sharma, Snigdha Singh, Jesus G. Romero, Vinoth Rajendran, Reagan M. Mogire, Mohammad Kashif, Jordan Beach, Walter Jeske, Poonam, Bernhards R. Ogutu, Stefan M. Kanzok, Hoseah M. Akala, Jennifer Legac, Philip J. Rosenthal, David J. Rademacher, Ravi Durvasula, Agam P. Singh, Brijesh Rathi, and Prakasha Kempaiah more...
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antimalarial ,multistage activity ,Ca2+ homeostasis ,transient receptor potential mucolipin like channel ,electron microscopy ,field isolates ,Pharmacy and materia medica ,RS1-441 - Abstract
Malaria elimination urgently needs novel antimalarial therapies that transcend resistance, toxicity, and high costs. Our multicentric international collaborative team focuses on developing multistage antimalarials that exhibit novel mechanisms of action. Here, we describe the design, synthesis, and evaluation of a novel multistage antimalarial compound, ‘Calxinin’. A compound that consists of hydroxyethylamine (HEA) and trifluoromethyl-benzyl-piperazine. Calxinin exhibits potent inhibitory activity in the nanomolar range against the asexual blood stages of drug-sensitive (3D7), multidrug-resistant (Dd2), artemisinin-resistant (IPC4912), and fresh Kenyan field isolated Plasmodium falciparum strains. Calxinin treatment resulted in diminished maturation of parasite sexual precursor cells (gametocytes) accompanied by distorted parasite morphology. Further, in vitro liver-stage testing with a mouse model showed reduced parasite load at an IC50 of 79 nM. A single dose (10 mg/kg) of Calxinin resulted in a 30% reduction in parasitemia in mice infected with a chloroquine-resistant strain of the rodent parasite P. berghei. The ex vivo ookinete inhibitory concentration within mosquito gut IC50 was 150 nM. Cellular in vitro toxicity assays in the primary and immortalized human cell lines did not show cytotoxicity. A computational protein target identification pipeline identified a putative P. falciparum membrane protein (Pf3D7_1313500) involved in parasite calcium (Ca2+) homeostasis as a potential Calxinin target. This highly conserved protein is related to the family of transient receptor potential cation channels (TRP-ML). Target validation experiments showed that exposure of parasitized RBCs (pRBCs) to Calxinin induces a rapid release of intracellular Ca2+ from pRBCs; leaving de-calcinated parasites trapped in RBCs. Overall, we demonstrated that Calxinin is a promising antimalarial lead compound with a novel mechanism of action and with potential therapeutic, prophylactic, and transmission-blocking properties against parasites resistant to current antimalarials. more...
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- 2022
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7. Plasmodium Perforin-Like Protein Pores on the Host Cell Membrane Contribute in Its Multistage Growth and Erythrocyte Senescence
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Swati Garg, Abhishek Shivappagowdar, Rahul S. Hada, Rajagopal Ayana, Chandramohan Bathula, Subhabrata Sen, Inderjeet Kalia, Soumya Pati, Agam P. Singh, and Shailja Singh
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perforin like proteins ,malaria ,erythrocyte ,anemia ,invasion ,egress ,Microbiology ,QR1-502 - Abstract
The pore forming Plasmodium Perforin Like Proteins (PPLP), expressed in all stages of the parasite life cycle are critical for completion of the parasite life cycle. The high sequence similarity in the central Membrane Attack Complex/ Perforin (MACPF) domain among PLPs and their distinct functional overlaps define them as lucrative target for developing multi-stage antimalarial therapeutics. Herein, we evaluated the mechanism of Pan-active MACPF Domain (PMD), a centrally located and highly conserved region of PPLPs, and deciphered the inhibitory potential of specifically designed PMD inhibitors. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to hemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence which can be hypothesized to account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing malaria anemia. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress processes and protected erythrocytes against rPMD induced senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage, transmission-blocking potential of these inhibitors. Concievably, our study has introduced a novel set of anti-PMD inhibitors with pan-inhibitory activity against all the PPLPs members which can be developed into potent cross-stage antimalarial therapeutics along with erythrocyte senescence protective potential to occlude PPLPs mediated anemia in severe malaria. more...
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- 2020
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8. Pathogenic Pore Forming Proteins of Plasmodium Triggers the Necrosis of Endothelial Cells Attributed to Malaria Severity
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Abhishek Shivappagowdar, Swati Garg, Akriti Srivastava, Rahul S. Hada, Inderjeet Kalia, Agam P. Singh, Lalit C. Garg, Soumya Pati, and Shailja Singh
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malaria ,Plasmodium falciparum ,perforin like proteins ,necrosis ,blebbing ,calcium ,Medicine - Abstract
Severe malaria caused by Plasmodium falciparum poses a major global health problem with high morbidity and mortality. P. falciparum harbors a family of pore-forming proteins (PFPs), known as perforin like proteins (PLPs), which are structurally equivalent to prokaryotic PFPs. These PLPs are secreted from the parasites and, they contribute to disease pathogenesis by interacting with host cells. The severe malaria pathogenesis is associated with the dysfunction of various barrier cells, including endothelial cells (EC). Several factors, including PLPs secreted by parasites, contribute to the host cell dysfunction. Herein, we have tested the hypothesis that PLPs mediate dysfunction of barrier cells and might have a role in disease pathogenesis. We analyzed various dysfunctions in barrier cells following rPLP2 exposure and demonstrate that it causes an increase in intracellular Ca2+ levels. Additionally, rPLP2 exposed barrier cells displayed features of cell death, including Annexin/PI positivity, depolarized the mitochondrial membrane potential, and ROS generation. We have further performed the time-lapse video microscopy of barrier cells and found that the treatment of rPLP2 triggers their membrane blebbing. The cytoplasmic localization of HMGB1, a marker of necrosis, further confirmed the necrotic type of cell death. This study highlights the role of parasite factor PLP in endothelial dysfunction and provides a rationale for the design of adjunct therapies against severe malaria. more...
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- 2021
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9. A potent candidate against Zika virus infection: Synthesis, bioactivity, radiolabeling and biodistribution studies
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Sumit Kumar, Neha Sharma, Willyenne Marilia Dantas, Jessica Catarine Frutuoso do Nascimento, Hannah Maus, Ronaldo Nascimento de Oliveira, Unnat Pandit, Agam P. Singh, Tanja Schirmeister, Puja Panwar Hazari, Lindomar Pena, null Poonam, and Brijesh Rathi more...
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Materials Chemistry ,General Chemistry ,Catalysis - Abstract
Compound VI exhibits potent activity against Zika virus infection combined with favorable cellular uptake and biodistribution without apparent cytotoxicity in a mouse model.
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- 2022
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10. Synthesis of the new analogs of morpholine and their antiplasmodial evaluation against the human malaria parasite Plasmodium falciparum
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Charu Upadhyay, Neha Sharma, Sumit Kumar, Prem Prakash Sharma, Diana Fontinha, Bhupender S. Chhikara, Budhaditya Mukherjee, Dhruv Kumar, Miguel Prudencio, Agam P. Singh, and null Poonam
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parasitic diseases ,Materials Chemistry ,General Chemistry ,Catalysis - Abstract
A series of morpholine analogs functionalized with hydroxyethylamine (HEA) pharmacophore was synthesized and assayed for the initial screening against Plasmodium falciparum 3D7 in culture, which suggested that analog 6k is a hit molecule with an inhibitory concentration of 5.059 ± 0.2036 μM. more...
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- 2022
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11. Plasmodium falciparum metacaspase-2 capture its natural substrate in a non-canonical way
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Dinesh Gupta, Ajay K. Saxena, Inderjeet Kalia, Rajan Pandey, E Srinivasan, Agam P. Singh, Kailash C. Pandey, R Rajaekaran, and Vandana
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Cell Death ,biology ,Chemistry ,Plasmodium falciparum ,Apoptosis ,General Medicine ,Substrate (biology) ,biology.organism_classification ,Biochemistry ,Metacaspase ,Non canonical ,Caspases ,Catalytic Domain ,Biophysics ,Humans ,Molecular Biology - Abstract
Programmed cell death (PCD) is a multi-step process initiated by a set of proteases, which interacts and cleaves diverse proteins, thus modulating their biochemical and cellular functions. In metazoans, PCD is mediated by proteolytic enzymes called caspases, which triggered cell death by proteolysis of human Tudor staphylococcus nuclease (TSN). Non-metazoans lack a close homologue of caspases but possess an ancestral family of cysteine proteases termed ‘metacaspases’. Studies supported that metacaspases are involved in PCD, but their natural substrates remain unknown. In this study, we performed the Plasmodium falciparum TSN (PfTSN) cleavage assay using wild and selected mutants of P. falciparum metacaspases-2 (PfMCA-2) in vitro and in vivo. Interestingly, PfMCA-2, cleaved a phylogenetically conserved protein, PfTSN at multiple sites. Deletion or substitution mutation in key interacting residues at the active site, Cys157 and His205 of PfMCA-2, impaired its enzymatic activity with the artificial substrate, z-GRR-AMC. However, the mutant Tyr224A did not affect the activity with z-GRR-AMC but abolished the cleavage of PfTSN. These results indicated that the catalytic dyad, Cys157 and His205 of PfMCA-2 was essential for its enzymatic activity with an artificial substrate, whereas Tyr224 and Cys157 residues were responsible for its interaction with the natural substrate and subsequent degradation of PfTSN. Our results suggested that MCA-2 interacts with TSN substrate in a non-canonical way using non-conserved or conformationally available residues for its binding and cleavage. In future, it would be interesting to explore how this interaction leads to the execution of PCD in the Plasmodium. more...
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- 2021
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12. Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti
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Agam P. Singh, Kavitha Govindasamy, Shreya Bhattacharya, Dabbu Kumar Jaijyan, and Jyoti Prakash Singh
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0301 basic medicine ,Transgene ,animal diseases ,030231 tropical medicine ,Genetic Vectors ,lcsh:Medicine ,Diseases ,Babesia microti ,Transfection ,Microbiology ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Genes, Reporter ,Babesiosis ,parasitic diseases ,Genetics ,Animals ,Plasmodium berghei ,Promoter Regions, Genetic ,lcsh:Science ,Gene ,Multidisciplinary ,biology ,Electroporation ,lcsh:R ,Promoter ,biology.organism_classification ,bacterial infections and mycoses ,Virology ,Reverse genetics ,Mice, Inbred C57BL ,030104 developmental biology ,lcsh:Q ,mCherry ,Biotechnology - Abstract
Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as β-actin, ef-1β, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development. more...
