Variable Surface Antigens of Plasmodium falciparum: Protein Families with Divergent Roles.

Malaria caused by Plasmodium falciparum (Pf) is an illness that contributes significantly to the global health burden. Pf makes significant alterations to the host cell to meet its metabolic demands and escape the immune response of the host. These include the export of a large number of parasite proteins to the infected Red Blood Cells (iRBC). Variable Surface Antigens (VSAs), which are highly polymorphic protein families with important roles in immune evasion, form an important component of the exported proteins. A total of five protein families constitute the VSAs, viz. PfEMP1 (Pf erythrocyte membrane protein 1), RIFIN (repetitive interspersed family), STEVOR (sub-telomeric open reading frame), SURFIN (surface-associated interspersed gene family), and PfMC-2TM (Pf Maurer's cleft two transmembrane). With orthologues present in various simian-infecting species, VSAs take up a variety of domain topologies and organizational structures while exhibiting differential expressions throughout the parasite life cycle. Their expression varies across clinical isolates and laboratory strains, which suggests their crucial role in host cell survival and defense. Members of VSAs are reported to contribute significantly to disease pathogenesis through immune evasion processes like cytoadherence, iRBC sequestration in the host vasculature, rosetting, reduced erythrocyte deformability, and direct immunosuppression. In this study, we have gathered information on various aspects of VSAs, like their orthologues, domain architecture, surface topology, functions and interactions, and three-dimensional structures, while emphasizing discoveries in the field. Considering the vast repertoire of Plasmodial VSAs with new emergent functions, a lot remains unknown about these families and, hence, malaria biology.

Molecular Players at the Sorting Stations of Malaria Parasite 'Plasmodium falciparum'.

The apicomplexan pathogenic parasite 'Plasmodium falciparum' (Pf) is responsible for most of the malaria related mortality. It resides in and refurbishes the infected red blood cells (iRBCs) for its own survival and to suffice its metabolic needs. Remodeling of host erythrocytes involves alteration of physical and biochemical properties of the membrane and genesis of new parasite induced structures within the iRBCs. The generated structures include knobs and solute ion channels on the erythrocyte surface and specialized organelles i.e. Maurer's clefts (MCs) in the iRBC cytosol. The above processes are mediated by exporting a large repertoire of proteins to the host cell, most of which are transported via MCs, the sorting stations in parasitized erythrocytes. Information about MC biogenesis and the molecules involved in maintaining MC architecture remains incompletely elucidated. Here, we have compiled a list of experimentally known MC resident proteins, several of which have roles in maintaining its architecture and function. Our short review covers available data on the domain organization, orthologues, topology and specific roles of these proteins. We highlight the current knowledge gaps in our understanding of MCs as crucial organelles involved in parasite biology and disease pathogenesis.

Analyzing Interaction of Rhodacyanine Inhibitor 'MKT-077' with Plasmodium falciparum HSP70s.

INTRODUCTION: MKT-077 and its derivatives are rhodacyanine inhibitors that hold potential in the treatment of cancer, neurodegenerative diseases and malaria. These allosteric drugs act by inhibiting the ATPase action of heat shock proteins of 70 kDa (HSP70). MKT-077 accumulates in the mitochondria and displays differential activity against HSP70 homologs. METHODS: The four Plasmodium falciparum HSP70s (PfHSP70) are present in various subcellular locations to perform distinct functions. In the present study, we have used bioinformatics tools to understand the interaction of MKT-077 at the ADP and HEW (2-amino 4 bromopyridine) binding sites on PfHSP70s. Our molecular docking experiments predict that the mitochondrial and endoplasmic reticulum PfHSP70 homologs are likely to bind MKT-077 with higher affinities at their ADP binding sites. RESULTS: Binding analysis indicates that the nature of the identified interactions is primarily hydrophobic. We have also identified specific residues of PfHSP70s that are involved in interacting with the ligand. CONCLUSION: Information obtained in this study may form the foundation for the design and development of MKT-077-based drugs against malaria.

