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Molecular mechanisms of red cell invasion by the Plasmodium vivax parasite remain obscure since information on receptor-ligand interaction is scarce. Our lab had identified Basigin as receptor molecule for one of the important members of Pvfam “a’ family protein i.e., PvTRAg38. Here, we demonstrate by using solid phase binding assay, and Surface Plasmon Resonance that seven out of ten-erythrocyte binding Pvfam ‘a’ family proteins of P. vivax interact with erythrocyte receptor Basigin. This receptor-ligand interaction seems to be important for parasite’s survival since each of these proteins interfered with the parasite growth in a heterologous culture system. Furthermore, a higher parasite growth inhibition rate was achieved with combination of proteins suggesting the significance of multiple parasite ligands interaction with the same erythrocyte receptor during invasion process. These results will be helpful in understanding P. vivax biology and developing the therapeutics for vivax malaria.

The ATP-dependent ClpQY system is a prokaryotic proteasome-like machinery in the mitochondrion of malaria parasite, Plasmodium. This protease system is identified as a validated target for malaria intervention. Here we have identified drug-like small chemical compounds, pyrimidotriazine derivatives, as specific inhibitors of P. falciparum ClpQ (PfClpQ) having effective parasiticidal efficacies on asexual and liver stages. Screening of the ‘Malaria Box’ compound library, using a robust in vitro activity assay, identified a hit compound targeting the PfClpQ protease with IC50 at ~750nM. Further, Structure-Activity Relationship (SAR) analysis, using a small library of derivatives of the hit compound, identified three potential lead compounds, ICGEB-L1, ICGEB-L2 and ICGEB-L3, which inhibited enzyme activity with IC50 in 100-200nM ranges. The three lead compounds effectively inhibited in vitro asexual stage parasite growth in similar concentration ranges with ~100-fold selectivity over mammalian cell line; in addition, these compounds effectively inhibited in vitro growth and maturation of hepatocytic stage parasites with EC50 in low nano-molar ranges. These compounds were found to effectively block the development of trophozoite stages to schizont stage, concurrently arresting development and segregation of parasite mitochondria, in asexual as well as hepatocytic stages of the parasite. These morphological and developmental effects of the drugs on parasites mimic the data from genetic ablation of PfClpQ. Importantly, all three compounds showed significant in vivo anti-parasitic efficacies on blood stages of P. berghei mouse malaria model. Overall these results suggest that these PfClpQ targeting compounds are promising candidates to develop new anti-malarials.

Malaria, a deadly disease caused by pathogen Plasmodium, is a global problem. After discovery of Plasmodium as a causative agent of malaria, the understanding of the malaria biology using conventional techniques was at slower pace as compared to the variation in the parasite, e.g., drug resistance. In the era of omics technologies, e.g., proteomics, genomics, and metabolomics, newer strategies to combat this infection have evolved. Also, these different technologies have helped in better understanding of Plasmodium parasite biology in detail. Omics tools involving high-throughput technologies, automation, and data mining have helped in better understanding of the pathways and key proteins required in the life cycle of the parasite and hence pathogenesis of this disease in faster, reliable, and affordable mode. In the recent times, advance in the field of omics displayed high potential in accelerating malaria research to fight the diseases. This chapter will highlight the role of omics tools in deciphering mysteries of malaria parasite biology and their applications in diagnosis, treatment, and eradication of the disease.

In 2020, a novel strain of coronavirus (COVID-19) has led to a significant morbidity and mortality worldwide. As of the date of this writing, a total of 116 M cases has been diagnosed worldwide leading to 2.5 M deaths. The number of mortalities is directly correlated with the rise of innate immune cells (especially macrophages) in the lungs that secrete inflammatory cytokines (IL-1β and IL-6) leading to the development of "Cytokine Storm Syndrome" (CSS), multi-organ-failure and death. Given that currently the treatment of this condition is rare and release of effective vaccine might be months away, here, we review the plants and their pharmacologically active-compounds as potential phytopharmaceuticals for the virus induced inflammatory response. Experimental validation of the effectiveness of these natural compounds to prevent or reduce the cytokine storm might be beneficial as an adjunct treatment of SARS-CoV-2.

