Hemozoin (beta-hematin) formation inhibitors; A promising target for the development of new antimalarials: Current update and A future prospect

Background: Malaria is responsible for a social and an economic burden in most low-income malaria-affected countries. Thus, newer antimalarials are needed to tackle morbidities and mortalities associated with the drug-resistant malarial strains. Haemoglobin digestion inside the food vacuole of malarial parasite would lead to producing redox-active and toxic-free heme. The detoxification process adopted by Plasmodium sp. would give rise to hemozoin (Hz) (beta-hematin) formation. Targeting the pathway of hemozoin formation is considered as a validated target for the discovery of newer antimalarials. Objective: This study aims to collect detailed information about aspects of hemozoin (Hz) (beta-hematin) inhibitors. Methods: A systemic search has been carried out using PubMed, Google Scholar, CNKI, etc., for relevant studies having the keyword, ' hemozoin or beta-hematin' for almost the last 2 decades (2000-2021). Results: This mini-review tries to summarize all the recent advancements made for the developments of synthetic, natural isolated phytoconstituents and plant extracts inhibiting the hemozoin (beta-hematin) formation. Conclusion: thus, would act as promising antimalarial candidates in near future.

An essential vesicular-trafficking phospholipase mediates neutral lipid synthesis and contributes to hemozoin formation in Plasmodium falciparum

Background Plasmodium falciparum is the pathogen responsible for the most devastating form of human malaria. As it replicates asexually in the erythrocytes of its human host, the parasite feeds on haemoglobin uptaken from these cells. Heme, a toxic by-product of haemoglobin utilization by the parasite, is neutralized into inert hemozoin in the food vacuole of the parasite. Lipid homeostasis and phospholipid metabolism are crucial for this process, as well as for the parasite’s survival and propagation within the host. P. falciparum harbours a uniquely large family of phospholipases, which are suggested to play key roles in lipid metabolism and utilization. Results Here, we show that one of the parasite phospholipase (P. falciparum lysophospholipase, PfLPL1) plays an essential role in lipid homeostasis linked with the haemoglobin degradation and heme conversion pathway. Fluorescence tagging showed that the PfLPL1 in infected blood cells localizes to dynamic vesicular structures that traffic from the host-parasite interface at the parasite periphery, through the cytosol, to get incorporated into a large vesicular lipid rich body next to the food-vacuole. PfLPL1 is shown to harbour enzymatic activity to catabolize phospholipids, and its transient downregulation in the parasite caused a significant reduction of neutral lipids in the food vacuole-associated lipid bodies. This hindered the conversion of heme, originating from host haemoglobin, into the hemozoin, and disrupted the parasite development cycle and parasite growth. Detailed lipidomic analyses of inducible knock-down parasites deciphered the functional role of PfLPL1 in generation of neutral lipid through recycling of phospholipids. Further, exogenous fatty-acids were able to complement downregulation of PfLPL1 to rescue the parasite growth as well as restore hemozoin levels. Conclusions We found that the transient downregulation of PfLPL1 in the parasite disrupted lipid homeostasis and caused a reduction in neutral lipids essentially required for heme to hemozoin conversion. Our study suggests a crucial link between phospholipid catabolism and generation of neutral lipids (TAGs) with the host haemoglobin degradation pathway.

Involvement of translocon complex in hemoglobin import from infected erythrocyte cytoplasm into the Plasmodium parasite

Haemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in great detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP), a protein localized in the parasitophorous vacuole, parasite food vacuole and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP-GFP fusion protein from a transgenic line using anti-GFP antibody and of Plasmodium parasite extract using anti-human Hb antibodies respectively, showed the association of PfHDP/Hb with each other as well as with the members of PTEX translocon complex. Some of these associations such as PfHDP/Hb and PfHDP/Pfexp-2 interactions were confirmed by in vitro protein-protein interaction tools. To know the roles of PfHDP and translocon complex in Hb import into the parasites, we next studied the Hb uptake by the parasite in PfHDP knock-down line using the GlmS ribozyme strategy. PfHDP knock-down significantly reduced the Hb uptake in these parasites in comparison to the wild type parasites. Further, the transient knock-down of one of the members of the translocon complex; PfHSP101 showed considerable reduction in Hb uptake. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Together, we implicate the translocon complex in the trafficking of PfHDP/Hb complex in the parasite and suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.

Calcium in the Backstage of Malaria Parasite Biology

The calcium ion (Ca2+) is a ubiquitous second messenger involved in key biological processes in prokaryotes and eukaryotes. In Plasmodium species, Ca2+ signaling plays a central role in the parasite life cycle. It has been associated with parasite development, fertilization, locomotion, and host cell infection. Despite the lack of a canonical inositol-1,4,5-triphosphate receptor gene in the Plasmodium genome, pharmacological evidence indicates that inositol-1,4,5-triphosphate triggers Ca2+ mobilization from the endoplasmic reticulum. Other structures such as acidocalcisomes, food vacuole and mitochondria are proposed to act as supplementary intracellular Ca2+ reservoirs. Several Ca2+-binding proteins (CaBPs) trigger downstream signaling. Other proteins with no EF-hand motifs, but apparently involved with CaBPs, are depicted as playing an important role in the erythrocyte invasion and egress. It is also proposed that a cross-talk among kinases, which are not members of the family of Ca2+-dependent protein kinases, such as protein kinases G, A and B, play additional roles mediated indirectly by Ca2+ regulation. This statement may be extended for proteins directly related to invasion or egress, such as SUB1, ERC, IMC1I, IMC1g, GAP45 and EBA175. In this review, we update our understanding of aspects of Ca2+-mediated signaling correlated to the developmental stages of the malaria parasite life cycle.