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- 2020
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13. Identification and characterization of protective CD8 + T‐epitopes in a malaria vaccine candidate SLTRiP
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Inderjeet Kalia, Mohammad Kashif, Agam P. Singh, and Afshana Quadiri
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lcsh:Immunologic diseases. Allergy ,0301 basic medicine ,biology ,T‐cell epitopes ,Malaria vaccine ,Protein subunit ,Immunology ,biology.organism_classification ,medicine.disease ,Plasmodium ,Virology ,Epitope ,In vitro ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Immune system ,Antigen ,vaccine ,medicine ,Immunology and Allergy ,liver‐stage malaria ,lcsh:RC581-607 ,Malaria ,030215 immunology - Abstract
Introduction Efforts are required at developing an effective vaccine that can inhibit malaria prevalence and transmission. Identifying the critical immunogenic antigens and understanding their interactions with host proteins forms a major focus of subunit vaccine development. Previously, our laboratory showed that SLTRiP conferred protection to the liver stage of Plasmodium growth in rodents. In the follow‐up of earlier research, we demonstrate that SLTRiP‐mediated protection is majorly concentrated in specific regions of protein. Method To identify particular protective regions of protein, we synthesized multiple nonoverlapping fragments from SLTRiP protein. From this, we designed a panel of 8‐20mer synthetic peptides, which were predicted using T‐epitope‐based prediction algorithm. We utilized the IFN‐γ enzyme‐linked immunosorbent spot assay to identify immunodominant peptides. The latter were used to immunize mice, and these mice were challenged to assess protection. Results The protective polypeptide fragment SLTRiP C3 and SLTRiP C4 were identified, by expressing and testing multiple fragments of PbSLTRiP protein. The immune responses generated by these fragments were compared to identify the immunodominant fragment. The T‐epitopes were predicted from SLTRiP protein using computer‐based algorithms. The in vitro immune responses generated by these peptides were compared with each other to identify the immunodominant T‐epitope. Immunization using these peptides showed significant reduction in parasite numbers during liver stage. Conclusion Our findings show that the protective efficacy shown by SLTRiP is localized in particular protein fragments. The peptides designed from such regions showed protective efficacy equivalent to whole protein. The sequence conservation analysis with human Plasmodium species also showed that these peptides were conserved. In conclusion, these peptides or their equivalent from other Plasmodium species could impart protection against malaria in their respective hosts too. Our studies provide a basis for the inclusion of these peptides in clinical vaccine constructs against malaria. more...
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- 2020
- Full Text
- View/download PDF
14. Potentin vivoantimalarial activity of water-soluble artemisinin nano-preparations
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Deepak Gaur, Praveesh Valissery, Roshni Thapa, Jyoti Prakash Singh, Agam P. Singh, Jaydeep Bhattacharya, and Suman Kumar Dhar
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0303 health sciences ,biology ,Chemistry ,General Chemical Engineering ,Plasmodium falciparum ,02 engineering and technology ,General Chemistry ,Pharmacology ,021001 nanoscience & nanotechnology ,biology.organism_classification ,Bioavailability ,03 medical and health sciences ,Pyrimethamine ,In vivo ,Chloroquine ,parasitic diseases ,medicine ,Plasmodium berghei ,Nanocarriers ,Artemisinin ,0210 nano-technology ,030304 developmental biology ,medicine.drug - Abstract
Artemisinin is a remarkable compound whose derivatives and combinations with multiple drugs have been utilized at the forefront of malaria treatment. However, the inherent issues of the parent compound such as poor bioavailability, short serum half-life, and high first-pass metabolism partially limit further applications of this drug. In this study, we enhanced the aqueous phase solubility of artemisinin by encapsulating it in two nanocarriers based on the polymer polycaprolactone (ART-PCL) and lipid-based Large Unilamellar Vesicles (ART-LIPO) respectively. Both nanoformulations exhibit in vitro parasite killing activity against Plasmodium falciparum with the ART-LIPO performing at comparable efficacy to the control drug solubilized in ethanol. These water-soluble formulations showed potent in vivo antimalarial activity as well in the mouse model of malaria at equivalent doses of the parent drug. Additionally, the artemisinin-PCL nanoformulation used in combination with either pyrimethamine or chloroquine increased the survival of the Plasmodium berghei infected mice for more than 34 days and effectively cured the mice of the infection. We highlight the potential for polymer and liposome-based nanocarriers in improving not only the aqueous phase solubility of artemisinin but also concomitantly retaining its therapeutic efficacy in vivo as well. more...
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- 2020
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15. Multistage antiplasmodial activity of hydroxyethylamine compounds, in vitro and in vivo evaluations
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Vladimir Potemkin, Agam P. Singh, Snigdha Singh, Poonam, Raman Mathur, Prateek Pathak, Meenakshi Bansal, Brijesh Rathi, Maria Grishina, Jyoti Prakash Singh, Prakasha Kempaiah, Mohammad Kashif, Mohd Shahbaaz, Vinoth Rajendran, Yash Gupta, and Neha Sharma more...
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0303 health sciences ,education.field_of_study ,Chemistry ,General Chemical Engineering ,Population ,General Chemistry ,Pharmacology ,medicine.disease ,01 natural sciences ,In vitro ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,03 medical and health sciences ,Plasmepsin II ,In vivo ,parasitic diseases ,medicine ,Gametocyte ,education ,Cytotoxicity ,IC50 ,Malaria ,030304 developmental biology - Abstract
Malaria, a global threat to the human population, remains a challenge partly due to the fast-growing drug-resistant strains of Plasmodium species. New therapeutics acting against the pathogenic asexual and sexual stages, including liver-stage malarial infection, have now attained more attention in achieving malaria eradication efforts. In this paper, two previously identified potent antiplasmodial hydroxyethylamine (HEA) compounds were investigated for their activity against the malaria parasite's multiple life stages. The compounds exhibited notable activity against the artemisinin-resistant strain of P. falciparum blood-stage culture with 50% inhibitory concentrations (IC50) in the low micromolar range. The compounds' cytotoxicity on HEK293, HepG2 and Huh-7 cells exhibited selective killing activity with IC50 values > 170 μM. The in vivo efficacy was studied in mice infected with P. berghei NK65, which showed a significant reduction in the blood parasite load. Notably, the compounds were active against liver-stage infection, mainly compound 1 with an IC50 value of 1.89 μM. Mice infected with P. berghei sporozoites treated with compound 1 at 50 mg kg−1 dose had markedly reduced liver stage infection. Moreover, both compounds prevented ookinete maturation and affected the developmental progression of gametocytes. Further, systematic in silico studies suggested both the compounds have a high affinity towards plasmepsin II with favorable pharmacological properties. Overall, the findings demonstrated that HEA and piperidine possessing compounds have immense potential in treating malarial infection by acting as multistage inhibitors. more...
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- 2020
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16. The Multistage Antimalarial Compound Calxinin Modulates Calcium Homeostasis Targeting a Unique Calcium Channel Involved in Subcellular Calcium Storage in P. falciparum
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Yash Gupta, Neha Sharma, Snigdha Singh, Jesus G. Romero, Vinoth Rajendran, Reagan M. Mogire, Raman Mathur, Mohammad Kashif, Jordan Beach, Walter Jeske, Poonam, Bernhards Ogutu, Stefan M. Kanzok, Hoseah M. Akala, Jennifer Legac, Philip J. Rosenthal, David J. Rademacher, Ravi Durvasula, Agam P. Singh, Brijesh Rathi, and Prakasha Kempaiah more...
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History ,Polymers and Plastics ,Business and International Management ,Industrial and Manufacturing Engineering - Published
- 2022
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17. Corrigendum: Liver-stage specific response among endemic populations: diet and immunity
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Sarat Kumar Dalai, Naveen eYadav, Manoj ePatidar, Hardik ePatel, and Agam Prasad Singh
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sterile protection ,plasmodia ,liver-stage immunity ,natural habit ,Chloroquine and chemoprophylaxis ,Immunologic diseases. Allergy ,RC581-607 - Published
- 2015
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18. Cytokines induce effector T-helper cells during invasive aspergillosis; what we have learned about T-helper cells?
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Raman eThakur, Rajesh eAnand, Shraddha eTiwari, Agam P Singh, Bhupendra N Tiwary, and Jata eShankar
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Aspergillus ,Cytokines ,Dendritic Cells ,antigen presenting cells ,T-helper cells ,invasive aspergillosis ,Microbiology ,QR1-502 - Abstract
Invasive aspergillosis caused by Aspergillus species (Aspergillus fumigatus, A. flavus and A. terreus) is life-threatening infections in immunocompromised patients. Understanding the innate and adaptive immune response particularly T-helper cells (TH-cells) against these Aspergillus species and how the different sub-set of TH-cells are regulated by differentiating cytokines at primary target organ site like lung, kidney and brain is of great significance to human health. This review focuses on presentation of Aspergillus through Antigen presenting cells (APCs) to the naive CD4+ T-cells in the host. The production of differentiating/effector cytokines that activate following TH-cells e.g., TH1, TH2, TH9 and TH17 has been reported in association or alone in allergic or invasive aspergillosis. Chemokines (CXCL1, CXCL2, CCL1 and CCL20) and their receptors associated to these TH-cells have also been observed in invasive aspergillosis. Thus, further study of these TH-cells in invasive aspergillosis and other elements of adaptive immune response with Aspergillus species are required in order to have a better understanding of host response for safer and effective therapeutic outcome. more...
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- 2015
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19. Liver Stage specific response among Endemic Populations: Diet & Immunity
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Sarat Kumar Dalai, Naveen eYadav, Manoj ePatidar, Hardik ePatel, and Agam Prasad Singh
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sterile protection ,plasmodia ,liver-stage immunity ,natural habit ,Chloroquine and chemoprophylaxis ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Developing effective anti-malarial vaccine has been a challenge for long. Various factors including complex life cycle of parasite and lack of knowledge of stage specific critical antigens are some of the reasons. Moreover, inadequate understanding of the immune responses vis-à-vis sterile protection induced naturally by Plasmodia infection has further compounded the problem. It has been shown that people living in endemic areas take years to develop protective immunity to blood stage infection. But hardly anyone believes that immunity to liver-stage infection could be developed. Various experimental model studies using attenuated parasite suggest that liver stage immunity might exist among endemic populations. This could be induced because of the attenuation of parasite in liver by various compounds present in the diet of endemic populations. more...