In late 2019, SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) infection started in Hubei province of China and now it has spread like a wildfire in almost all parts of the world except some. WHO named the disease caused by SARS-CoV-2 as COVID-19 (CoronaVirus Disease-2019). It is very intriguing to see a mild trend of infection in some countries which could be attributed to mitigation efforts, lockdown strategies, health infrastructure, demographics and cultural habits. However, the lower rate of infection and death rates in mostly developing countries, which are not placed at higher levels in terms of healthcare facilities, is a very surprising observation. To address this issue, we hypothesize that this lower rate of infection is majorly been observed in countries which have a higher transmission/prevalence of protozoan parasite borne disease, malaria. We compared the COVID-19 spread and malaria endemicity of 108 countries which have shown at least 200 cases of COVID-19 till 18th April 2020. We found that the number of COVID-19 cases per million population correlates negatively with the malaria endemicity of respective countries. The malaria free countries not only have higher density of COVID-19 infections but also the higher case fatality rates as compared to highly malaria endemic countries. We also postulate that this phenomenon is due to natural immune response against malaria infection, which is providing a heterologous protection against the virus. Unfortunately, there is no licensed vaccine against SARS-CoV-2 yet, but this information will be helpful in design of future strategies against fast spreading COVID-19 disease.

Cytoadherence-linked asexual gene 9 (Clag9), a conserved Plasmodium protein expressed during the asexual blood stages, is involved in the cytoadherence of infected red blood cells (RBCs) to the endothelial lining of blood vessels. Here, we show that Plasmodium falciparum Clag9 (PfClag9) is a component of the PfClag9-RhopH complex that is involved in merozoite binding to human erythrocytes. To characterize PfClag9, we expressed four fragments of PfClag9, encompassing the entire protein. Immunostaining analysis using anti-PfClag9 antibodies showed expression and localization of PfClag9 at the apical end of the merozoites. Mass spectrometric analysis of merozoite extracts after immunoprecipitation using anti-PfClag9 antibody identified P. falciparum rhoptry-associated protein 1 (PfRAP1), PfRAP2, PfRAP3, PfRhopH2, and PfRhopH3 as associated proteins. The identified rhoptry proteins were expressed, and their association with PfClag9 domains was assessed by using protein-protein interaction tools. We further showed that PfClag9 binds human RBCs by interacting with the glycophorin A-band 3 receptor-coreceptor complex. In agreement with its cellular localization, PfClag9 was strongly recognized by antibodies generated during natural infection. Mice immunized with the C-terminal domain of PfClag9 were partially protected against a subsequent challenge infection with Plasmodium berghei, further supporting a biological role of PfClag9 during natural infection. Taken together, these results provide direct evidence for the existence of a PfRhopH-Clag9 complex on the Plasmodium merozoite surface that binds to human RBCs.

Complement system is the first line of human defence against intruding pathogens and is recognized as a potentially useful therapeutic target. Human malaria parasite Plasmodium employs a series of intricate mechanisms that enables it to evade different arms of immune system, including the complement system. Here, we show the expression of a multi-domain Plasmodium Complement Control Protein 1, PfCCp1 at asexual blood stages and its binding affinity with C3b as well as C4b proteins of human complement cascade. Using a biochemical assay, we demonstrate that PfCCp1 binds with complement factors and inhibits complement activation. Active immunization of mice with PfCCp1 followed by challenge with Plasmodium berghei resulted in the loss of biphasic growth of parasites and early death in comparison to the control group. The study also showed a role of PfCCp1 in modulating Toll-like receptor (TLR)-mediated signalling and effector responses on antigen-presenting cells. PfCCp1 binds with dendritic cells that down-regulates the expression of signalling molecules and pro-inflammatory cytokines, thereby dampening the TLR2-mediated signalling; hence acting as a potent immuno-modulator. In summary, PfCCp1 appears to be an important component of malaria parasite directed immuno-modulating strategies that promote the adaptive fitness of pathogens in the host.

