Conserved Plasmodium Protein (PF3D7_0406000) of Unknown Function: In-silico Analysis and Cellular Localization.

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.

A novel quinoline-appended chalcone derivative as potential Plasmodium falciparum gametocytocide.

BACKGROUND & OBJECTIVES: Malaria has remained a global health problem despite the effective control and treatment measures. In the backdrop of drug resistance, developing novel hybrid molecules targeting the sexual stages (gametocytes) of the human malaria parasite Plasmodium falciparum is of great significance. Recently, chalcone- based polyphenols have generated a great interest in the malaria research community worldwide due to their ease of synthesis and significant biological activity. The primary objective of this study was to investigate the interaction of a newly synthesized quinoline-appended chalcone derivative (ADMQ) with gametocyte specific proteins, Pfg 27 and Pfs 25 and explore its in vitro gametocytocidal potential. METHODS: The characterization of ligand-protein interactions at the atomistic level was done by a simulation strategy that combines molecular docking and molecular dynamics (MD) simulation in a coherent workflow. The X-ray crystal structure of Pfg 27 was retrieved from protein data bank and Pfs 25 was built using the Iterative Threading ASSembly Refinement (I-TASSER) server. The detailed interaction of both ADMQ and a known gametocytocidal agent, methylene blue (MB) (used as a positive control) with gametocyte proteins Pfg 27 and Pfs 25 was studied with a 50 ns explicit MD simulation. The ligand binding pose in terms of glide score, molecular mechanics-generalized born surface area (MM-GBSA) binding energies, protein-ligand root-mean-square-deviation (RMSD) and secondary structure elements (SSE) changes were analyzed accordingly. The direct effect of ADMQ on structural integrity of P. falciparum gametocytes was also examined using in vitro microscopy. RESULTS: The analogous Glide score and MM-GBSA free energy of binding indicated stable interactions for both ADMQ and MB harboured in the active site of targeted gametocyte proteins, Pfg 27 and Pfs 25, separately. Explicit MD simulation by Desmond software package indicated similar distinguishable conformational changes in the active site of target polypeptide chain due to the specific accommodation of ADMQ molecule. The simulation also manifested comparable mechanistic profile in terms of protein-ligand RMSD and changes in secondary structure elements (SSE). Further, ADMQ treatment was found to adversely affect the structural integrity of gametocytes, which resulted in appearance of vesicles protruding from the gametocytes. INTERPRETATION & CONCLUSION: The consolidated in silico molecular modeling and in vitro study described herein may give an insight into the interaction patterns of quinoline-chalcone hybrids with critical gametocyte proteins in the mosquito. This study will possibly pave the way for further exploration of similar heterocyclic quinoline-chalcone hybrids to open up new avenues in drug candidate development against P. falciparum gametocytes.

Molecular analysis of the cytoadherence phenotype of a Plasmodium falciparum field isolate that binds intercellular adhesion molecule-1.

The ability of Plasmodium falciparum-infected erythrocytes to adhere to endothelial receptors and sequester in diverse host organs is an important pathogenic mechanism. Cytoadherence is mediated by variant surface antigens, which are referred to as PfEMP-1 and are encoded by var genes. The extracellular regions of PfEMP-1 contain multiple conserved cysteine-rich domains that are referred to as Duffy-binding-like (DBL) domains. Here, we analyze the adhesive phenotype of an Indian P. falciparum field isolate, JDP8, which binds ICAM-1 but does not bind CD36. This is a unique cytoadherence phenotype because P. falciparum strains that bind ICAM-1 described thus far usually also bind CD36. Moreover, binding to both receptors is thought to be important for static adhesion under flow. The ICAM-1 binding population of P. falciparum JDP8 adheres to endothelial cells under flow despite poor binding to CD36. We have also identified an expressed var gene, JDP8Icvar, which mediates the ICAM-1 binding phenotype of JDP8. Expression of different regions of JDP8Icvar on the surface of COS-7 cells followed by binding assays demonstrates that the ICAM-1 binding domain maps to the DBL2betaC2 domain of JDP8Icvar. Sequence comparison with two previously identified ICAM-1 binding domains of PfEMP-1, which also map to DBLbetaC2 domains, suggests that diverse P. falciparum isolates use a structurally conserved domain to bind ICAM-1. It thus appears that functional constraints may place limits on the extent of sequence diversity in receptor-binding domains of PfEMP-1.

Bacterially expressed and refolded receptor binding domain of Plasmodium falciparum EBA-175 elicits invasion inhibitory antibodies.