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- 2015
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20. Molecular characterization and expression profile of an alternate proliferating cell nuclear antigen homolog PbPCNA2 in Plasmodium berghei
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Sabyasachi Pradhan, Inderjeet Kalia, Tridibes Adak, Suman Kumar Dhar, Om P. Singh, Sourav Singha Roy, and Agam P. Singh
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DNA Replication ,0301 basic medicine ,Plasmodium berghei ,DNA damage ,DNA polymerase ,Plasmodium falciparum ,Clinical Biochemistry ,Protozoan Proteins ,DNA-Directed DNA Polymerase ,Biochemistry ,Gene Knockout Techniques ,03 medical and health sciences ,0302 clinical medicine ,Proliferating Cell Nuclear Antigen ,Genetics ,Animals ,Humans ,Molecular Biology ,Gene ,Gene knockout ,Genome ,biology ,DNA replication ,Cell Biology ,biology.organism_classification ,Proliferating cell nuclear antigen ,Cell biology ,030104 developmental biology ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,biology.protein ,Homologous recombination ,DNA Damage - Abstract
Proliferative cell nuclear antigen (PCNA) is the processivity factor for various DNA polymerases and it functions in response to DNA damage in eukaryotic system. Plasmodium falciparum contains two PCNAs, while PCNA1 has been attributed to DNA replication, the role of PCNA2 has been assigned to DNA damage response in erythrocytic developmental stages. Although a recent transposon mediated knockout strategy qualified pcna2 as a nonessential gene in Plasmodium berghei, a conventional homologous recombination-based knockout strategy has not been employed for this gene yet. Moreover, the cellular dynamics of PCNA2 in extraerythrocytic stages still remain elusive in Plasmodium. We attempted multiple times to knock out PbPCNA2 from the parasite genome using homologous recombination strategy without much success. However, we were able to generate PbPCNA2-GFP tagged transgenic parasites confirming that the pcna2 locus is amenable to genetic manipulation. The GFP-tagged parasites showed similar growth phenotype, compared to wild-type parasites, in both erythrocytic and sporogonic cycle, suggesting that tagging had no effect on parasite physiology. PbPCNA2 expression was also observed during the sporogonic cycle in midgut oocyst and salivary gland sporozoites. The PbPCNA2 expression was upregulated in the presence of DNA damaging agents like hydroxyurea and methyl methanesulphonate. Our inability to knock out PCNA2 suggested its essentiality in the parasite development and elevated expression during DNA damaging condition hint at a critical role of the protein in parasite physiology. © 2019 IUBMB Life, 71(9):1293-1301, 2019. more...
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- 2019
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21. Novel Antiplasmodial Compounds Leveraged with Multistage Potency against the Parasite
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Neha, Sharma, Mohammad, Kashif, Vigyasa, Singh, Diana, Fontinha, Budhaditya, Mukherjee, Dhruv, Kumar, Shailja, Singh, Miguel, Prudencio, Agam P, Singh, and Brijesh, Rathi
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Membrane Potential, Mitochondrial ,Mice, Inbred BALB C ,Molecular Structure ,Plasmodium berghei ,Plasmodium falciparum ,Molecular Dynamics Simulation ,Piperazines ,Malaria ,Mitochondria ,Mice, Inbred C57BL ,Molecular Docking Simulation ,Antimalarials ,Structure-Activity Relationship ,Parasitic Sensitivity Tests ,Ethanolamines ,Animals ,Aspartic Acid Endopeptidases ,Protein Binding - Abstract
Hydroxyethylamine (HEA)-based novel compounds were synthesized and their activity against
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- 2021
22. SLTRiP induces long lasting and protective T-cell memory response
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Inderjeet Kalia, Agam P. Singh, Mohammad Kashif, and Afshana Quadiri
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Long lasting ,biology ,T cell ,medicine.disease ,biology.organism_classification ,Plasmodium ,medicine.anatomical_structure ,Immune system ,Immunization ,Immunology ,medicine ,Parasite hosting ,Secretion ,Malaria - Abstract
Major developments have been made in the past many years to characterize and explore potential vaccine candidates that can induce host immune responses against parasite. These advances were based on the fact that the induction of host immune responses could effectively target parasite at different stages of its life cycle and thus, abrogate Plasmodium infections. The role of T-cells against malaria comes from initial studies on rodents showing these cells could inhibit parasite development during pre-erythrocytic stages. Since then, the importance of the cellular immune responses against malaria has been increasingly emphasized, especially for vaccine development against pre-erythrocytic stages. Previous work in our laboratory has confirmed that SLTRiP confers protection against the pre-erythrocytic stage of Plasmodium growth in rodents. Here we report that the protection is mainly due to cell mediated immune responses and PbSLTRiP specific cellular memory responses could be efficiently recalled in mice challenged with P. berghei parasites even after a year following immunization. Our results thereby, highlight the role of the T cell response involved in protection. Characterization of T-cells by intracellular cytokine staining (ICS) revealed that the induced T cells were polyfunctional and involved in secretion of pro-inflammatory cytokines which mediate anti-parasitic activity. The findings contribute to our understanding of the immunological mechanisms underlying the protective vaccines. more...
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- 2021
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23. Pathogenic Pore Forming Proteins of Plasmodium Triggers the Necrosis of Endothelial Cells Attributed to Malaria Severity
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Agam P. Singh, Abhishek Shivappagowdar, Soumya Pati, Inderjeet Kalia, Swati Garg, Akriti Srivastava, Shailja Singh, Lalit C. Garg, and Rahul S. Hada
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blebbing ,Cell Membrane Permeability ,Erythrocytes ,Necrosis ,Health, Toxicology and Mutagenesis ,Protozoan Proteins ,lcsh:Medicine ,Apoptosis ,Video microscopy ,Toxicology ,Madin Darby Canine Kidney Cells ,necrosis ,Pathogenesis ,HMGB1 Protein ,Malaria, Falciparum ,Endothelial dysfunction ,HMGB1 ,0303 health sciences ,biology ,030302 biochemistry & molecular biology ,Recombinant Proteins ,Cell biology ,Plasmodium falciparum ,Blood-Brain Barrier ,perforin like proteins ,Mitochondrial Membranes ,medicine.symptom ,Intracellular ,Programmed cell death ,Cell Survival ,malaria ,Article ,Cell Line ,03 medical and health sciences ,Dogs ,Human Umbilical Vein Endothelial Cells ,medicine ,Animals ,Humans ,030304 developmental biology ,calcium ,Perforin ,lcsh:R ,Endothelial Cells ,biology.organism_classification ,medicine.disease ,biology.protein ,Reactive Oxygen Species ,Biomarkers - Abstract
Severe malaria caused by Plasmodium falciparum poses a major global health problem with high morbidity and mortality. P. falciparum harbors a family of pore-forming proteins (PFPs), known as perforin like proteins (PLPs), which are structurally equivalent to prokaryotic PFPs. These PLPs are secreted from the parasites and, they contribute to disease pathogenesis by interacting with host cells. The severe malaria pathogenesis is associated with the dysfunction of various barrier cells, including endothelial cells (EC). Several factors, including PLPs secreted by parasites, contribute to the host cell dysfunction. Herein, we have tested the hypothesis that PLPs mediate dysfunction of barrier cells and might have a role in disease pathogenesis. We analyzed various dysfunctions in barrier cells following rPLP2 exposure and demonstrate that it causes an increase in intracellular Ca2+ levels. Additionally, rPLP2 exposed barrier cells displayed features of cell death, including Annexin/PI positivity, depolarized the mitochondrial membrane potential, and ROS generation. We have further performed the time-lapse video microscopy of barrier cells and found that the treatment of rPLP2 triggers their membrane blebbing. The cytoplasmic localization of HMGB1, a marker of necrosis, further confirmed the necrotic type of cell death. This study highlights the role of parasite factor PLP in endothelial dysfunction and provides a rationale for the design of adjunct therapies against severe malaria. more...
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- 2021
24. STK35L1 regulates Plasmodium infection and expression of cell cycle genes during liver stage of malaria
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Mohammad Kashif, Akhil Agarwal, Pankaj Goyal, Phulwanti Kumari Sharma, Kirti Raj Chahar, Daniela Brünnert, Afshana Quadiri, Vibha Kaushik, Agam P. Singh, and Inderjeet Kalia
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Liver stage ,parasitic diseases ,medicine ,Biology ,biology.organism_classification ,medicine.disease ,Virology ,Plasmodium ,Cell Cycle Gene ,Malaria - Abstract
Protein kinases of both the parasite and host are crucial in parasite invasion and survival and might be drug targets against drug-resistant malaria. STK35L1 was among the top five hits in kinome-wide screening, suggesting a role in malaria’s liver stage. The function of STK35L1 in malaria is not known yet. We found that STK35L1 was highly upregulated during the infection of P. berghei in HepG2 cells and mice liver. Knockdown of STK35L1 remarkably suppressed the sporozoite infection in hepatocytes. STAT3 is upregulated and phosphorylated during P. berghei sporozoites infection. We found that STAT3 activation is required for both STK35L1 and STAT3 upregulation. Furthermore, ten cell cycle genes were upregulated in the sporozoite-infected hepatocytes. Knockdown of STK35L1 completely inhibited the upregulation of these genes. We identified STK35L1 as a host kinase that plays an obligatory role in malaria’s liver stage. It may be a potential drug target against drug-resistant malaria. more...