Elucidating the molecular mechanisms of the host-parasite interaction during red cell invasion by Plasmodium is important for developing newer antimalarial therapeutics. Recently, we have characterized a Plasmodium vivax tryptophan-rich antigen PvTRAg38, which is expressed by its merozoites, binds to host erythrocytes, and interferes with parasite growth. Interaction of this parasite ligand with the host erythrocyte occurs through its two regions present at amino acid positions 167–178 (P2) and 197–208 (P4). Each region recognizes its own erythrocyte receptor. Previously, we identified band 3 as the chymotrypsin-sensitive erythrocyte receptor for the P4 region, but the other receptor, binding to P2 region, remained unknown. Here, we have identified basigin as the second erythrocyte receptor for PvTRAg38, which is resistant to chymotrypsin. The specificity of interaction between PvTRAg38 and basigin was confirmed by direct interaction where basigin was specifically recognized by P2 and not by the P4 region of this parasite ligand. Interaction between P2 and basigin is stabilized through multiple amino acid residues, but Gly-171 and Leu-175 of P2 were more critical. These two amino acids were also critical for parasite growth. Synthetic peptides P2 and P4 of PvTRAg38 interfered with the parasite growth independently but had an additive effect if combined together indicating involvement of both the receptors during red cell invasion. In conclusion, PvTRAg38 binds to two erythrocyte receptors basigin and band 3 through P2 and P4 regions, respectively, to facilitate parasite growth. This advancement in our knowledge on molecular mechanisms of host-parasite interaction can be exploited to develop therapeutics against P. vivax malaria.

Autophagy has a direct and indirect role in health and disease. By definition, it is an exceedingly complex process which degrades and modifies damaged and surplus macromolecules in the cell. Autophagy has also been defined as a process which plays a role in dynamics of organelles using enzymes in lysosomes. Malaria parasite, i.e. Plasmodium, is a deadly parasite which encounters various conditions during its life cycle, ranging from temperature fluctuations to drug pressures. Role of autophagy in coping with these changes in Plasmodium is not well defined; however, there is growing evidence for role of this mechanism in life cycle of the parasite. This chapter highlights the link between different homologues of ATG protein present in Plasmodium and explores the mechanisms underlying these connections and their implications for cell physiology and survival of the parasite.

Malaria remains a global threat with millions of deaths annually. Emergence of parasite strains resistant to widely used antimalarials, including the artemisinin combination therapy (ACT), and the absence of an effective vaccine makes treatment of malaria difficult than ever before. The need of the hour is to re-evaluate the chemotherapeutic approach and to identify new drug targets and develop new pharmacophores against the parasite. An important approach for antimalarial drug discovery is to understand critical metabolic pathways in the parasite which may help us to identify critical targets in the parasites and design specific inhibitors for these targets. Here, we have discussed proteins and pathways in different parasite organelles, i.e. apicoplast, mitochondrial and food vacuole, which have been suggested as potential drug targets; these unique parasite proteins can be targeted to develop new and novel antimalarials. In addition, we have also discussed several antimalarial projects currently under different stages of drug development pipeline. These promising antimalarial compounds have the potential to overcome multidrug resistance. Ongoing global efforts to develop new antimalarials and to identify drug targets suggest a promising future on malaria elimination and eradication.

Tryptophan-rich antigens of malarial parasites interact with host molecules and play an important role in parasite survival. Merozoite expressed Plasmodium vivax tryptophan-rich antigen PvTRAg38 binds to human erythrocytes and facilitates parasite growth in a heterlologous Plasmodium falciparum culture system. Recently, we identified band 3 in human erythrocytes as one of its receptors, although the receptor-ligand binding mechanisms remain unknown. In the present study, using synthetic mutated peptides of PvTRAg38, we show that multiple amino acid residues of its 12 amino acid domain (KWVQWKNDKIRS) at position 197-208 interact with three different ectodomains of band 3 receptor on human erythrocytes. Our findings may help in the design of new therapeutic approaches for malaria.