Understanding the Molecular Basis for Enhanced Glutenase Activity of Actinidin using Structural Bioinformatics.

BACKGROUND: Management of gluten intolerance is currently possible only by consumption of a gluten-free diet (GFD) for a lifetime. The scientific community has been searching for alternatives to GFD, like the inclusion of natural proteases with meals or pre-treatment of gluten-containing foods with glutenases. Actinidin from kiwifruit has shown considerable promise in digesting immunogenic gliadin peptides compared to other plant-derived cysteine proteases. METHODS: In this study, we aimed to understand the structural basis for the elevated protease action of actinidin against gliadin peptides by using an in silico approach. RESULTS: Docking experiments revealed key differences between the binding of gliadin peptide to actinidin and papain, which may be responsible for their differential digestive action. CONCLUSION: Sequence comparison of different plant cysteine proteases highlights amino acid residues surrounding the active site pocket of actinidin that are unique to this molecule and hence likely to contribute to its digestive properties.

Purification and characterization of a high molecular weight serine protease from Microbacterium paraoxydans sp. SKS10.

Alkaline proteases from microbial sources have been found suitable for diverse industrial applications, with serine proteases being the most common enzymes used in the detergent industry. In the present study, we have purified and characterized an extracellular alkaline serine protease from Microbacterium paraoxydans sp. SKS10. The protease was purified using ammonium sulfate precipitation followed by different chromatography techniques (fold purification 6.919). Km and Vmax for the protease were determined to be 0.183 mg/mL and 4.904 U/mL, respectively. This enzyme is a thermostable high molecular weight (∼109.4 kDa) protease which has maximal activity at 60°C, and above pH 10. Inhibitor assays revealed the enzyme to be a serine protease whose activity increased by 2.5-fold in the presence of EDTA. This enzyme remained active in the presence of various metal salts and organic solvents and was compatible with commercially available laundry detergents highlighting its potential for use in the detergent industry.

Plasmodium helical interspersed subtelomeric family-an enigmatic piece of the Plasmodium biology puzzle.

Plasmodium falciparum (Pf) refurbishes the infected erythrocytes by exporting a myriad of parasite proteins to the host cell. A novel exported protein family 'Plasmodium Helical Interspersed Subtelomeric' (PHIST) has gained attention for its significant roles in parasite biology. Here, we have collected and analysed available information on PHIST members to enhance understanding of their functions, varied localization and structure-function correlation. Functional diversity of PHIST proteins is highlighted by their involvement in PfEMP1 (Pf erythrocyte membrane protein 1) expression, trafficking and switching. This family also contributes to cytoadherence, gametocytogenesis, host cell modification and generation of extracellular vesicles. While the PHIST domain forms the hallmark of this family, existence and functions of additional domains (LyMP, TIGR01639) and the MEC motif underscores its diversity further. Since specific PHIST proteins seem to form pairs with PfEMP1 members, we have used in silico tools to predict such potential partners in Pf. This information and our analysis of structural data on a PHIST member provide important insights into their functioning. This review overall enables readers to view the PHIST family comprehensively, while highlighting key knowledge gaps in the field.

CX3CL1 binding protein-2 (CBP2) of Plasmodium falciparum binds nucleic acids.

Several exported Plasmodium falciparum (Pf) proteins contribute to malaria biology through their involvement in cytoadherence, immune evasion and host cell remodelling. Many of these exported proteins and other host molecules are present in iRBC (infected red blood cell) generated extracellular vesicles (EVs), which are responsible for host cell modification and parasite development. CX3CL1 binding proteins (CBPs) present on the surface of iRBCs have been reported to contribute to cytoadhesion by binding with the chemokine 'CX3CL1' via their extracellular domains. Here, we have characterized the cytoplasmic domain of CBP2 to understand its function in parasite biology using biochemical and biophysical methods. Recombinant cytoplasmic CBP2 (cCBP2) binds nucleic acids showing interaction with DNA/RNA. cCBP2 shows dimer formation under non-reducing conditions highlighting the role of disulphide bonds in its oligomerization while ATP binding leads to structural changes in the protein. In vitro interaction studies depict its binding with a Maurer's cleft resident protein 'PfSBP1', which is influenced by ATP binding of cCBP2. Our results suggest CBP2 as a two-transmembrane (2TM) receptor responsible for targeting EVs and delivering cargo to host endothelial cells. We propose CBP2 as an important molecule having roles in cytoadherence and immune modulation through its extracellular and cytoplasmic domains respectively.