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- 2020
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25. A nonpeptidyl molecule modulates apoptosis-like cell death by inhibiting P. falciparum metacaspase-2
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Sudha Sankar, Vandana Kumari, Inderjeet Kalia, Rajkishor Rai, Agam P. Singh, Mohammad Kashif, Kona Madhavinadha Prasad, and Kailash C. Pandey
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Male ,Proteases ,Programmed cell death ,Cell Survival ,Plasmodium falciparum ,Apoptosis ,Cysteine Proteinase Inhibitors ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Antimalarials ,Mice ,Bacterial Proteins ,Drug Discovery ,medicine ,Animals ,Humans ,Malaria, Falciparum ,Molecular Biology ,030304 developmental biology ,Membrane Potential, Mitochondrial ,0303 health sciences ,Mice, Inbred BALB C ,biology ,030302 biochemistry & molecular biology ,Cell Biology ,Dipeptides ,Hep G2 Cells ,Ketones ,biology.organism_classification ,Amides ,In vitro ,Cell biology ,Cysteine Endopeptidases ,Oxidative Stress ,chemistry ,Fatty Acids, Unsaturated ,Female ,Piperic acid ,Intracellular ,Oxidative stress - Abstract
Metacaspases are novel cysteine proteases found in apicomplexan whose function is poorly understood. Our earlier studies on Plasmodium falciparum metacaspase-2 (PfMCA-2) revealed that the caspase inhibitor, Z-FA-FMK efficiently inhibited PfMCA-2 activity and, expression, and significantly blocked in vitro progression of the parasite developmental cycle via apoptosis-like parasite death. Building on these findings, we synthesized a set of novel inhibitors based on structural modification of Z-FA-FMK with the amides of piperic acid and investigated their effect on PfMCA-2. One of these analogues, SS-5, specifically inhibited the activity and expression of PfMCA-2. The activities of some other known malarial proteases (falcipains, plasmepsins, & vivapain), and human cathepsins-B, D and L, and caspase-3 and -7 were not inhibited by SS-5. SS-5 blocked the development of P. falciparumin vitro (IC50 1µM) and caused prominent morphological distortions. Incubation with SS-5 led to persistent parasite oxidative stress accompanied by depolarization of mitochondrial potential and accumulation of intracellular Ca2+. SS-5 also inhibited the development of P. berghei in a murine model. Our results suggest that the inhibition of PfMCA-2 results in oxidative stress, leading to apoptosis-like parasite death. Thus, SS-5 offers a starting point for optimization of new antimalarials, and PfMCA-2 could be a novel target for antimalarial drug discovery. more...
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- 2020
26. Plasmodium Perforin-Like Protein Pores on the Host Cell Membrane Contribute in Its Multistage Growth and Erythrocyte Senescence
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Soumya Pati, Swati Garg, Abhishek Shivappagowdar, Inderjeet Kalia, Rahul S. Hada, Agam P. Singh, R. Ayana, Shailja Singh, Subhabrata Sen, and Chandramohan Bathula
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0301 basic medicine ,Microbiology (medical) ,Senescence ,Plasmodium ,Erythrocytes ,Plasmodium falciparum ,030106 microbiology ,Immunology ,lcsh:QR1-502 ,malaria ,Protozoan Proteins ,Microbiology ,lcsh:Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Cellular and Infection Microbiology ,Escherichia coli ,Original Research ,Host cell membrane ,MACPF ,atomic force microscopy ,biology ,Perforin ,Cell Membrane ,invasion ,biology.organism_classification ,anemia ,egress ,Cell biology ,030104 developmental biology ,Infectious Diseases ,Dextran ,chemistry ,perforin like proteins ,Raman spectroscopy ,biology.protein ,erythrocyte ,Hemoglobin ,Complement membrane attack complex - Abstract
The pore forming Plasmodium Perforin Like Proteins (PPLP), expressed in all stages of the parasite life cycle are critical for completion of the parasite life cycle. The high sequence similarity in the central Membrane Attack Complex/ Perforin (MACPF) domain among PLPs and their distinct functional overlaps define them as lucrative target for developing multi-stage antimalarial therapeutics. Herein, we evaluated the mechanism of Pan-active MACPF Domain (PMD), a centrally located and highly conserved region of PPLPs, and deciphered the inhibitory potential of specifically designed PMD inhibitors. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to hemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence which can be hypothesized to account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing malaria anemia. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress processes and protected erythrocytes against rPMD induced senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage, transmission-blocking potential of these inhibitors. Concievably, our study has introduced a novel set of anti-PMD inhibitors with pan-inhibitory activity against all the PPLPs members which can be developed into potent cross-stage antimalarial therapeutics along with erythrocyte senescence protective potential to occlude PPLPs mediated anemia in severe malaria. ispartof: Frontiers In Cellular And Infection Microbiology vol:10 ispartof: location:Switzerland status: Published online more...
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- 2020
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27. Fast-Acting Small Molecules Targeting Malarial Aspartyl Proteases, Plasmepsins, Inhibit Malaria Infection at Multiple Life Stages
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Angela O. Achieng, Kailash C. Pandey, Amit Singh, Mansi Pandit, Poonam, Brajendra K. Singh, Daniel E. Goldberg, Ben M. Dunn, Ramesh Chandra, Brijesh Rathi, Prakasha Kempaiah, Latha Narayanan, Snigdha Singh, Jiang He, Afshana Quadiri, Akansha Pant, Agam P. Singh, Armiyaw S. Nasamu, Prahlad C. Ghosh, Vandana, Vinoth Rajendran, Jyoti Prakash Singh, and Nikesh Gupta more...
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0301 basic medicine ,Plasmodium berghei ,Plasmodium falciparum ,030106 microbiology ,Plasmodium vivax ,Phthalimides ,Plasmodium ,Microbiology ,Antimalarials ,Inhibitory Concentration 50 ,Mice ,03 medical and health sciences ,In vivo ,Drug Discovery ,parasitic diseases ,Ethylamines ,medicine ,Gametocyte ,Animals ,Aspartic Acid Endopeptidases ,Life Cycle Stages ,biology ,Chloroquine ,biology.organism_classification ,medicine.disease ,In vitro ,030104 developmental biology ,Infectious Diseases ,Malaria - Abstract
The eradication of malaria remains challenging due to the complex life cycle of Plasmodium and the rapid emergence of drug-resistant forms of Plasmodium falciparum and Plasmodium vivax. New, effective, and inexpensive antimalarials against multiple life stages of the parasite are urgently needed to combat the spread of malaria. Here, we synthesized a set of novel hydroxyethylamines and investigated their activities in vitro and in vivo. All of the compounds tested had an inhibitory effect on the blood stage of P. falciparum at submicromolar concentrations, with the best showing 50% inhibitory concentrations (IC50) of around 500 nM against drug-resistant P. falciparum parasites. These compounds showed inhibitory actions against plasmepsins, a family of malarial aspartyl proteases, and exhibited a marked killing effect on blood stage Plasmodium. In chloroquine-resistant Plasmodium berghei and P. berghei ANKA infected mouse models, treating mice with both compounds led to a significant decrease in blood parasite load. Importantly, two of the compounds displayed an inhibitory effect on the gametocyte stages (III-V) of P. falciparum in culture and the liver-stage infection of P. berghei both in in vitro and in vivo. Altogether, our findings suggest that fast-acting hydroxyethylamine-phthalimide analogs targeting multiple life stages of the parasite could be a valuable chemical lead for the development of novel antimalarial drugs. more...
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- 2018
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28. STK35L1 regulates host cell cycle-related genes and is essential for Plasmodium infection during the liver stage of malaria
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Inderjeet Kalia, Kirti Raj Chahar, Afshana Quadiri, Vibha Kaushik, Daniela Brünnert, Agam P. Singh, Pankaj Goyal, Phulwanti Kumari Sharma, Mohammad Kashif, and Akhil Agrawal
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STAT3 Transcription Factor ,Plasmodium berghei ,Cell Cycle Proteins ,Protein Serine-Threonine Kinases ,Plasmodium ,Microbiology ,Mice ,Downregulation and upregulation ,parasitic diseases ,medicine ,Animals ,Humans ,Gene knockdown ,biology ,Kinase ,Hep G2 Cells ,Cell Biology ,Cell cycle ,medicine.disease ,biology.organism_classification ,Cell Cycle Gene ,Malaria ,Mice, Inbred C57BL ,Gene Expression Regulation ,Liver ,Sporozoites ,Female - Abstract
Protein kinases of both the parasite and the host are crucial in parasite invasion and survival and might act as drug targets against drug-resistant malaria. STK35L1 was among the top five hits in kinome-wide screening, suggesting its role in malaria's liver stage. However, the role of host STK35L1 in malaria remains elusive. In this study, we found that STK35L1 was highly upregulated during the infection of Plasmodium berghei (P. berghei) in HepG2 cells and mice liver, and knockdown of STK35L1 remarkably suppressed the sporozoites' infection in HepG2 cells. We showed that STAT3 is upregulated and phosphorylated during P. berghei sporozoites' infection, and STAT3 activation is required for both the upregulation of STK35L1 and STAT3. Furthermore, we found that ten cell cycle genes were upregulated in the sporozoite-infected hepatocytes. Knockdown of STK35L1 inhibited the basal expression of these genes except CDKN3 and GTSE1 in HepG2 cells. Thus, we identified STK35L1 as a host kinase that plays an obligatory role in malaria's liver stage and propose that it may serve as a potential drug target against drug-resistant malaria. more...
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- 2021
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29. Conserved Plasmodium Protein (PF3D7_0406000) of Unknown Function: In-silico Analysis and Cellular Localization
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Afshana Quadiri, Isha Pandey, Ashis Das, C R Pillai, Agam P. Singh, and Ishan Wadi
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0301 basic medicine ,Microbiology (medical) ,In silico ,Plasmodium falciparum ,030106 microbiology ,Protozoan Proteins ,Microbiology ,Plasmodium ,Antibodies ,Mice ,03 medical and health sciences ,parasitic diseases ,Genetics ,medicine ,Gametocyte ,Animals ,Humans ,Malaria, Falciparum ,Molecular Biology ,Gene ,Ecology, Evolution, Behavior and Systematics ,Cellular localization ,biology ,biology.organism_classification ,medicine.disease ,Up-Regulation ,030104 developmental biology ,Infectious Diseases ,biology.protein ,Antibody ,Peptides ,Transcriptome ,Malaria - Abstract
In spite of a decrease in malaria cases, the threat of malaria due to Plasmodium falciparum still prevails. The sequencing of Plasmodium falciparum reveals that approximately 60% of the Plasmodium genes code for hypothetical/putative proteins. Here we report an in silico characterization and localization of one such protein. This was encoded by one of the hub genes, in a weighted gene co-expression based systems network, from in-vivo samples of patients suffering from uncomplicated malaria or complicated malaria disease like jaundice and jaundice with renal failure. Interestingly, the protein PF3D7_0406000 (PFD0300w) is classified as a conserved protein of unknown function and shows no identity with any protein from the human host. The transcriptomic data shows up-regulation of transcripts in cases of malaria induced disease complications. PFD0300w peptide antibody based immunolocalization studies using a, gametocyte producing P. falciparum strain RKL-9, shows presence of the protein in the cytoplasm of both asexual and sexual stage parasites. more...