Clearance of non-infected red blood cells (nRBCs) is one of the main components of anemia associated with Plasmodium vivax malaria. Recently, we have shown that anemic patients with P. vivax infection had elevated levels of anti-RBCs antibodies, which could enhance in vitro phagocytosis of nRBCs and decrease their deformability. Using immunoproteomics, here we characterized erythrocytic antigens that are differentially recognized by autoantibodies from anemic and non-anemic patients with acute vivax malaria. Protein spots exclusively recognized by anemic P. vivax-infected patients were identified by mass spectrometry revealing band 3 and spectrin as the main targets. To confirm this finding, antibody responses against these specific proteins were assessed by ELISA. In addition, an inverse association between hemoglobin and anti-band 3 or anti-spectrin antibodies levels was found. Anemic patients had higher levels of IgG against both band 3 and spectrin than the non-anemic ones. To determine if these autoantibodies were elicited because of molecular mimicry, we used in silico analysis and identified P. vivax proteins that share homology with human RBC proteins such as spectrin, suggesting that infection drives autoimmune responses. These findings suggest that band 3 and spectrin are potential targets of autoantibodies that may be relevant for P. vivax malaria-associated anemia.

Complement system is the first line of human defence against intruding pathogens and is recognized as a potentially useful therapeutic target. Human malaria parasite

Plasmodium falciparum merozoite surface protein 3 (MSP3) is an abundantly expressed secreted merozoite surface protein and a leading malaria vaccine candidate antigen. However, it is unclear how MSP3 is retained on the surface of merozoites without a glycosylphosphatidylinositol (GPI) anchor or a transmembrane domain. In the present study, we identified an MSP3-associated network on the Plasmodium merozoite surface by immunoprecipitation of Plasmodium merozoite lysate using antibody to the N terminus of MSP3 (anti-MSP3N) followed by mass spectrometry analysis. The results suggested the association of MSP3 with other merozoite surface proteins: MSP1, MSP6, MSP7, RAP2, and SERA5. Protein-protein interaction studies by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis showed that MSP3 complex consists of MSP1, MSP6, and MSP7 proteins. Immunological characterization of MSP3 revealed that MSP3N is strongly recognized by hyperimmune serum from African and Asian populations. Furthermore, we demonstrate that human antibodies, affinity purified against recombinant MSP3N (rMSP3N), promote opsonic phagocytosis of merozoites in cooperation with monocytes. At nonphysiological concentrations, anti-MSP3N antibodies inhibited the growth of P. falciparum in vitro Together, the data suggest that MSP3 and especially its N-terminal region containing known B/T cell epitopes are targets of naturally acquired immunity against malaria and also comprise an important candidate for a multisubunit malaria vaccine.

The human malaria parasite, Plasmodium falciparum, takes up numerous host cytosolic components and exogenous nutrients through endocytosis during the intra-erythrocytic stages. Eps15 homology domain-containing proteins (EHDs) are conserved NTPases, which are implicated in membrane remodeling and regulation of specific endocytic transport steps in eukaryotic cells. In the present study, we have characterized the dynamin-like C-terminal Eps15 homology domain containing protein of P. falciparum (PfEHD). Using a GFP-targeting approach, we studied localization and trafficking of PfEHD in the parasite. The PfEHD-GFP fusion protein was found to be a membrane bound protein that associates with vesicular network in the parasite. Time-lapse microscopy studies showed that these vesicles originate at parasite plasma membrane, migrate through the parasite cytosol and culminate into a large multi-vesicular like structure near the food-vacuole. Co-staining of food vacuole membrane showed that the multi-vesicular structure is juxtaposed but outside the food vacuole. Labeling of parasites with neutral lipid specific dye, Nile Red, showed that this large structure is neutral lipid storage site in the parasites. Proteomic analysis identified endocytosis modulators as PfEHD associated proteins in the parasites. Treatment of parasites with endocytosis inhibitors obstructed the development of PfEHD-labeled vesicles and blocked their targeting to the lipid storage site. Overall, our data suggests that the PfEHD is involved in endocytosis and plays a role in the generation of endocytic vesicles at the parasite plasma membrane, that are subsequently targeted to the neutral lipid generation/storage site localized near the food vacuole.