Exploration of carbohydrate binding behavior and anti-proliferative activities of Arisaema tortuosum lectin.

BACKGROUND: Lectins have come a long way from being identified as proteins that agglutinate cells to promising therapeutic agents in modern medicine. Through their specific binding property, they have proven to be anti-cancer, anti-insect, anti-viral agents without affecting the non-target cells. The Arisaema tortuosum lectin (ATL) is a known anti-insect and anti-cancer candidate, also has interesting physical properties. In the present work, its carbohydrate binding behavior is investigated in detail, along with its anti-proliferative property. RESULTS: The microcalorimetry of ATL with a complex glycoprotein asialofetuin demonstrated trivalency contributed by multiple binding sites and enthalpically driven spontaneous association. The complex sugar specificity of ATL towards multiple sugars was also demonstrated in glycan array analysis in which the trimannosyl pentasaccharide core N-glycan [Manα1-6(Manα1-3)Manß1-4GlcNAcß1-4GlcNAcß] was the highest binding motif. The high binding glycans for ATL were high mannans, complex N-glycans, core fucosylated N-glycans and glycans with terminal lactosamine units attached to pentasaccharide core. ATL induced cell death in IMR-32 cells was observed as time dependent loss in cell number, formation of apoptotic bodies and DNA damage. As a first report of molecular cloning of ATL, the in silico analysis of its cDNA revealed ATL to be a ß-sheet rich heterotetramer. A homology model of ATL showed beta prism architecture in each monomer with 85% residues in favoured region of Ramachandran plot. CONCLUSIONS: Detailed exploration of carbohydrate binding behavior indicated ATL specificity towards complex glycans, while no binding to simple sugars, including mannose. Sequence analysis of ATL cDNA revealed that during the tandem evolutionary events, domain duplication and mutations lead to the loss of mannose specificity, acquiring of new sugar specificity towards complex sugars. It also resulted in the formation of a two-domain single chain polypeptide with both domains having different binding sites due to mutations within the consensus carbohydrate recognition sites [QXDXNXVXY]. This unique sugar specificity can account for its significant biological properties. Overall finding of present work signifies anti-cancer, anti-insect and anti-viral potential of ATL making it an interesting molecule for future research and/or theragnostic applications.

Cholesterol bound Plasmodium falciparum co-chaperone 'PFA0660w' complexes with major virulence factor 'PfEMP1' via chaperone 'PfHsp70-x'.

Lethality of Plasmodium falciparum caused malaria results from 'cytoadherence', which is mainly effected by exported Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. Several exported P. falciparum proteins (exportome) including chaperones alongside cholesterol rich microdomains are crucial for PfEMP1 translocation to infected erythrocyte surface. An exported Hsp40 (heat shock protein 40) 'PFA0660w' functions as a co-chaperone of 'PfHsp70-x', and these co-localize to specialized intracellular mobile structures termed J-dots. Our studies attempt to understand the function of PFA0660w-PfHsp70-x chaperone pair using recombinant proteins. Biochemical assays reveal that N and C-terminal domains of PFA0660w and PfHsp70-x respectively are critical for their activity. We show the novel direct interaction of PfHsp70-x with the cytoplasmic tail of PfEMP1, and binding of PFA0660w with cholesterol. PFA0660w operates both as a chaperone and lipid binding molecule via its separate substrate and cholesterol binding sites. PfHsp70-x interacts with cholesterol bound PFA0660w and PfEMP1 simultaneously in vitro to form a complex. Collectively, our results and the past literature support the hypothesis that PFA0660w-PfHsp70-x chaperone pair assists PfEMP1 transport across the host erythrocyte through cholesterol containing 'J-dots'. These findings further the understanding of PfEMP1 export in malaria parasites, though their in vivo validation remains to be performed.