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- 2021
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30. Identification and characterization of protective CD8
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Afshana, Quadiri, Inderjeet, Kalia, Mohammad, Kashif, and Agam P, Singh
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Male ,T‐cell epitopes ,Plasmodium falciparum ,Epitopes, T-Lymphocyte ,Antigens, Protozoan ,CD8-Positive T-Lymphocytes ,Peptide Fragments ,Malaria ,Mice, Inbred C57BL ,Mice ,vaccine ,Malaria Vaccines ,Animals ,Female ,Amino Acid Sequence ,liver‐stage malaria ,Epitope Mapping ,Original Research - Abstract
Introduction Efforts are required at developing an effective vaccine that can inhibit malaria prevalence and transmission. Identifying the critical immunogenic antigens and understanding their interactions with host proteins forms a major focus of subunit vaccine development. Previously, our laboratory showed that SLTRiP conferred protection to the liver stage of Plasmodium growth in rodents. In the follow‐up of earlier research, we demonstrate that SLTRiP‐mediated protection is majorly concentrated in specific regions of protein. Method To identify particular protective regions of protein, we synthesized multiple nonoverlapping fragments from SLTRiP protein. From this, we designed a panel of 8‐20mer synthetic peptides, which were predicted using T‐epitope‐based prediction algorithm. We utilized the IFN‐γ enzyme‐linked immunosorbent spot assay to identify immunodominant peptides. The latter were used to immunize mice, and these mice were challenged to assess protection. Results The protective polypeptide fragment SLTRiP C3 and SLTRiP C4 were identified, by expressing and testing multiple fragments of PbSLTRiP protein. The immune responses generated by these fragments were compared to identify the immunodominant fragment. The T‐epitopes were predicted from SLTRiP protein using computer‐based algorithms. The in vitro immune responses generated by these peptides were compared with each other to identify the immunodominant T‐epitope. Immunization using these peptides showed significant reduction in parasite numbers during liver stage. Conclusion Our findings show that the protective efficacy shown by SLTRiP is localized in particular protein fragments. The peptides designed from such regions showed protective efficacy equivalent to whole protein. The sequence conservation analysis with human Plasmodium species also showed that these peptides were conserved. In conclusion, these peptides or their equivalent from other Plasmodium species could impart protection against malaria in their respective hosts too. Our studies provide a basis for the inclusion of these peptides in clinical vaccine constructs against malaria., SLTRiP protective polypeptide fragment was identified by expressing and testing multiple fragments of PbSLTRiP protein and protective T‐epitopes were identified from a curated list of peptides. ELISpot assay was performed to identify the immunodominant T‐epitope. Sporozoite challenge assay done with immunized mice, was used to pinpoint protective CD8 + T‐epitopes. more...
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- 2019
31. Supression of Perforin-like Protein pores inhibitPlasmodiummultistage-growth, transmission and erythrocyte senescence
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Rahul S. Hada, Soumya Pati, Inderjeet Kalia, R. Ayana, Agam P. Singh, Swati Garg, Chandramohan Bathula, Shakti Nath Singh, Subhabrata Sen, and Abhishek Shivappagowdar
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Senescence ,0303 health sciences ,MACPF ,biology ,biology.organism_classification ,Plasmodium ,3. Good health ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Dextran ,chemistry ,Perforin ,030220 oncology & carcinogenesis ,biology.protein ,Parasite hosting ,Complement membrane attack complex ,Function (biology) ,030304 developmental biology - Abstract
The pore formingPlasmodiumperforin like proteins (PPLP), expressed in all stages of the parasite life cycle are central drivers for host interactions critical for completion of parasite life cycle and high transmission rates. The high sequence similarity in the central membrane attack complex/ perforin (MACPF) domain and consequent functional overlaps defines them as an attractive target for the development of multi-stage antimalarials. Herein we evaluated the mechanism of pan active function of central, highly conserved region of PPLPs, MACPF domain (PMD) and inhibitory potential of specifically designed anti-PMD chemo. TheE. coliexpressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to haemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence at 48 hours which can account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing anemia during malaria infection. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress of merozoites and protecting against erythrocyte senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage and transmission-blocking potential of these inhibitors. Additionally, the erythrocyte senescence protective potential of PMD inhibitors can be used to occlude PPLPs mediated severe malarial anemia. Further these inhibitors can be developed with a potential to protect against severity of the disease.Author SummaryMalaria continues to be a major global health threat despite of several exciting improvements in the treatment and prevention of the disease. One of the major concerns in the development of therapy is the emergence of the drug resistance. But for the efficient treatment regime, targeting multiple stages including host and vector would serve as an ideal therapy. Perforin like proteins (PLPs) are eukaryotic pore forming proteins that are highly conserved in the apicomplexan parasites. These play crucial roles in entry and exit of parasites from the host cells and establish infection at multiple stages ofPlasmodium spp.life cycle. Understanding the mechanism of pore formation by smaller, functional, pan-active scaffold of PLPs can serve as a target for development of cross stage protection. Here, using various biochemical, biophysical and pharmacological evidences, we validate the activity and characterize the pore formation of PLPs on erythrocytes. Further, our specifically designed inhibitors could restrict this pore formation and impede the exit/entry of the parasites. Moreover, these inhibitors could also exert multiple stage inhibition and rescue the uninfected erythrocytes from death. Together, this study highlights the mechanism of pore formation by PPLPs and evaluates their potential for the development of pan-active inhibitors to provide both symptomatic and transmission blocking cure for malaria. more...
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- 2019
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32. Metacaspase-3 of Plasmodium falciparum: An atypical trypsin-like serine protease
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Kailash C. Pandey, Mohammad Abid, Ajay K. Saxena, Mohammad Kashif, Ruby Sharma, Sonia Verma, Atul, Rajnikant Dixit, Agam P. Singh, Bhumika Kumar, and Veena Pande
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medicine.medical_treatment ,Plasmodium falciparum ,02 engineering and technology ,Biochemistry ,Serine ,03 medical and health sciences ,Structural Biology ,Catalytic Domain ,medicine ,Amino Acid Sequence ,Molecular Biology ,Caspase ,030304 developmental biology ,Serine protease ,0303 health sciences ,Protease ,biology ,Chemistry ,Serine Endopeptidases ,Temperature ,General Medicine ,Hydrogen-Ion Concentration ,021001 nanoscience & nanotechnology ,Trypsin ,biology.organism_classification ,Caspase Inhibitors ,Metacaspase ,Kinetics ,Caspases ,biology.protein ,Biocatalysis ,0210 nano-technology ,medicine.drug ,Cysteine - Abstract
Metacaspases are clan CD cysteine peptidases found in plants, fungi and protozoa that possess a conserved Peptidase_C14 domain, homologous to the human caspases and a catalytic His/Cys dyad. Earlier reports have indicated the role of metacaspases in cell death; however, metacaspases of human malaria parasite remains poorly understood. In this study, we aimed to functionally characterize a novel malarial protease, P. falciparum metacaspase-3 (PfMCA3). Unlike other clan CD peptidases, PfMCA3 has an atypical active site serine (Ser1865) residue in place of canonical cysteine and it phylogenetically forms a distinct branch across the species. To investigate whether this domain retains catalytic activity, we expressed, purified and refolded the Peptidase_C14 domain of PfMCA3 which was found to express in all asexual stages. PfMCA3 exhibited trypsin-like serine protease activity with ser1865 acting as catalytic residue to cleave trypsin oligopeptide substrate. PfMCA3 is inhibited by trypsin-like serine protease inhibitors. Our study found that PfMCA3 enzymatic activity was abrogated when catalytic serine1865 (S1865A) was mutated. Moreover, PfMCA3 was found to be inactive against caspase substrate. Overall, our study characterizes a novel metacaspase of P. falciparum, different from human caspases and not responsible for the caspase-like activity, therefore, could be considered as a potential chemotherapeutic target. more...
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- 2019
33. Tryptophan-kynurenine pathway attenuates β-catenin-dependent pro-parasitic role of STING-TICAM2-IRF3-IDO1 signalosome in Toxoplasma gondii infection
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Md. Arafat Hussain, Namita Chauhan, Shibnath Mazumder, Arun K. Haldar, Shagun Sharma, Ruchi Bharti, Amit Kumar Sahu, Tanmay Majumdar, Agam P. Singh, Vipin Singh Rana, Inderjeet Kalia, and Manmohan Kumar more...