The prokaryotic ATP-dependent ClpP protease, localized in the relict plastid of malaria parasite, represents a potential drug target. In the present study, we utilized in silico structure-based screening and medicinal chemistry approaches to identify a novel pyrimidine series of compounds inhibiting P. falciparum ClpP protease activity and evaluated their antiparasitic activities. Structure-activity relationship indicated that morpholine moiety at C2, an aromatic substitution at N3 and a 4-oxo moiety on the pyrimidine are important for potent inhibition of ClpP enzyme along with antiparasiticidal activity. Compound 33 exhibited potent antiparasitic activity (EC₅₀ 9.0±0.2μM), a 9-fold improvement over the antiparasitic activity of the hit molecule 6. Treatment of blood stage P. falciparum cultures with compound 33 caused morphological and developmental abnormalities in the parasites; further, compound 33 treatment hindered apicoplast development indicating the targeting of apicoplast.

Background The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface. Methods Recombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-165 were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte binding assay with 6-cys proteins were undertaken to find out their possible role in host-parasite infection and seropositivity was assessed using Indian and Liberian sera. Results Immunoprecipitation of parasite-derived polypeptides, followed by LC–MS/MS analysis, identified a large Pfs38 complex comprising of 6-cys proteins: Pfs41, Pfs38, Pfs12 and other merozoite surface proteins: GLURP, SERA5 and MSP-1. The existence of such a complex was further corroborated by several protein–protein interaction tools, co-localization and co-sedimentation analysis. Pfs38 protein of Pfs38 complex binds to host red blood cells (RBCs) directly via glycophorin A as a receptor. Seroprevalence analysis showed that of the six antigens, prevalence varied from 40 to 99%, being generally highest for MSP-165 and GLURP proteins. Conclusions Together the data show the presence of a large Pfs38 protein-associated complex on the parasite surface which is involved in RBC binding. These results highlight the complex molecular interactions among the P. falciparum merozoite surface proteins and advocate the development of a multi-sub-unit malaria vaccine based on some of these protein complexes on merozoite surface. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1716-0) contains supplementary material, which is available to authorized users.

Elucidation of molecular mechanisms of receptor-ligand biology during host-parasite interaction helps in developing therapeutic targets. Several Pv-fam-a family proteins of Plasmodium vivax bind to host erythrocytes but their erythrocyte receptors remains to be explored. Here, we show that three merozoite proteins (PvTRAg36, PvATRAg74, and PvTRAg38) of this family interact with Band 3 on human erythrocytes through its three exofacial loops (loop 1, loop 3, and loop 6). These parasite proteins also interfered with the parasite growth in in-vitro, and the inhibition rate seems to be associated with their binding affinity to Band 3. This redundancy in receptor-ligand interaction could be one of the probable mechanism parasite utilizes to invade the host erythrocyte more efficiently.

Plasmodium tryptophan-rich proteins are involved in host-parasite interaction and thus potential drug/vaccine targets. Recently, we have described several P. vivax tryptophan-rich antigens (PvTRAgs), including merozoite expressed PvTRAg38, from this noncultivable human malaria parasite. PvTRAg38 is highly immunogenic in humans and binds to host erythrocytes, and this binding is inhibited by the patient sera. This binding is also affected if host erythrocytes were pretreated with chymotrypsin. Here, Band 3 has been identified as the chymotrypsin-sensitive erythrocyte receptor for this parasite protein. Interaction of PvTRAg38 with Band 3 has been mapped to its three different ectodomains (loops 1, 3, and 6) exposed at the surface of the erythrocyte. The binding region of PvTRAg38 to Band3 has been mapped to its sequence, KWVQWKNDKIRSWLSSEW, present at amino acid positions 197-214. The recombinant PvTRAg38 was able to inhibit the parasite growth in in vitro Plasmodium falciparum culture probably by competing with the ligand(s) of this heterologous parasite for the erythrocyte Band 3 receptor. In conclusion, the host-parasite interaction at the molecular level is much more complicated than known so far and should be considered during the development of anti-malarial therapeutics.

