Insights into the early liver stage biology of Plasmodium.

Even though malaria is preventable and curable, it has become a serious threat to mankind. In 2016, there were an estimated 216 million cases of malaria across the world. The biology of its causative agent, i.e. Plasmodium parasite is full of complex mechanisms. There are five Plasmodium species responsible for malaria in humans, viz. Plasmodium falciparum, P. vivax, P. malariae, P. ovale and recently identified P. knowlesi that normally infect apes. In humans, malaria is spread by the injection of Plasmodium sporozoites through the bite of infectious Anopheles' female mosquito during their blood meal. From the time of entry into human skin till the development into the asexual forms, the parasite undergoes several transformations. This review attempts to understand the science behind the pre-erythrocytic liver stage of Plasmodium. Research articles explaining parasite biology, cell-traversal, transformation stages, cell-egress process, etc. were retrieved from PubMed and google scholar database. Various known and unknown mechanisms and strategies used by the malaria parasite P. berghei in rodent models have been discussed in this review. Limited or no information was available for humans, due to technical feasibility and complexity of parasite's life cycle. Hence, it was concluded that there is an urgent need to investigate the hepatic invasion, traversal and egress mechanism of P. falciparum and P. vivax for developing novel therapeutics to fight against malaria.

Biochemical and structural characterization of a thermostable ß-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis.

Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial ß-glucosidases (BGL) with significant ß-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific ß-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal ß-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 °C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are ß-D-Galp-(1→6)-D-Lac (6GALA) and ß-D-Galp-(1→3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophile Halothermothrix orenii.

A ribokinase gene (rbk) from the anaerobic halothermophilic bacterium Halothermothrix orenii was cloned and overexpressed in Escherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 45.6, b = 61.1, c = 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.

Expression, purification and preliminary crystallographic analysis of the recombinant ß-glucosidase (BglA) from the halothermophile Halothermothrix orenii.

The ß-glucosidase A gene (bglA) has been cloned from the halothermophilic bacterium Halothermothrix orenii and the recombinant enzyme (BglA; EC 3.2.1.21) was bacterially expressed, purified using metal ion-affinity chromatography and subsequently crystallized. Orthorhombic crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystal structure with two molecules in the asymmetric unit was solved by molecular replacement using a library of known glucosidase structures. Attempts to collect higher resolution diffraction data from crystals grown under different conditions and structure refinement are currently in progress.

Glutamate dehydrogenase: a novel candidate to diagnose Plasmodium falciparum through rapid diagnostic test in blood specimen from fever patients.

In recent years, Plasmodium falciparum histidine-rich protein 2 gene deletion has been reported in India. Such isolates are prone to selective transmission and thus form a challenge to case management. As most of the rapid malaria diagnostic tests are based on the detection of HRP2 protein in the blood, we attempted to use Glutamate Dehydrogenase (GDH) as a biomarker for the diagnosis of P. falciparum. Recombinant PfGDH was successfully cloned, expressed and purified using the Ni-NTA approach. Polyclonal antibodies were raised against full-length rPfGDH and its peptides. Antibodies for rPfGDH showed a strong immune response against the recombinant protein. However, antibody showed no affinity towards the peptides, which suggests they failed as antigen. Antibodies for rPfGDH significantly detected the GDH in human blood specimens. This is the first report where P. falciparum GDH was detected in malaria cases from various parts of India. The raised polyclonal antibodies had shown an affinity for PfGDH in quantitative ELISA and are capable to be exploited for RDTs. This research needs further statistical validation on a large number and different sample types from candidates infected with P. falciparum and other species.

High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with α-L-arabinofuranosidase activity.

A gene from the heterotrophic, halothermophilic marine bacterium Halothermothrix orenii has been cloned and overexpressed in Escherichia coli. This gene encodes the only glycoside hydrolase of family 43 (GH43) produced by H. orenii. The crystal structure of the H. orenii glycosidase was determined by molecular replacement and refined at 1.10 Šresolution. As for other GH43 members, the enzyme folds as a five-bladed ß-propeller. The structure features a metal-binding site on the propeller axis, near the active site. Based on thermal denaturation data, the H. orenii glycosidase depends on divalent cations in combination with high salt for optimal thermal stability against unfolding. A maximum melting temperature of 76°C was observed in the presence of 4 M NaCl and Mn(2+) at pH 6.5. The gene encoding the H. orenii GH43 enzyme has previously been annotated as a putative α-L-arabinofuranosidase. Activity was detected with p-nitrophenyl-α-L-arabinofuranoside as a substrate, and therefore the name HoAraf43 was suggested for the enzyme. In agreement with the conditions for optimal thermal stability against unfolding, the highest arabinofuranosidase activity was obtained in the presence of 4 M NaCl and Mn(2+) at pH 6.5, giving a specific activity of 20-36 µmol min(-1) mg(-1). The active site is structurally distinct from those of other GH43 members, including arabinanases, arabinofuranosidases and xylanases. This probably reflects the special requirements for degrading the unique biomass available in highly saline aqueous ecosystems, such as halophilic algae and halophytes. The amino-acid distribution of HoAraf43 has similarities to those of mesophiles, thermophiles and halophiles, but also has unique features, for example more hydrophobic amino acids on the surface and fewer buried charged residues.