REVIEW: PERAN NANOPARTIKEL DALAM MENGHAMBAT PERTUMBUHAN PARASIT Plasmodium PENYEBAB MALARIA

Review: The Role of Nanoparticles in Inhibiting the Growth of the Plasmodium Parasite Causing Malarial Disease Malaria is a health problem in Indonesia with the most cases in eastern parts of Indonesia. This study provides an overview of the potential of nanoparticles in inhibiting malaria vectors and the growth of Plasmodium parasites that causes malaria based on the latest literature as reference materials and future research ideas. Nanoparticle can be synthesized using three methods i.e. physical, chemical and biological synthesis. The use of nanoparticles with biological method is highly recommended because they are practicable, environmentally friendly, non-toxic, and easy to reproduce compared to physico-chemically synthesized nanoparticles. Nanoparticles synthesized from several plants can inhibit the growth of Plasmodium parasites with IC50 3–78 g mL–1. This activity is classified as high to moderate in inhibiting the growth of the Plasmodium parasite that causes malaria. The mechanism of inhibition of Plasmodium growth is by increasing the pH of food vacuole due to the reaction of nanoparticles with Ferriprotoporphyrin IX. The high pH in the food vacuole will interfere with metabolic activity by inhibiting the activity of aspartate and cysteine ??protease enzymes so that the parasites will die. Malaria merupakan masalah kesehatan yang dihadapi Indonesia khususnya di beberapa wilayah timur Indonesia. Kajian ini memberikan gambaran potensi nanopartikel dalam menghambat vektor malaria maupun pertumbuhan parasit Plasmodium penyebab malaria berdasarkan literatur terbaru sebagai bahan acuan maupun ide-ide penelitian di masa mendatang. Nanopartikel dapat disintesis menggunakan tiga metode yaitu fisika, kimia dan biologi. Penggunaan nanopartikel dengan metode biologi sangat direkomendasikan karena lebih mudah diterapkan, ramah lingkungan, bersifat non-toksik, dan mudah diperbanyak dibandingkan dengan nanopartikel yang disintensis dari fisiko-kimia. Nanopartikel yang disintesis dari beberapa tanaman dapat menghambat pertumbuhan parasit Plasmodium dengan IC50 3–78 g mL–1. Aktivitas ini tergolong tinggi hingga sedang dalam menghambat pertumbuhan parasit Plasmodium penyebab malaria. Mekanisme penghambatan pertumbuhan Plasmodium dengan cara meningkatkan pH vakuola makanan akibat reaksi nanopartikel dengan feriprotoporpirin IX. Tingginya pH pada vakuola makanan akan mengganggu aktivitas metabolisme dengan cara menghambat aktivitas enzim aspartat dan sistein protease sehingga parasit akan mati.

GlmS mediated knock-down of a phospholipase expedite alternate pathway to generate phosphocholine required for phosphatidylcholine synthesis in Plasmodium falciparum

Phospholipid synthesis is crucial for membrane proliferation in malaria parasites during the entire cycle in the host cell. The major phospholipid of parasite membranes, phosphatidylcholine (PC), is mainly synthesized through the Kennedy pathway. The phosphocholine required for this synthetic pathway is generated by phosphorylation of choline derived from catabolism of the lyso-phosphatidylcholine (LPC) scavenged from the host milieu. Here we have characterized a Plasmodium falciparum lysophospholipase (PfLPL20) which showed enzymatic activity on LPC substrate to generate choline. Using GFP- targeting approach, PfLPL20 was localized in vesicular structures associated with the neutral lipid storage bodies present juxtaposed to the food-vacuole. The C-terminal tagged glmS mediated inducible knock-down of PfLPL20 caused transient hindrance in the parasite development, however, the parasites were able to multiply efficiently, suggesting that PfLPL20 is not essential for the parasite. However, in PfLPL20 depleted parasites, transcript levels of enzyme of SDPM pathway (Serine Decarboxylase-Phosphoethanolamine Methyltransferase) were altered along with upregulation of phosphocholine and SAM levels; these results show upregulation of alternate pathway to generate the phosphocholine required for PC synthesis through the Kennedy pathway. Our study highlights presence of alternate pathways for lipid homeostasis/membrane-biogenesis in the parasite; these data could be useful to design future therapeutic approaches targeting phospholipid metabolism in the parasite.