Intercellular adhesion molecules (ICAMs) belong to the immunoglobulin superfamily and participate in diverse cellular processes including host-pathogen interactions. ICAM-1 is expressed on various cell types including macrophages, whereas ICAM-4 is restricted to red blood cells. Here we report the identification of an 11-kDa synthetic protein, M5, that binds to human ICAM-1 and ICAM-4, as shown by in vitro interaction studies, surface plasmon resonance and immunolocalization. M5 greatly inhibits the invasion of macrophages and erythrocytes by Mycobacterium tuberculosis and Plasmodium falciparum, respectively. Pharmacological and siRNA-mediated inhibition of ICAM-1 expression also results in reduced M. tuberculosis invasion of macrophages. ICAM-4 binds to P. falciparum merozoites, and the addition of recombinant ICAM-4 to parasite cultures blocks invasion of erythrocytes by newly released merozoites. Our results indicate that ICAM-1 and ICAM-4 play roles in host cell invasion by M. tuberculosis and P. falciparum, respectively, either as receptors or as crucial accessory molecules.

The Central Himalayan region experiences severe ecological problems as a result of continuous deforestation, which threatens the subsistence population of the region. In the present study, changes in the forest cover of a catchment over a period of 16 years have been quantified. Baliya catchment (7783 ha) contains 2865·47 ha of different types of forest, i.e. sal ( Shorea robusta ), mixed sal, pine ( Pinus roxburghii ), pine-mixed broadleaf, mixed oak ( Quercus spp.), cupressus ( Cupressus torulosa ). The process of fragmentation has been studied through changes in forest area and biomass. There has been a sharp decline in biomass, although only a marginal reduction in forest area (3·5%) has occurred. Decline in the stocking density has also been substantial. Forest area with higher crown cover (>60%) declined from 736·32 ha to 292 ha during 1973–1989, indicating the intensity of human interference.

Summary The ATP-dependent ClpQY system is a prokaryotic proteasome-like multi-subunit machinery localized in the mitochondrion of malaria parasite. The ClpQY machinery consists of ClpQ threonine pro- tease and ClpY ATPase. In the present study, we have assessed cellular effects of transient interference of PfClpQ protease activity in Plas- modium falciparum using a trans-dominant nega- tive approach combined with FKBP degradation domain system. A proteolytically inactive mutant PfClpQ protein (PfClpQ(mut)) fused with FKBP degradation domain was expressed in parasites, which gets stabilized by Shield1 drug treatment. We show that the inactive PfClpQ(mut) interacts with wild-type PfClpQ and associates within multi- subunit complex in the parasite. Stabilization of the PfClpQ(mut) and its association in the protease machinery caused dominant negative effect in the transgenic parasites, which disrupted the growth cycle of asexual blood stage parasites. The mito- chondria in these parasites showed abnormal mor- phology, these mitochondria were not able to grow and divide in the parasite. We further show that the dominant negative effect of PfClpQ(mut) disrupted transcription of mitochondrial genome encoded genes, which in turn blocked normal development and functioning of the mitochondria.

Malaria remains a major parasitic disease in the tropical and sub-tropical countries mainly due to dramatic increase in parasite lines resistant to commonly used anti-malarials. Characterization of novel metabolic pathways in the parasites and understanding their functional role is a prerequisite to design new anti-malarial strategies. Parasite proteases play key role in growth and differentiation of all the developmental stages across the parasite life cycle and present the most promising targets to develop new drugs against malaria. In Plasmodium falciparum genome database a total of 123 proteases are identified; these proteases belong to five different clans: Cysteine, Aspartic, Serine, Metallo-, and Threonine. Some of the most studied parasite proteases are those that are functional in the asexual blood stage cycle. Starting with the processing of key parasite ligand in merozoite, the invasive form of blood stage parasite, degradation of host hemoglobin in food-vacuole, regulation of levels of key metabolic pathways in cytosol and cellular organelles, degradation of misfolded and unused proteins, and rupture of host membrane for egress of daughter merozoites is mediated by these proteases. Here we discuss roles of some of the parasite proteases involved in various steps of the parasite intra-erythrocytic cycle.

SUMMARY The dwindling forests of the Central Himalaya are suffering from serious losses, threatening the subsistence population of the region. This study analyses the rate of consumption of fodder and firewood in a Central Himalayan catchment and estimates the carrying capacity of the catchment for these resources. Estimates indicate that the carrying capacity of the natural ecosystems and agroecosystems, in terms of availability of fodder and fuelwood resources, has already been exceeded.