Inner membrane complex 1l protein of Plasmodium falciparum links membrane lipids with cytoskeletal element 'actin' and its associated motor 'myosin'.

The inner membrane complex (IMC) is a defining feature of apicomplexans comprising of lipid and protein components involved in gliding motility and host cell invasion. Motility of Plasmodium parasites is accomplished by an actin and myosin based glideosome machinery situated between the parasite plasma membrane (PPM) and IMC. Here, we have studied in vivo expression and localization of a Plasmodium falciparum (Pf) IMC protein 'PfIMC1l' and characterized it functionally by using biochemical assays. We have identified cytoskeletal protein 'actin' and motor protein 'myosin' as novel binding partners of PfIMC1l, alongside its interaction with the lipids 'cholesterol' and 'phosphatidyl-inositol 4, 5 bisphosphate' (PIP2). While actin and myosin compete for interaction with PfIMC1l, actin and either of the lipids (cholesterol or PIP2) simultaneously bind PfIMC1l. Interestingly, PfIMC1l showed enhanced binding with actin in the presence of calcium ions, and displayed direct binding with calcium. Based on our in silico analysis and experimental data showing PfIMC1l-actin/myosin and PfIMC1l-lipid interactions, we propose that this protein may anchor the IMC membrane with the parasite gliding apparatus. Considering its binding with key proteins involved in motility viz. myosin and actin (with calcium dependence), we suggest that PfIMC1l may have a role in the locomotion of Plasmodium.

Plasmodium falciparum protein 'PfJ23' hosts distinct binding sites for major virulence factor 'PfEMP1' and Maurer's cleft marker 'PfSBP1'.

Plasmodium falciparum (Pf) proteins exported to infected erythrocytes are key effectors of malaria pathogenesis. These include the PfEMP1 (Pf erythrocyte membrane protein 1) protein family that affects malaria-related mortality through cytoadhesion and parasite immune evasion. Parasites also induce membranous structures called Maurer's clefts (MC) in infected erythrocytes to compensate the lack of host protein synthetic and export machinery. PfEMP1 export is mediated by a myriad of proteins including Pf skeleton binding protein 1 (PfSBP1) and PF70, a hypothetical 16 family member. Here, we aim to understand the function of the only other exported PEXEL-positive hyp16 member 'PfJ23'. Our in vitro and in silico data suggest this protein to be mostly α-helical while displaying different oligomeric forms under reducing and non-reducing conditions. We show coherent expression, partial co-localization and direct interaction of purified PfSBP1 with recombinant and native PfJ23. Recombinant and parasite-expressed PfJ23 also bind to the cytoplasmic tail of PfEMP1, and they seem to partly co-localize during parasite development. Both novel binding partners interact simultaneously with PfJ23 in vitro to form a complex. Our results suggest a probable role for PfJ23 in export of PEXEL-negative proteins like PfSBP1 and PfEMP1, furthering our understanding of malaria biology.

'2TM proteins': an antigenically diverse superfamily with variable functions and export pathways.