Biosynthetic heme of malaria parasite induces cerebral pathogenesis by regulating hemozoin formation and griseofulvin can prevent cerebral malaria

ABSTRACTHeme-biosynthetic pathway of malaria parasite is dispensable for asexual stages, but essential for sexual and liver stages. Despite having backup mechanisms to acquire hemoglobin-heme, pathway intermediates and/or enzymes from the host, asexual parasites express heme pathway enzymes and synthesize heme. Here we show heme synthesized in asexual stages promotes cerebral pathogenesis by enhancing hemozoin formation. Hemozoin is a parasite molecule associated with inflammation, aberrant host-immune responses, disease severity and cerebral pathogenesis. The heme pathway knockout parasites synthesize less hemozoin, and mice infected with knockout parasites are completely protected from cerebral malaria and death due to anaemia is delayed. Biosynthetic heme regulates food vacuole integrity and the food vacuoles from knockout parasites are compromised in pH, lipid unsaturation and proteins, essential for hemozoin formation. Targeting parasite heme synthesis by griseofulvin - a FDA-approved drug, prevents cerebral malaria in mice and provides a new adjunct therapeutic option for cerebral and severe malaria.

Plasmodium falciparum Atg18 localizes to the food vacuole via interaction with the multi-drug resistance protein 1 and phosphatidylinositol 3-phosphate

Autophagy, a lysosome-dependent degradative process, does not appear to be a major degradative process in malaria parasites and has a limited repertoire of genes. To better understand the autophagy process, we investigated Plasmodiumfalciparum Atg18 (PfAtg18), a PROPPIN family protein, whose members like S. cerevisiae Atg18 (ScAtg18) and human WIPI2 bind PI3P and play an essential role in autophagosome formation. Wild type and mutant PfAtg18 were expressed in P. falciparum and assessed for localization, the effect of various inhibitors and antimalarials on PfAtg18 localization, and identification of PfAtg18-interacting proteins. PfAtg18 is expressed in asexual erythrocytic stages and localized to the food vacuole, which was also observed with other Plasmodium Atg18 proteins, indicating that food vacuole localization is likely a shared feature. Interaction of PfAtg18 with the food vacuole-associated PI3P is essential for localization, as PfAtg18 mutants of PI3P-binding motifs neither bound PI3P nor localized to the food vacuole. Interestingly, wild type ScAtg18 interacted with PI3P, but its expression in P. falciparum showed complete cytoplasmic localization, indicating additional requirement for food vacuole localization. The food vacuole multi-drug resistance protein 1 (MDR1) was consistently identified in the immunoprecipitates of PfAtg18 and P. berghei Atg18, and also interacted with PfAtg18. In contrast to PfAtg18, ScAtg18 did not interact with MDR1, which, in addition to PI3P, could play a critical role in localization of PfAtg18. Chloroquine and amodiaquine caused cytoplasmic localization of PfAtg18, suggesting that these target PfAtg18 transport pathway. Thus, PI3P and MDR1 are critical mediators of PfAtg18 localization.

CARD-FISH in the Sequencing Era: Opening a New Universe of Protistan Ecology

Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on in situ abundance, feeding modes, grazing preferences, and growth rates of specific lineages can be reliably obtained only using microscopy-based molecular methods, such as Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that Paraphysomonas or Spumella-like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages (e.g., CRY1), cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo- and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists.

Plasmodium falciparum Atg18 localization to the food vacuole is phosphatidylinositol 3- phosphate-dependent and quinoline-sensitive

Malaria parasites exhibit atypical, but essential, autophagy process, which is also associated with resistance to chloroquine and artemisinin. To better understand the autophagy process and its association with drug resistance, we investigated P. falciparum Atg18 (PfAtg18), a PROPPIN family protein, whose members like S. cerevisiae Atg18 and human WIPI2 are essential for autophagy. PfAtg18 and its mutants were expressed in P. falciparum and assessed for localization and co-localization, the effect of various inhibitors and antimalarials on PfAtg18 localization, and to identify PfAtg18-interacting proteins. PfAtg18 is expressed in asexual erythrocytic stages and localized to the food vacuole, which was also observed with other Plasmodium Atg18 proteins, indicating that food vacuole localization is a conserved feature. Interaction of PfAtg18 with the food vacuole-associated phosphatidylinositol 3-phosphate (PI3P) is essential for localization, as PfAtg18 mutants of PI3P-binding motifs neither bound PI3P nor localized to the food vacuole. PfAtg18 also interacted with the food vacuole multi-drug resistance protein 1, which, in addition to PI3P, could play a major role in localization of PfAtg18. PfAtg18 interacted and co-localized with P. falciparum Atg8 at a peri-food vacuole site, which may be a site for generation of PfAtg8 puncta. PfAtg18 localization was greatly affected upon treatment with chloroquine and amodiaquine, suggesting that these quinolines target PfAtg18 or the proteins that might be involved in its localization. Food vacuole localization, altered localization upon treatment with chloroquine and amodiaquine, and interaction with the multi-drug resistance protein 1 present PfAtg18 as a potential link between autophagy and the associated drug resistance.