The ATP-dependent caseinolytic protease, ClpP, is highly conserved in bacteria and in the organelles of different organisms. In cyanobacteria, plant plastids, and the apicoplast of the genus Plasmodium, a noncatalytic paralog of ClpP, termed ClpR, has been identified. ClpRs are found to form heterocomplexes with ClpP resulting in a ClpRP tetradecameric cylinder having less than 14 catalytic triads. The exact role of ClpR in such a complex remains enigmatic. Here we describe the x-ray crystal structure of ClpR protein heptamer from Plasmodium falciparum (PfClpR). This is the first structure of a ClpR protein. The structure shows that the PfClpR monomer adopts a fold similar to that of ClpP, but has a unique motif, which we named the R-motif, forming a β turn located near the inactive catalytic triad in a three-dimensional space. The PfClpR heptamer exhibits a more open and flat ring than a ClpP heptamer. PfClpR was localized in the P. falciparum apicoplast as is the case of PfClpP. However, biochemical and structural data suggest that, contrary to what has been observed in other organisms, PfClpP and PfClpR do not form a stable heterocomplex in the apicoplast of P. falciparum.

The ATP-dependent ClpQY system is a prokaryotic proteasome-like multi-subunit machinery localized in the mitochondrion of malaria parasite. The ClpQY machinery consists of ClpQ threonine protease and ClpY ATPase. In the present study, we have assessed cellular effects of transient interference of PfClpQ protease activity in Plasmodium falciparum using a trans-dominant negative approach combined with FKBP degradation domain system. A proteolytically inactive mutant PfClpQ protein [PfClpQ(mut)] fused with FKBP degradation domain was expressed in parasites, which gets stabilized by Shield1 drug treatment. We show that the inactive PfClpQ(mut) interacts with wild-type PfClpQ and associates within multi-subunit complex in the parasite. Stabilization of the PfClpQ(mut) and its association in the protease machinery caused dominant negative effect in the transgenic parasites, which disrupted the growth cycle of asexual blood stage parasites. The mitochondria in these parasites showed abnormal morphology, these mitochondria were not able to grow and divide in the parasite. We further show that the dominant negative effect of PfClpQ(mut) disrupted transcription of mitochondrial genome encoded genes, which in turn blocked normal development and functioning of the mitochondria.

The prokaryotic ATP-dependent protease machineries such as CIpQY and CIpAP in the malaria parasite may represent potential drug targets. In the present study, we show that the orthologue of cyanobacterial CIpP protease in Plasmodium falciparum (PfCIpP) is expressed in the asexual blood stages and possesses serine protease activity. The PfCIpP was localized in the apicoplast using a GFP-targeting approach, immunoelectron microscopy and by immunofluorescence assays. A set of cell permeable β-lactones, which specifically bind with the active site of prokaryotic ClpP, were screened using an in vitro protease assay of PfClpP. A PfClpP-specific protease inhibitor was identified in the screen, labelled as U1-lactone. In vitro growth of the asexual stage parasites was significantly inhibited by U1-lactone treatment. The U1-treated parasites showed developmental arrest at the late-schizont stage. We further show that the U1-lactone treatment resulted in formation of abnormal apicoplasts which were not able to grow and segregate in the parasite progeny; these effects were also evident by blockage in the replication of the apicoplast genome. Overall, our data show that the PfClpP protease has confirmed localization in the apicoplast and it plays important role in development of functional apicoplasts.

The prokaryotic ATP-dependent protease machineries such as ClpQY and ClpAP in the malaria parasite may represent potential drug targets. In the present study, we show that the orthologue of cyanobacterial ClpP protease in Plasmodium falciparum (PfClpP) is expressed in the asexual blood stages and possesses serine protease activity. The PfClpP was localized in the apicoplast using a GFP-targeting approach, immunoelectron microscopy and by immunofluorescence assays. A set of cell permeable β-lactones, which specifically bind with the active site of prokaryotic ClpP, were screened using an in vitro protease assay of PfClpP. A PfClpP-specific protease inhibitor was identified in the screen, labelled as U1-lactone. In vitro growth of the asexual stage parasites was significantly inhibited by U1-lactone treatment. The U1-treated parasites showed developmental arrest at the late-schizont stage. We further show that the U1-lactone treatment resulted in formation of abnormal apicoplasts which were not able to grow and segregate in the parasite progeny; these effects were also evident by blockage in the replication of the apicoplast genome. Overall, our data show that the PfClpP protease has confirmed localization in the apicoplast and it plays important role in development of functional apicoplasts.