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0301 basic medicine ,Cancer Research ,Kynurenine pathway ,Toluidines ,Transcription, Genetic ,Immunology ,Hydroxybutyrates ,Apoptosis ,Transfection ,Article ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Gene Knockout Techniques ,Mice ,Nitriles ,Animals ,Humans ,Indoleamine-Pyrrole 2,3,-Dioxygenase ,lcsh:QH573-671 ,Kinase activity ,Phosphorylation ,Protein kinase B ,Kynurenine ,beta Catenin ,Adaptor Proteins, Signal Transducing ,030102 biochemistry & molecular biology ,biology ,lcsh:Cytology ,Chemistry ,Tryptophan ,Toxoplasma gondii ,Membrane Proteins ,Tyrosine phosphorylation ,Cell Biology ,biology.organism_classification ,Cell biology ,030104 developmental biology ,RAW 264.7 Cells ,Crotonates ,Interferon Regulatory Factor-3 ,Caco-2 Cells ,IRF3 ,Proto-Oncogene Proteins c-akt ,Toxoplasma ,Toxoplasmosis - Abstract
Recent studies have documented the diverse role of host immunity in infection by the protozoan parasite, Toxoplasma gondii. However, the contribution of the β-catenin pathway in this process has not been explored. Here, we show that AKT-mediated phosphorylated β-catenin supports T. gondii multiplication which is arrested in the deficiency of its phosphorylation domain at S552 position. The β-catenin-TCF4 protein complex binds to the promoter region of IRF3 gene and initiates its transcription, which was also abrogated in β-catenin knockout cells. TBK-independent phosphorylation of STING(S366) and its adaptor molecule TICAM2 by phospho-AKT(T308S473) augmented downstream IRF3-dependent IDO1 transcription, which was also dependent on β-catenin. But, proteasomal degradation of IDO1 by its tyrosine phosphorylation (at Y115 and Y253) favoured parasite replication. In absence of IDO1, tryptophan was catabolized into melatonin, which supressed cellular reactive oxygen species (ROS) and boosted parasite growth. Conversely, when tyrosine phosphorylation was abolished by phosphosite mutations, IDO1 escaped its ubiquitin-mediated proteasomal degradation system (UPS) and the stable IDO1 prevented parasite replication by kynurenine synthesis. We propose that T. gondii selectively utilizes tryptophan to produce the antioxidant, melatonin, thus prolonging the survival of infected cells through functional AKT and β-catenin activity for better parasite replication. Stable IDO1 in the presence of IFN-γ catabolized tryptophan into kynurenine, promoting cell death by suppressing phospho-AKT and phospho-β-catenin levels, and circumvented parasite replication. Treatment of infected cells with kynurenine or its analogue, teriflunomide suppressed kinase activity of AKT, and phosphorylation of β-catenin triggering caspase-3 dependent apoptosis of infected cells to inhibit parasite growth. Our results demonstrate that β-catenin regulate phosphorylated STING-TICAM2-IRF3-IDO1 signalosome for a cell-intrinsic pro-parasitic role. We propose that the downstream IRF3-IDO1-reliant tryptophan catabolites and their analogues can act as effective immunotherapeutic molecules to control T. gondii replication by impairing the AKT and β-catenin axis. more...
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- 2018
34. A Sporozoite- and Liver Stage-expressed Tryptophan-rich Protein Plays an Auxiliary Role in Plasmodium Liver Stage Development and Is a Potential Vaccine Candidate
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Dabbu Kumar Jaijyan, Himanshu Singh, and Agam P. Singh
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Plasmodium berghei ,T-Lymphocytes ,Molecular Sequence Data ,Protozoan Proteins ,Gene Expression ,Immunofluorescence ,Microbiology ,Biochemistry ,Plasmodium ,Rats, Sprague-Dawley ,Transcriptome ,Mice ,parasitic diseases ,Anopheles ,Malaria Vaccines ,Escherichia coli ,medicine ,Animals ,Parasite hosting ,Amino Acid Sequence ,Molecular Biology ,Immunity, Cellular ,Mice, Inbred BALB C ,biology ,medicine.diagnostic_test ,Antibody titer ,Cell Biology ,medicine.disease ,biology.organism_classification ,Virology ,Recombinant Proteins ,Insect Vectors ,Malaria ,Rats ,Mice, Inbred C57BL ,Liver ,Parasitology ,Sporozoites ,biology.protein ,Female ,Immunization ,Antibody ,Sequence Alignment - Abstract
The liver stages of the malaria parasite are clinically silent and constitute ideal targets for causal prophylactic drugs and vaccines. Cellular and molecular events responsible for liver stage development are poorly characterized. Here, we show that sporozoite, liver stage tryptophan-rich protein (SLTRiP) forms large multimers. Mice immunized with a purified recombinant SLTRiP protein gave high antibody titers in both inbred and outbred mice. Immunized mice showed highly significant levels of protection upon challenge with sporozoites and exhibited 10,000-fold fewer parasite 18S-rRNA copy numbers in their livers. The protection offered by immunization with SLTRiP came mainly from T-cells, and antibodies had little role to play despite their high titers. Immunofluorescence assays showed that SLTRiP is expressed in the sporozoite and early to late liver stages of malaria parasites. SLTRiP protein is exported to the cytosol of infected host cells during the early hours of parasite infection. Parasites deficient in SLTRiP were moderately defective in liver stage parasite development. A transcriptome profile of SLTRiP-deficient parasite-infected hepatocytes highlighted that SLTRiP interferes with multiple pathways in the host cell. We have demonstrated a role for SLTRiP in sporozoites and the liver stage of malaria parasites. more...
- Published
- 2015
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35. Aspergillus flavus induces granulomatous cerebral aspergillosis in mice with display of distinct cytokine profile
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Bhupendra N. Tiwary, Agam P. Singh, Rajesh Anand, and Jata Shankar
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Pathology ,medicine.medical_specialty ,Necrosis ,medicine.medical_treatment ,Immunology ,Central nervous system ,Enzyme-Linked Immunosorbent Assay ,Aspergillus flavus ,Aspergillosis ,Interleukin-23 ,Biochemistry ,Interferon-gamma ,Mice ,medicine ,Animals ,Immunology and Allergy ,Molecular Biology ,Neuroaspergillosis ,Neurons ,Mice, Inbred BALB C ,Aspergillus ,Granuloma ,biology ,Interleukin-12 Subunit p40 ,Interleukin-6 ,Tumor Necrosis Factor-alpha ,Interleukin-17 ,Brain ,Hematology ,biology.organism_classification ,medicine.disease ,Interleukin-12 ,Disease Models, Animal ,Kinetics ,medicine.anatomical_structure ,Cytokine ,Cytokines ,Interleukin-4 ,medicine.symptom ,Pyknosis - Abstract
Aspergillus flavus is one of the leading Aspergillus spp. resulting in invasive aspergillosis of central nervous system (CNS) in human beings. Immunological status in aspergillosis of central nervous system remains elusive in case of both immunocompetent and immunocompromised patients. Since cytokines are the major mediators of host response, evaluation of disease pathology along with cytokine profile in brain may provide snapshots of neuro-immunological response. An intravenous model of A. flavus infection was utilized to determine the pathogenicity of infection and cytokine profile in the brain of male BALB/c mice. Enumeration of colony forming units and histopathological analyses were performed on the brain tissue at distinct time periods. The kinetics of cytokines (TNF-α, IFN-γ, IL-12/IL-23p40, IL-6, IL-23, IL-17A and IL-4) was evaluated at 6, 12, 24, 48, 72 and 96h post infection (hPI) in brain homogenates using murine cytokine specific enzyme linked immunosorbent assay. Histological analysis exhibited the hyphae with leukocyte infiltrations leading to formation of granulomata along with ischemia and pyknosis of neurons in the brain of infected mice. Diseased mice displayed increased secretion of IFN-γ, IL-12p40 and IL-6 with a concomitant reduction in the secretion of Th2 cytokine IL-4, and Th17 promoting cytokine, IL-23 during the late phase of infection. A.flavus induced inflammatory granulomatous cerebral aspergillosis in mice, characterized by a marked increase in the Th1 cytokines and neurons undergoing necrosis. A marked increase in necrosis of neurons with concurrent inflammatory responses might have led to the host mortality during late phase of infection. more...
- Published
- 2015
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36. Biochemical characterization of unusual cysteine protease of P. falciparum, metacaspase-2 (MCA-2)
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Ajay K. Saxena, Agam P. Singh, Rajnikant Dixit, Vandana, Kailash C. Pandey, Jitendra Singh, Mymoona Akhter, Ruby Sharma, Brijesh Rathi, Anju Katyal, and Pradyumna Kumar Mishra
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0301 basic medicine ,Proteases ,Proteolysis ,030106 microbiology ,Plasmodium falciparum ,Cysteine Proteinase Inhibitors ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Cysteine Proteases ,Leucine ,parasitic diseases ,medicine ,Molecular Biology ,Caspase ,medicine.diagnostic_test ,biology ,Gene Expression Profiling ,Dipeptides ,Ketones ,Cysteine protease ,Metacaspase ,Z-FA-FMK ,030104 developmental biology ,chemistry ,Biochemistry ,Apoptosis ,biology.protein ,DNA fragmentation ,Parasitology - Abstract
Earlier studies on Plasmodium apoptosis revealed the presence of proteases with caspases like- activity, which are known as “metacaspases”. Although this family of cysteine proteases is structurally similar to caspases with Cys-His dyad but their evolutionary significance and functional relevance remains largely unknown. These proteases are considered to be an important target against malaria due to their absence in humans. In this report, we have biochemically characterized metacaspase-2 (PfMCA-2) of P.falciparum. Enzymatic assay showed that PfMCA-2 efficiently cleaved arginine/lysine specific peptide, but not caspase-specific substrate. Consistently, PfMCA-2 activity was sensitive to effector caspases inhibitor, Z-FA-FMK, and mildly inhibited by aprotinin and E-64. However, general caspase inhibitors such as Z-VAD-FMK and Z-DEVD-FMK had no effect on PfMCA-2 activity. Z-FA-FMK inhibits parasite growth with an IC50 value of 2.7 μM along with the notable morphological changes. PfMCA-2 specifically expressed in schizonts and gametocyte stages and there was a notable depletion of PfMCA-2 expression in Z-FA-FMK treated schizonts and gametocytes stages of parasite. Notably, PfMCA-2 cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease) and the proteolysis of PfTSN did not occur after treatment with the Z-FA-FMK. The production of large amount of reactive oxygen species in presence of Z-FA-FMK caused oxidative stress which in turn leads to loss of cell viability. The oxidative stress further generates positive feedback for the occurrence of cell death in term of phosphatidylserine externalization and DNA fragmentation in vitro. more...
- Published
- 2017
37. Evaluation of aflatoxin B1 biosynthesis in A. flavus isolates from central india and identification of atoxigenic isolates
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Tarun Patel, Agam P. Singh, Jata Shankar, Rajesh Anand, and Bhupendra N. Tiwary
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Aflatoxin ,Electrospray ionization ,Selected reaction monitoring ,Biomedical Engineering ,food and beverages ,Bioengineering ,Aspergillus flavus ,Biology ,Contamination ,Tandem mass spectrometry ,Mass spectrometry ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Microbiology ,chemistry.chemical_compound ,Biosynthesis ,chemistry ,Biotechnology - Abstract
Present study describes the effect of temperature and pH on biosynthesis of aflatoxin B1 (AFB1) to identify atoxigenic (aflatoxin non-producer) isolates of Aspergillus flavus. An indigenous detection method for AFB1 was developed using tandem mass spectrometric method. Detection of AFB1 was carried out in positive polarity using triple quadrapole mass spectrometer with electrospray ionization interface in Multiple Reaction Monitoring (MRM) mode. A total of four A. flavus isolates were selected for screening of AFB1 biosynthesis at pH 4.0, 5.5, 7.0, and 8.5 at 27℃. Highest AFB1 biosynthesis in MTCC11866 was found at pH 6.5 while in MTCC9367 it was at pH 7.0. On screening AFB1 biosynthesis in A. flavus culture at various temperatures a significant suppression in AFB1 biosynthesis was found at 37℃ in comparison to 24 and 27℃ in MTCC11866 and MTCC9367. AFB1 was not detected in other two A. flavus isolates MTCC11580 and MTCC11588 at any temperature and pH tested. AFB1 nonproducing isolates, MTCC11580 and MTCC11588 could be used as potent biocontrol agent. Additionally, present standardized method for AFB1 detection may find its application in qualitative and quantitative analysis of AFB1 contamination in food crop and other products. more...