Malaria parasites make specific receptor-ligand interactions to invade erythrocytes. A 175 kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) binds sialic acid residues on glycophorin A during invasion of human erythrocytes. The receptor-binding domain of EBA-175 lies in a conserved, amino-terminal, cysteine-rich region, region F2 of EBA-175 (PfF2), that is homologous to the binding domains of other erythrocyte binding proteins such as Plasmodium vivax Duffy binding protein. We have developed methods to produce recombinant PfF2 in its functional form. Recombinant PfF2 was expressed in Escherichia coli, purified from inclusion bodies, renatured by oxidative refolding and purified to homogeneity by ion-exchange and gel filtration chromatography. Refolded PfF2 has been characterized using biochemical and biophysical methods and shown to be pure, homogenous and functional in that it binds human erythrocytes with specificity. Immunization with refolded PfF2 yields high titre antibodies that efficiently inhibit P. falciparum invasion of erythrocytes in vitro. Importantly, antibodies raised against PfF2 block invasion by a P. falciparum field isolate that invades erythrocytes using multiple pathways. These observations support the development of recombinant PfF2 as a vaccine candidate for P. falciparum malaria.

Role of macrophages in experimental malaria: VII--studies on adoptive transfer of macrophages.

Adoptive transfer of purified macrophages harvested from normal, Plasmodium berghei infected and latent/cured mice and also macrophages exposed to parasites in vitro were carried out to see the role of macrophages in transferring immunity against P. berghei infection. Macrophages obtained from mice having high parasitaemia at a dose of one million cells/animal showed significant increase in survival period (SP) and K values, compared to controls. Macrophages exposed to low parasite density conferred significant K values only. There was a decrease in prepatent period (PP) in the animal which received macrophages from animals cured 7-11 months compared to controls. The adoptive transfer studies with macrophages conditioned in vitro to parasite contributed towards increased protection of host against P. berghei as expressed by K values only. These studies showed that the macrophages harvested from infected mice were capable of acting as immunogen against P. berghei infection.

Role of macrophages in experimental malaria: VI--Effect of Freund's complete adjuvant in Plasmodium berghei infected mice.

Freund's complete adjuvant (FCA) treated group of mice when challenged with lethal Plasmodium berghei showed increased survival value; survival period (SP) and median survival day (MSD) compared to their respective control groups. K values were affected and mean parasitaemia during infection period was lower than that of control. In general survival rate after 35 days of infection was 10.5% in FCA recipients. The survival rate in a particular group of animals which received 0.2 ml FCA 3 days before challenge was 22.7%. FCA was found to contribute to increased survival of the host against P. berghei infection. The study indicates that adjuvants, like FCA induce protective immunity and future studies should include non-specific immunization against human malaria.

Role of macrophages in experimental malaria: V--Effect of ethyl palmitate on macrophages in Plasmodium berghei infected mice.

Ethyl palmitate (EP) was used as a macrophage cytotoxin. The response of P. berghei after exposing the macrophage to EP was opposite to what was seen with other agents like Silica, Antimacrophage serum and Freund's complete adjuvant. EP at dose of 5 mg and above decreased the survival period (SP), median survival day (MSD) and parasite density 24 hrs. before death (K values). Prepatent period (PP) was lower at doses 10 mg and 20 mg per day for 5 days before challenge compared to their corresponding controls. EP at a dose of 5 mg and above was found to be toxic to host, mice. EP in dosage of 3 mg per mouse administered 48 hrs. before challenge resulted in an increase in the mean survival period, survival rate (30%) and decrease in the mean parasitaemia per day when compared with the corresponding control. The interfering agents affected differently both the host and/or parasite. A proper modulation of the macrophage during the course of infection may help the host in surviving this lethal infection.

High prevalence of chloroquine resistant Plasmodium falciparum infection in Rajasthan epidemic.

Plasmodium falciparum is the main killer among all human malaria parasites. In 1994, there was a falciparum malaria epidemic in Rajasthan, India, with many deaths. We have investigated active falciparum malaria cases from this epidemic and found that most of the parasite isolates (95%) were resistant to chloroquine. Nevertheless, all the tested isolates from the epidemic, were sensitive to mefloquine and quinine and ninety percent were also susceptible to sulfadoxine/pyrimethamine. Most individuals had moderate levels of TNF-alpha (20-220 pg/ml) and anti-parasite IgM antibodies compared to IgG levels which were relatively lower. In conclusion, the high transmission rate of the chloroquine resistant P. falciparum parasite could be the probable cause of the disease epidemic in Rajasthan. The timely drug sensitivity test and availability of appropriate antimalarial drugs are, therefore, warranted.

Role of macrophages in experimental malaria: I. Development of immunobioassay indicators.

The role of macrophages in immunogenic mechanisms of malaria was studied. The first part of the study aimed at development of indicators for assessing immunobioassay. Accordingly, data on the natural course of lethal Plasmodium berghei infection in mice were collected, and baseline estimates of a set of indicators were made. The indicators along with their estimated means are: prepatent period (PP), 2.57 +/- 0.06 days; survival period (SP), 17.63 +/- 0.29 days; median survival day (MSD), 17.20 days; and parasite density 24 h before death (K), 3582.6 infected RBC/10(4)RBCs. The probable role of immunogenic mechanisms judged indirectly by course of parasitaemia in different phases is discussed.