Multivariate PLS Modeling of Apicomplexan FabD-Ligand Interaction Space for Mapping Target-Specific Chemical Space and Pharmacophore Fingerprints

Biomolecular recognition underlying drug-target interactions is determined by both binding affinity and specificity. Whilst, quantification of binding efficacy is possible, determining specificity remains a challenge, as it requires affinity data for multiple targets with the same ligand dataset. Thus, understanding the interaction space by mapping the target space to model its complementary chemical space through computational techniques are desirable. In this study, active site architecture of FabD drug target in two apicomplexan parasites viz. Plasmodium falciparum (PfFabD) and Toxoplasma gondii (TgFabD) is explored, followed by consensus docking calculations and identification of fifteen best hit compounds, most of which are found to be derivatives of natural products. Subsequently, machine learning techniques were applied on molecular descriptors of six FabD homologs and sixty ligands to induce distinct multivariate partial-least square models. The biological space of FabD mapped by the various chemical entities explain their interaction space in general. It also highlights the selective variations in FabD of apicomplexan parasites with that of the host. Furthermore, chemometric models revealed the principal chemical scaffolds in PfFabD and TgFabD as pyrrolidines and imidazoles, respectively, which render target specificity and improve binding affinity in combination with other functional descriptors conducive for the design and optimization of the leads.

Besnoitia besnoiti and Toxoplasma gondii: two apicomplexan strategies to manipulate the host cell centrosome and Golgi apparatus

Besnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.

Purine nucleoside phosphorylase: A potential target for the development of drugs to treat T-cell- and apicomplexan parasite-mediated diseases

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and thus drugs that inhibit human PNP activity have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Besides, the purine salvage pathway is the only possible way for apicomplexan parasites to obtain the building blocks for RNA and DNA synthesis, which makes PNP from these parasites an attractive target for drug development against diseases such as malaria. Hence, a number of research groups have made efforts to elucidate the mechanism of action of PNP based on structural and kinetic studies. It is conceivable that the mechanism may be different for PNPs from diverse sources, and influenced by the oligomeric state of the enzyme in solution. Furthermore, distinct transition state structures can make possible the rational design of specific inhibitors for human and apicomplexan enzymes. Here, we review the current status of these research efforts to elucidate the mechanism of PNP-catalyzed chemical reaction, focusing on the mammalian and Plamodium falciparum enzymes, targets for drug development against, respectively, T-Cell and Apicomplexan parasites-mediated diseases.

Galactose Recognition by the Apicomplexan Parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.

Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.

Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii

Apicomplexan parasites such as Toxoplasma gondii contain a primitive plastid, the apicoplast, whose genome consists of a 35-kb circular DNA related to the plastid DNA of plants. Plants synthesize fatty acids in their plastids. The first committed step in fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACC). This enzyme is encoded in the nucleus, synthesized in the cytosol, and transported into the plastid. In the present work, two genes encoding ACC from T. gondii were cloned and the gene structure was determined. Both ORFs encode multidomain proteins, each with an N-terminal extension, compared with the cytosolic ACCs from plants. The N-terminal extension of one isozyme, ACC1, was shown to target green fluorescent protein to the apicoplast of T. gondii. In addition, the apicoplast contains a biotinylated protein, consistent with the assertion that ACC1 is localized there. The second ACC in T. gondii appears to be cytosolic. T. gondii mitochondria also contain a biotinylated protein, probably pyruvate carboxylase. These results confirm the essential nature of the apicoplast and explain the inhibition of parasite growth in cultured cells by herbicides targeting ACC.

Cellular and molecular interactions between the apicomplexan parasites Plasmodium and Theileria and their host cells

Apicomplexan parasites of the genera Theileria and Plasmodium have complicated life cycles including infection of a vertebrate intermediate host and an arthropod definitive host. As the Plasmodium parasite progresses through its life cycle, it enters a number of different cell types, both in its mammalian and mosquito hosts. The fate of these cells varies greatly, as do the parasite and host molecules involved in parasite-host interactions. In mammals, Plasmodium parasites infect hepatocytes and erythrocytes whereas Theileria infects ruminant leukocytes and erythrocytes. Survival of Plasmodium-infected hepatocytes and Theileria-infected leukocytes depends on parasite-mediated inhibition of host cell apoptosis but only Theileria-infected cells exhibit a fully transformed phenotype. As the development of both parasites progresses towards the merozoite stage, the parasites no longer promote the survival of the host cell and the infected cell is finally destroyed to release merozoites. In this review we describe similarities and differences of parasite-host cell interactions in Plasmodium-infected hepatocytes and Theileria-infected leukocytes and compare the observed phenotypes to other parasite stages interacting with host cells.