- Published
- 2014
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38. A novel FIKK kinase regulates the development of mosquito and liver stages of the malaria
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Dabbu Kumar Jaijyan, Praveen Kumar Verma, and Agam P. Singh
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0301 basic medicine ,Plasmodium berghei ,Plasmodium falciparum ,Protein Serine-Threonine Kinases ,Biology ,Article ,Transcriptome ,Gene Knockout Techniques ,Mice ,03 medical and health sciences ,Immune system ,Anopheles ,Animals ,Protein phosphorylation ,Amino Acid Sequence ,Phosphorylation ,Protein kinase A ,Mice, Inbred BALB C ,Multidisciplinary ,030102 biochemistry & molecular biology ,Kinase ,Wild type ,Cell cycle ,Malaria ,Cell biology ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,Liver ,Sporozoites ,Signal transduction ,Protein Processing, Post-Translational ,Signal Transduction - Abstract
Protein phosphorylation is the most important post-translational event in the regulation of various essential signaling pathways in a cell. Here, we show the functional characterization of a FIKK family protein kinase of the rodent malaria parasite (PbMLFK), which is expressed only in mosquito and liver stages and contains two functional C-terminal PEXEL motifs. We demonstrate that this protein plays a role in mosquito and liver stages of parasite growth. The oocysts of PbMLFK-deficient parasites produced 4-fold fewer sporozoites. In the liver of infected mice, PbMLFK-deficient parasites grew 100-fold less than did wild type parasites. We also show that the C-terminal domain of this protein has a functional serine-threonine kinase and that its activity was inhibited by a known PKA inhibitor. Transcriptome analysis of infected host cells suggests that in absence of this protein expression of the 288 host mRNAs are perturbed which are primarily associated with the immune system, cell cycle and metabolism. more...
- Published
- 2016
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39. Cytokines induce effector T-helper cells during invasive aspergillosis; what we have learned about T-helper cells?
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Rajesh Anand, Raman Thakur, Shraddha Tiwari, Jata Shankar, Bhupendra N. Tiwary, and Agam P. Singh
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Microbiology (medical) ,Chemokine ,Mini Review ,lcsh:QR1-502 ,CCL1 ,antigen presenting cells ,Aspergillosis ,T-helper cells ,Microbiology ,lcsh:Microbiology ,Aspergillus fumigatus ,medicine ,Antigen-presenting cell ,skin and connective tissue diseases ,Aspergillus ,invasive aspergillosis ,biology ,Effector ,Dendritic Cells ,biology.organism_classification ,medicine.disease ,Acquired immune system ,Immunology ,biology.protein ,Cytokines - Abstract
Invasive aspergillosis caused by Aspergillus species (Aspergillus fumigatus, A. flavus, and A. terreus) is life-threatening infections in immunocompromised patients. Understanding the innate and adaptive immune response particularly T-helper cells (TH-cells) against these Aspergillus species and how the different sub-set of TH-cells are regulated by differentiating cytokines at primary target organ site like lung, kidney and brain is of great significance to human health. This review focuses on presentation of Aspergillus through Antigen presenting cells (APCs) to the naive CD4(+) T-cells in the host. The production of differentiating/effector cytokines that activate following TH-cells, e.g., TH1, TH2, TH9, and TH17 has been reported in association or alone in allergic or invasive aspergillosis. Chemokines (CXCL1, CXCL2, CCL1, and CCL20) and their receptors associated to these TH-cells have also been observed in invasive aspergillosis. Thus, further study of these TH-cells in invasive aspergillosis and other elements of adaptive immune response with Aspergillus species are required in order to have a better understanding of host response for safer and effective therapeutic outcome. more...
- Published
- 2015
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40. Targeted deletion ofPlasmodium knowlesiDuffy binding protein confirms its role in junction formation during invasion
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Clemens H. M. Kocken, Alan W. Thomas, Agam P. Singh, Hastings Ozwara, Sunil K. Puri, and Chetan E. Chitnis
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Genetics ,Mutation ,biology ,Binding protein ,Duffy binding proteins ,biology.organism_classification ,medicine.disease_cause ,Microbiology ,Plasmodium ,Cell biology ,Apicomplexa ,Antigen ,Plasmodium knowlesi ,parasitic diseases ,medicine ,Molecular Biology ,Gene - Abstract
Red cell invasion by Plasmodium merozoites involves multiple steps such as attachment, apical reorientation, junction formation and entry into a parasitophorous vacuole. These steps are mediated by specific molecular interactions. P. vivax and the simian parasite P. knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes. P. vivax and P. knowlesi Duffy binding proteins (PvDBP and PkDBP), which bind the Duffy antigen during invasion, share regions of sequence homology and belong to a family of erythrocyte binding proteins (EBPs). By deletion of the gene that encodes PkDBP, we demonstrate that interaction of PkDBP with the Duffy antigen is absolutely necessary for invasion of human erythrocytes by P. knowlesi. Electron microscopy studies reveal that PkDBP knockout parasites are unable to form a junction with human erythrocytes. The interaction of PkDBP with the Duffy antigen is thus necessary for the critical step of junction formation during invasion. These studies provide support for development of intervention strategies that target EBPs to inhibit junction formation and block erythrocyte invasion by malaria parasites. more...
- Published
- 2005
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41. Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC)
- Author
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Hyeryun Choe, Michael Farzan, Rushdi Shakri, Agam P. Singh, Chetan E. Chitnis, Christopher M. Owens, Paulette L. Wright, Natalya Vasilieva, Wenhui Li, and Michael Moore
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Chemokine ,biology ,Binding protein ,Plasmodium vivax ,Duffy binding proteins ,biology.organism_classification ,Microbiology ,Virology ,Chemokine receptor ,Antigen ,Plasmodium knowlesi ,parasitic diseases ,biology.protein ,Tyrosine ,Molecular Biology - Abstract
Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC-expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP-1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino-terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax. more...
- Published
- 2005
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42. Antibodies raised against receptor-binding domain of Plasmodium knowlesi Duffy binding protein inhibit erythrocyte invasion
- Author
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Agam P. Singh, Chetan E. Chitnis, and Sunil K. Puri
- Subjects
Erythrocytes ,Plasmodium vivax ,Protozoan Proteins ,Antibodies, Protozoan ,Antigens, Protozoan ,Receptors, Cell Surface ,Spodoptera ,Host-Parasite Interactions ,chemistry.chemical_compound ,Antigen ,Malaria Vaccines ,parasitic diseases ,Animals ,Humans ,Glycophorin ,Plasmodium knowlesi ,Molecular Biology ,Cells, Cultured ,biology ,Binding protein ,Plasmodium falciparum ,biology.organism_classification ,Macaca mulatta ,Molecular biology ,Recombinant Proteins ,Malaria ,Sialic acid ,chemistry ,biology.protein ,Parasitology ,Rabbits ,Antibody ,Carrier Proteins ,Duffy Blood-Group System - Abstract
Erythrocyte invasion by malaria parasites requires specific receptor-ligand interactions. Plasmodium vivax and Plasmodium knowlesi are completely dependent on binding the Duffy blood group antigen to invade human erythrocytes. P. knowlesi invades rhesus erythrocytes by multiple pathways using the Duffy antigen as well as alternative receptors. Plasmodium falciparum binds sialic acid residues on glycophorin A as well as other sialic acid-independent receptors to invade human erythrocytes. Parasite proteins that mediate these interactions belong to a family of erythrocyte binding proteins, which includes the P. vivax Duffy binding protein, 175 kDa P. falciparum erythrocyte binding antigen (EBA-175), P. knowlesi a protein, which binds human and rhesus Duffy antigens, and P. knowlesi b and g proteins, which bind Duffy-independent receptors on rhesus erythrocytes. The receptor-binding domains of these proteins lie in conserved, N-terminal, cysteine-rich regions that are referred to as region II. Here, we have examined the feasibility of inhibiting erythrocyte invasion with antibodies directed against receptor-binding domains of erythrocyte binding proteins. Region II of P. knowelsi a protein (PkaRII), which binds the Duffy antigen, was expressed as a secreted protein in insect cells and purified from culture supernatants. Rabbit antibodies raised against recombinant PkaRII were tested for inhibition of erythrocyte binding and invasion. Antibodies raised against PkaRII inhibit P. knowlesi invasion of both human and rhesus erythrocytes. These data provide support for the development of recombinant vaccines based on the homologous binding domains of P. vivax Duffy binding protein and P. falciparum EBA-175. # 2002 Elsevier Science B.V. All rights reserved. more...
- Published
- 2002
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43. Apicoplast triose phosphate transporter (TPT) gene knockout is lethal for Plasmodium
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Namita Surolia, Dabbu Kumar Jaijyan, Tanushree Banerjee, Avadhesha Surolia, and Agam P. Singh
- Subjects
chemistry.chemical_classification ,Organelles ,Apicoplast ,Sugar phosphates ,Genes, Essential ,Cell Survival ,Plasmodium berghei ,Wild type ,Transporter ,Biology ,biology.organism_classification ,Gene Knockout Techniques ,chemistry ,Biochemistry ,Trioses ,parasitic diseases ,Phosphate Transport Proteins ,heterocyclic compounds ,Parasitology ,Sugar Phosphates ,Phosphoenolpyruvate carboxykinase ,Molecular Biology ,Gene knockout - Abstract
The C3, C5, C6 type sugar phosphate transporters bring sugars inside apicoplast, thus providing energy, reducing power and elements like carbon to apicoplast. Plasmodium berghei has two C3 type sugar phosphate transporters in the membrane of apicoplast: triose phosphate transporter (TPT) and phosphoenolpyruvate transporter (PPT). Here we report that P. berghei TPT knockout parasites failed to survive. However, PPT knockout parasite behaved similar to the wild type in the blood stages. The absence of PPT in other life stages, leads to defects in the development of parasite and was required at both mosquito as well as liver stages. This study also underlines the essentiality of triose transporters for apicoplast and its downstream pathways. more...