A novel class of phthalimides functionalized with privileged scaffolds was designed, synthesized and evaluated as potential inhibitors of plasmepsin 2 (Ki: 0.99 ± 0.1 μM for 6u) and plasmepsin 4 (Ki: 3.3 ± 0.3 μM for 6t), enzymes found in the digestive vacuole of the plasmodium parasite and considered as crucial drug targets. Three compounds were identified as potential candidates for further development. The listed compounds were also assayed for their antimalarial efficacy against chloroquine (CQ) sensitive strain (3D7) of Plasmodium falciparum. Assay of twenty seven hydroxyethylamine derivatives revealed four (5e, 6j, 6o and 6s) as strongly active, which were further evaluated against CQ resistant strain (7GB) of P. falciparum. Compound 5e possessing the piperidinopiperidine moiety exhibited promising antimalarial activity with an IC50 of 1.16 ± 0.04 μM. Further, compounds 5e, 6j, 6o and 6s exhibited low cytotoxic effect on MCF-7 cell line. Compound 6s possessing C 2 symmetry was identified as the least cytotoxic with significant antimalarial activity (IC50: 1.30 ± 0.03 μM). The combined presence of hydroxyethylamine and cyclic amines (piperazines and piperidines) was observed as crucial for the activity. The current studies suggest that hydroxyethylamine based molecules act as potent antimalarial agent and may be helpful in drug development.

A regulated protein turnover machinery in the cell is essential for effective cellular homeostasis; any interference with this system induces cellular stress and alters the normal functioning of proteins important for cell survival. In this study, we show that persistent cellular stress and organelle dysfunction because of disruption of cellular homeostasis in human malaria parasite Plasmodium falciparum, leads to apoptosis-like cell death. Quantitative global proteomic analysis of the stressed parasites before onset of cell death, showed upregulation of a number of proteins involved in cellular homeostasis; protein network analyses identified upregulated metabolic pathways that may be associated with stress tolerance and pro-survival mechanism. However, persistent stress on parasites cause structural abnormalities in endoplasmic reticulum and mitochondria, subsequently a cascade of reactions are initiated in parasites including rise in cytosolic calcium levels, loss of mitochondrial membrane potential and activation of VAD-FMK-binding proteases. We further show that activation of VAD-FMK-binding proteases in the parasites leads to degradation of phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), a known target of metacaspases, as well as degradation of other components of spliceosomal complex. Loss of spliceosomal machinery impairs the mRNA splicing, leading to accumulation of unprocessed RNAs in the parasite and thus dysregulate vital cellular functions, which in turn leads to execution of apoptosis-like cell death. Our results establish one of the possible mechanisms of instigation of cell death by organelle stress in Plasmodium.

The ATP-dependent ClpQY protease system in Plasmodium falciparum is a prokaryotic machinery in the parasite. In the present study, we have identified the complete ClpQY system in P. falciparum and elucidated its functional importance in survival and growth of asexual stage parasites. We characterized the interaction of P. falciparum ClpQ protease (PfClpQ) and PfClpY ATPase components, and showed that a short stretch of residues at the C terminus of PfClpY has an important role in this interaction; a synthetic peptide corresponding to this region antagonizes this interaction and interferes with the functioning of this machinery in the parasite. Disruption of ClpQY function by this peptide caused hindrance in the parasite growth and maturation of asexual stages of parasites. Detailed analyses of cellular effects in these parasites showed features of apoptosis-like cell death. The peptide-treated parasites showed mitochondrial dysfunction and loss of mitochondrial membrane potential. Dysfunctioning of mitochondria initiated a cascade of reactions in parasites, including activation of VAD–FMK-binding proteases and nucleases, which resulted in apoptosis-like cell death. These results show functional importance of mitochondrial proteases in the parasite and involvement of mitochondria in programmed cell death in the malaria parasites.