Intracellular survival of apicomplexan parasites and host cell modification

The intracellular stages of apicomplexan parasites are known to extensively modify their host cells to ensure their own survival. Recently, considerable progress has been made in understanding the molecular details of these parasite-dependent effects for Plasmodium-, Toxoplasma- and Theileria-infected cells. We have begun to understand how Plasmodium liver stage parasites protect their host hepatocytes from apoptosis during parasite development and how they induce an ordered cell death at the end of the liver stage. Toxoplasma parasites are also known to regulate host cell survival pathways and it has been convincingly demonstrated that they block host cell major histocompatibility complex (MHC)-dependent antigen presentation of parasite epitopes to avoid cell-mediated immune responses. Theileria parasites are the masters of host cell modulation because their presence immortalises the infected cell. It is now accepted that multiple pathways are activated to induce Theileria-dependent host cell transformation. Although it is now known that similar host cell pathways are affected by the different parasites, the outcome for the infected cell varies considerably. Improved imaging techniques and new methods to control expression of parasite and host cell proteins will help us to analyse the molecular details of parasite-dependent host cell modifications.

Estudo da expressão e participação de osteopontina durante a interação taquizoíto de Toxoplasma gondii célula hospedeira.

Toxoplasma gondii é um parasito do filo Apicomplexa que infecta uma grande variedade de hospedeiros, incluindo os humanos. O parasito invade a célula hospedeira por penetração ativa, com a participação das proteínas de suas organelas secretoras durante esse processo. Até o momento, somente um número limitado de proteínas secretoras tem sido descoberto, além disso, as moléculas efetoras envolvidas na invasão e sobrevivência do parasito não estão completamente compreendidas. A osteopontina (OPN) é uma glicofosfoproteína adesiva secretada, multifuncional, que contém o domínio arginina-glicina-ácido aspártico (RGD) de ligação à integrina, que está envolvida em uma variedade de eventos fisiológicos e patológicos, incluindo sinalização e sobrevivência celular. Pela primeira vez, nós demonstramos pelas técnicas de imunofluorescência e imunocitoquímica ultraestrutural que há uma intensa marcação para uma proteína OPN-like nos grânulos densos de taquizoítos de T. gondii extracelulares. O western blotting e o RT-PRC confirmaram a expressão de OPN-like nos taquizoítos. Nossos resultados também mostram que após a invasão dos macrófagos, a proteína OPN-like está localizada na membrana do vacúolo parasitóforo. Esses dados sugerem que os grânulos densos secretam uma proteína OPN-like, e nós podemos especular que essa proteína participa durante o processo de interação do parasito com as células hospedeiras. .

Lethal Parasites in Oysters from Coastal Georgia with Discussion of Disease and Management Implications

Extensive mortalities of oysters, Crassostrea virginica, occurred from 1985 through 1987 in coastal waters of Georgia. Fluid thioglycolate cultures of oysters collected from 16 of 17 locations revealed infections by the apicomplexan parasite Perkinsus marinus. An ascetosporan parasite, Haplosporidium nelsoni, was also observed in histopathological examination of oysters from 4 of the locations. While the range of H. nelsoni currently is recognized as the east coast of the United States from Maine to Florida, this is the first report of the parasite in Georgia waters. This paper documents the occurrence of these two lethal parasites in oysters from coastal waters of Georgia, along with potential disease and management implications. Results of an earlier independent and previously unpublished survey are also discussed which document the presence of P. marinus in Georgia as early as 1966.

Identification of plasmodium vivax proteins with potential role in invasion using sequence redundancy reduction and profile hidden markov models

Background: This study describes a bioinformatics approach designed to identify Plasmodium vivax proteins potentially involved in reticulocyte invasion. Specifically, different protein training sets were built and tuned based on different biological parameters, such as experimental evidence of secretion and/or involvement in invasion-related processes. A profile-based sequence method supported by hidden Markov models (HMMs) was then used to build classifiers to search for biologically-related proteins. The transcriptional profile of the P. vivax intra-erythrocyte developmental cycle was then screened using these classifiers. Results: A bioinformatics methodology for identifying potentially secreted P. vivax proteins was designed using sequence redundancy reduction and probabilistic profiles. This methodology led to identifying a set of 45 proteins that are potentially secreted during the P. vivax intra-erythrocyte development cycle and could be involved in cell invasion. Thirteen of the 45 proteins have already been described as vaccine candidates; there is experimental evidence of protein expression for 7 of the 32 remaining ones, while no previous studies of expression, function or immunology have been carried out for the additional 25. Conclusions: The results support the idea that probabilistic techniques like profile HMMs improve similarity searches. Also, different adjustments such as sequence redundancy reduction using Pisces or Cd-Hit allowed data clustering based on rational reproducible measurements. This kind of approach for selecting proteins with specific functions is highly important for supporting large-scale analyses that could aid in the identification of genes encoding potential new target antigens for vaccine development and drug design. The present study has led to targeting 32 proteins for further testing regarding their ability to induce protective immune responses against P. vivax malaria.

Functional characterization by genetic complementation of aroB-Encoded dehydroquinate synthase from Mycobactetium tuberculosis H37Rv and its heterologous expression and purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.