Malaria is a disease that affects millions of people annually. An intracellular habitat and lack of protein synthesizing machinery in erythrocytes pose numerous difficulties for survival of the human pathogen Plasmodium falciparum. The parasite refurbishes the infected red blood cell (iRBC) by synthesis and export of several proteins in an attempt to suffice its metabolic needs and evade the host immune response. Immune evasion is largely mediated by surface display of highly polymorphic protein families known as variable surface antigens. These include the two trans-membrane (2TM) superfamily constituted by multicopy repetitive interspersed family (RIFINs), subtelomeric variable open reading frame (STEVORs) and Plasmodium falciparum Maurer's cleft two trans-membrane proteins present only in P. falciparum and some simian infecting Plasmodium species. Their hypervariable region flanked by 2TM domains exposed on the iRBC surface is believed to generate antigenic diversity. Though historically named "2TM superfamily," several A-type RIFINs and some STEVORs assume one trans-membrane topology. RIFINs and STEVORs share varied functions in different parasite life cycle stages like rosetting, alteration of iRBC rigidity and immune evasion. Additionally, a member of the STEVOR family has been implicated in merozoite invasion. Differential expression of these families in laboratory strains and clinical isolates propose them to be important for host cell survival and defense. The role of RIFINs in modulation of host immune response and presence of protective antibodies against these surface exposed molecules in patient sera highlights them as attractive targets of antimalarial therapies and vaccines. 2TM proteins are Plasmodium export elements positive, and several of these are exported to the infected erythrocyte surface after exiting through the classical secretory pathway within parasites. Cleaved and modified proteins are trafficked after packaging in vesicles to reach Maurer's clefts, while information regarding delivery to the iRBC surface is sparse. Expression and export timing of the RIFIN and Plasmodium falciparum erythrocyte membrane protein1 families correspond to each other. Here, we have compiled and comprehended detailed information regarding orthologues, domain architecture, surface topology, functions and trafficking of members of the "2TM superfamily." Considering the large repertoire of proteins included in the 2TM superfamily and recent advances defining their function in malaria biology, a surge in research carried out on this important protein superfamily is likely.

PHISTc protein family members localize to different subcellular organelles and bind Plasmodium falciparum major virulence factor PfEMP-1.

Plasmodium falciparum encodes a novel repertoire of the Plasmodium helical interspersed subtelomeric (PHIST) family of exported proteins, which play diverse roles in infected red blood cells, contributing to malaria pathogenesis. PHIST proteins are central to parasite biology and modify human erythrocytes by interacting with parasite and host proteins. Here, we have attempted to understand the localization and function of two unexplored proteins of the PHISTc subfamily, PFD1140w and PF11_0503, and compared these with a well-characterized member, PFI1780w. We demonstrate that Phist domains assume different oligomeric states owing to a distinct array of subunit interface residues. Colocalization of a Maurer's cleft signature protein, P. falciparum skeleton-binding protein-1 (PfSBP-1), and P. falciparum erythrocyte membrane protein-1 (PfEMP-1) revealed different subcellular destinations for these PHIST members. We further show the binding of recombinant PHIST proteins to the cytoplasmic tail of PfEMP-1 and a novel interaction with PfSBP-1. Interestingly, PFD1140w interacts with PfEMP-1 and PfSBP-1 simultaneously in vitro leading to formation of a complex. These two distant PHISTc members also bind PfEMP-1 on distinct sites, despite sharing the Phist domain. Our data re-emphasize a supportive role for PHIST proteins in cytoadhesion, and identify a new binding partner, PfSBP-1, for members of this family. This information therefore adds another chapter to the understanding of P. falciparum biology and highlights the significance of the unexplored PHIST family.

Sauromatum guttatum lectin: Spectral studies, lectin-carbohydrate interaction, molecular cloning and in silico analysis.

Non-immune carbohydrate binding proteins are broadly defined as lectins. Having been reported from all kingdoms of life, phytolectins are the most widely studied group of lectins. Sauromatum guttatum agglutinin (SGA) was isolated from the plant tubers and characterized for structural variations due to solvent perturbation using polarimetry, fluorescence and light scattering. For the ß-sheet rich SGA, a pH and temperature induced molten globule like intermediate was identified. In isothermal titration microcalorimetry, SGA demonstrated cooperative binding to a complex glycoprotein in enthalpically driven mechanism. Fine sugar specificity exploration identified core pentasaccharide as the most common and highest binding motif with complex N-glycans and fucosylated core N-glycans as additional motifs. Molecular cloning of SGA which has previously been demonstrated to have anti-cancer and anti-insect activities is being reported for the first time. Full length cDNA sequence was obtained with RACE-PCR based upon the conserved carbohydrate recognition site [QXDXNXVXY] present in all GNA-related lectins. Quaternary structure was proposed by homology modeling and an attempt was made to explain the structure-function relationship by in silico analysis.