- Published
- 2011
44. Cytokine milieu in renal cavities of immunocompetent mice in response to intravenous challenge of Aspergillus flavus leading to aspergillosis
- Author
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Rajesh Anand, Bhupendra N. Tiwary, Agam P. Singh, and Jata Shankar
- Subjects
Male ,Immunology ,Inflammation ,Aspergillus flavus ,Biology ,Aspergillosis ,Kidney ,Biochemistry ,Blastoconidium ,Microbiology ,Mice ,Immune system ,Th2 Cells ,medicine ,Immunology and Allergy ,Animals ,Molecular Biology ,Colony-forming unit ,Mice, Inbred BALB C ,Hematology ,medicine.disease ,biology.organism_classification ,medicine.anatomical_structure ,Antifungal innate immune response ,Cytokines ,medicine.symptom - Abstract
Investigations in mice have demonstrated that Aspergillus flavus is more virulent than all other Aspergillus species except A. tamari . However, there is a complete lack of information on the immune responses elicited by A. flavus in systemic model. This communication reports the progression of infection and cytokine profile in BALB/c mice in response to intravenous challenge of A. flavus . The pathogenesis of infection was evaluated morphologically and by the analysis of Colony Forming Units (CFUs) in kidney homogenates. The kinetics of regulated cytokines was determined in kidneys by cytokine-specific murine ELISA. During the initial phase of infection the rate of clearance of A. flavus was high, most likely through recruited neutrophils and the resident renal macrophages with concurrent significant release of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-12/IL-23p40, IL-6) indicating antifungal innate immune response to be active at the site. However, at 24 h PI there was a significant rise of IL-17 and IL-23 suggesting the activation of IL-17/IL-23 axis of inflammation resulting in rise of CFU. The lack of significant induction in the anti-inflammatory cytokines like IL-4 and IL-10 confirmed the absence of Th2 type of response. In the late phase, after 3 days post-infection, there was a rise in the number of pathogen in the kidneys as determined by histopathology and CFU counts. The A. flavus hyphae were evident in the renal pelvis and ureter and we propose the production of blastoconidia by metamorphosed hyphae. more...
- Published
- 2011
45. Triclosan inhibit the growth of the late liver-stage of Plasmodium
- Author
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Avadhesha Surolia, Agam P. Singh, and Namita Surolia
- Subjects
Primaquine ,Clinical Biochemistry ,Plasmodium falciparum ,Drug resistance ,Biology ,Biochemistry ,Plasmodium ,Parasite load ,Cell Line ,parasitic diseases ,Genetics ,medicine ,Parasite hosting ,Animals ,Humans ,Molecular Biology ,Cell Biology ,biology.organism_classification ,medicine.disease ,Virology ,Triclosan ,Liver ,Cerebral Malaria ,Malaria ,medicine.drug - Abstract
Annually, approximately two million human deaths are caused worldwide by malaria, most of them being children. Plasmodium falciparum is the leading cause of cerebral malaria, the most severe and fatal form of disease. Moreover, the emergence of resistant strains to the existing drugs has worsened the situation. Currently, primaquine is the only drug available for eliminating liver-stage parasites. Because of the emergence of resistant parasite strains, it becomes necessary to find new targets unique to the malaria parasites. In the Plasmodium species, the discovery of a distinct Type-II fatty-acid synthesis pathway has created an opportunity to target this pathway for the development of new inhibitors of malaria parasite growth. The present study explored the growth inhibition potential of triclosan in the case of liver-stage parasites. Liver-stage of Plasmodium is an excellent target for intervention due to very small parasite load as well as possibility of eliminating parasites before it can cause blood-stage infection. Here we report that triclosan inhibits the development of the Plasmodium liver-stage parasites. more...
- Published
- 2009
46. Targeted deletion of Plasmodium knowlesi Duffy binding protein confirms its role in junction formation during invasion
- Author
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Agam P, Singh, Hastings, Ozwara, Clemens H M, Kocken, Sunil K, Puri, Alan W, Thomas, and Chetan E, Chitnis
- Subjects
Microscopy, Electron ,Erythrocytes ,Protozoan Proteins ,Animals ,Humans ,Antigens, Protozoan ,Plasmodium knowlesi ,Receptors, Cell Surface ,Macaca mulatta ,Gene Deletion - Abstract
Red cell invasion by Plasmodium merozoites involves multiple steps such as attachment, apical reorientation, junction formation and entry into a parasitophorous vacuole. These steps are mediated by specific molecular interactions. P. vivax and the simian parasite P. knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes. P. vivax and P. knowlesi Duffy binding proteins (PvDBP and PkDBP), which bind the Duffy antigen during invasion, share regions of sequence homology and belong to a family of erythrocyte binding proteins (EBPs). By deletion of the gene that encodes PkDBP, we demonstrate that interaction of PkDBP with the Duffy antigen is absolutely necessary for invasion of human erythrocytes by P. knowlesi. Electron microscopy studies reveal that PkDBP knockout parasites are unable to form a junction with human erythrocytes. The interaction of PkDBP with the Duffy antigen is thus necessary for the critical step of junction formation during invasion. These studies provide support for development of intervention strategies that target EBPs to inhibit junction formation and block erythrocyte invasion by malaria parasites. more...
- Published
- 2005
47. Definition of structural elements in Plasmodium vivax and P. knowlesi Duffy-binding domains necessary for erythrocyte invasion
- Author
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Sunita Pandey, Chetan E. Chitnis, Saurabh K Singh, Syed Shams Yazdani, Amit Sharma, and Agam P. Singh
- Subjects
Erythrocytes ,Proteolysis ,Plasmodium vivax ,Molecular Sequence Data ,Protozoan Proteins ,Mutagenesis (molecular biology technique) ,Antigens, Protozoan ,Receptors, Cell Surface ,Biology ,Biochemistry ,Polymerase Chain Reaction ,Antigen ,medicine ,Escherichia coli ,Animals ,Humans ,Plasmodium knowlesi ,Trypsin ,Binding site ,Cloning, Molecular ,Receptor ,Molecular Biology ,DNA Primers ,Sequence Deletion ,Cloning ,Binding Sites ,medicine.diagnostic_test ,Base Sequence ,Cell Biology ,biology.organism_classification ,Molecular biology ,Peptide Fragments ,Recombinant Proteins ,Cell biology ,Mutagenesis ,Linker ,Cell Adhesion Molecules ,Research Article - Abstract
Plasmodium vivax and P. knowlesi use the Duffy antigen as a receptor to invade human erythrocytes. Duffy-binding ligands belong to a family of erythrocyte-binding proteins that bind erythrocyte receptors to mediate invasion. Receptor-binding domains in erythrocyte-binding proteins lie in conserved cysteine-rich regions called Duffy-binding-like domains. In the present study, we report an analysis of the overall three-dimensional architecture of P. vivax and P. knowlesi Duffy-binding domains based on mild proteolysis and supportive-functional assays. Our proteolysis experiments indicate that these domains are built of two distinct subdomains. The N-terminal region from Cys-1–4 (C1–C4) forms a stable non-functional subdomain. The region spanning C5–C12 forms another subdomain, which is capable of binding Duffy antigen. These subdomains are joined by a protease-sensitive linker. Results from deletion constructs, designed for expression of truncated proteins on COS cell surface, show that regions containing C5–C8 of the Duffy-binding domains are sufficient for the binding receptor. Therefore the central region of Duffy-binding domains, which is flanked by two non-functional regions, is responsible for receptor recognition. Moreover, the minimal Duffy-binding region identified here is capable of folding into a functionally competent module. These studies pave the way for understanding the architecture of Duffy-binding domains and their interactions with host receptors. more...
- Published
- 2003
48. A Plasmodium yoelii yoelii erythrocyte binding protein that uses Duffy binding-like domain for invasion: a rodent model for studying erythrocyte invasion
- Author
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Chetan E. Chitnis, Amit Sharma, Agam P. Singh, and C.Durga Prasad
- Subjects
Erythrocytes ,Erythrocyte binding ,Molecular Sequence Data ,Protozoan Proteins ,Antigens, Protozoan ,Receptors, Cell Surface ,Domain (software engineering) ,Host-Parasite Interactions ,Mice ,Antigen ,Animals ,Humans ,Amino Acid Sequence ,Receptor ,Molecular Biology ,Peptide sequence ,Mice, Knockout ,biology ,Rodent model ,Plasmodium yoelii ,biology.organism_classification ,Cell biology ,Malaria ,Disease Models, Animal ,Parasitology ,Rabbits - Published
- 2003
49. Plasmodium Circumsporozoite Protein Promotes the Development of the Liver Stages of the Parasite
- Author
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Beatriz M. A. Fontoura, Elizabeth A. Winzeler, Hodaka Fujii, John R. Walker, Victor Nussenzweig, Daniel R. Nussenzweig, Carlos A. Buscaglia, Agata Levay, Agam P. Singh, and Qian Wang
- Subjects
Plasmodium ,Cell ,Amino Acid Motifs ,Nuclear Localization Signals ,Protozoan Proteins ,HUMDISEASE ,Biology ,Karyopherins ,General Biochemistry, Genetics and Molecular Biology ,Rats, Sprague-Dawley ,Mice ,medicine ,NLS ,Parasite hosting ,Animals ,Humans ,Gene ,Cell Nucleus ,Life Cycle Stages ,Biochemistry, Genetics and Molecular Biology(all) ,NF-kappa B ,biology.organism_classification ,Virology ,Cell biology ,Malaria ,Rats ,Circumsporozoite protein ,Mice, Inbred C57BL ,Protein Transport ,medicine.anatomical_structure ,Liver ,Cytoplasm ,CELLIMMUNO ,Hepatocyte ,Hepatocytes ,Mutant Proteins ,CELLBIO ,Nuclear transport ,Nuclear localization sequence ,HeLa Cells - Abstract
Summary The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFκB nuclear import, thus downregulating the expression of many genes controlled by NFκB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite. more...
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- View/download PDF
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