Cerebral malaria--clinical manifestations and pathogenesis.

One of the most common central nervous system diseases in tropical countries is cerebral malaria (CM). Malaria is a common protozoan infection that is responsible for enormous worldwide mortality and economic burden on the society. Episodes of Plasmodium falciparum (Pf) caused CM may be lethal, while survivors are likely to suffer from persistent debilitating neurological deficits, especially common in children. In this review article, we have summarized the various symptoms and manifestations of CM in children and adults, and entailed the molecular basis of the disease. We have also emphasized how pathogenesis of the disease is effected by the parasite and host responses including blood brain barrier (BBB) disruption, endothelial cell activation and apoptosis, nitric oxide bioavailability, platelet activation and apoptosis, and neuroinflammation. Based on a few recent studies carried out in experimental mouse malaria models, we propose a basis for the neurological deficits and sequelae observed in human cerebral malaria, and summarize how existing drugs may improve prognosis in affected individuals.

Erythrocytic casein kinase II regulates cytoadherence of Plasmodium falciparum-infected red blood cells.

Plasmodium falciparum malaria is a major human health scourge and a key cause of mortality. Its pathogenicity partly results from the phenomenon of "cytoadherence" mediated by the PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) family. Extracellular domains of PfEMP1s are variable and bind various host endothelial receptors, whereas their cytoplasmic domains (VARCs) are relatively conserved. VARCs affix PfEMP1s in the human erythrocyte membrane by interacting with host cytoskeleton proteins and exported parasite proteins. Here, we provide in vitro and in vivo evidence for PfEMP1 phosphorylation (on VARC) and propose an important function for this modification. Specific inhibitors and enhancers have been used to identify erythrocytic casein kinase II (CKII) as the enzyme responsible for VARC modification activity. We have also delineated probable CKII target residues on VARC, which mainly reside in an N-terminal acidic cluster. Our data show that VARC phosphorylation alters its binding to parasite encoded knob-associated histidine-rich protein (KAHRP). Finally, we demonstrate reduced cytoadherence of infected RBCs to endothelial receptors like ICAM-1 and CSA (these contribute to cerebral and placental malaria, respectively) in response to their CKII inhibition. Collectively, this study furthers our understanding of VARC function, underscores the importance of erythrocytic CKII in cytoadherence, and suggests a possible new target for anti-cytoadherence molecules.

Structural basis for Duffy recognition by the malaria parasite Duffy-binding-like domain.

Molecular processes that govern pathogenic features of erythrocyte invasion and cytoadherence in malaria are reliant on Plasmodium-specific Duffy-binding-like domains (DBLs). These cysteine-rich modules recognize diverse host cell-surface receptors during pathogenesis. DBLs of parasite erythrocyte-binding proteins mediate invasion, and those from the antigenically variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) have been implicated in cytoadherence. The simian and human malarial parasites, P. knowlesi and P. vivax, invade human erythrocytes exclusively through the host DARC receptor (Duffy antigen receptor for chemokines). Here we present the crystal structure of the P. knowlesi DBL domain (Pkalpha-DBL), which binds to DARC during invasion of human erythrocytes. Pkalpha-DBL retains the overall fold observed in DBLs from P. falciparum erythrocyte-binding antigen (EBA)-175 (ref. 4). Mapping the residues that have previously been implicated in binding highlights a fairly flat but exposed site for DARC recognition in subdomain 2 of Pkalpha-DBL; this is in sharp contrast to receptor recognition by EBA-175 (ref. 4). In Pkalpha-DBL, the residues that contact DARC and the clusters of residues under immune pressure map to opposite surfaces of the DBL, and suggest a possible mechanism for immune evasion by P. vivax. Our comparative structural analysis of Pkalpha-DBL and P. falciparum EBA-175 provides a framework for the understanding of malaria parasite DBLs, and may affect the development of new prophylactic and therapeutic strategies.