Heat Shock Protein 90 regulates encystation in Entamoeba

Enteric protozoan Entamoeba histolytica is a major cause of debilitating diarrheal infection worldwide with high morbidity and mortality. Even though the clinical burden of this parasite is very high, this infection is categorized as a neglected disease. Parasite is transmitted through feco-oral route and exhibit two distinct stages namely - trophozoites and cysts. Mechanism and regulation of encystation is not clearly understood. Previous studies have established the role of Heat shock protein 90 (Hsp90) in regulating stage transition in various protozoan parasites like Giardia, Plasmodium, Leishmania, and Toxoplasma. Our study for the first time reports that Hsp90 plays a crucial role in life cycle of Entamoeba as well. We identify Hsp90 to be a negative regulator of encystation in Entamoeba. We also show that Hsp90 inhibition interferes with the process of phagocytosis in Entamoeba. Overall, we show that Hsp90 plays an important role in virulence and transmission of Entamoeba.

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.

Chemical contaminants, pathogen exposure and general health status of live and beach-cast Washington sea otters (Enhydra lutris kenyoni)

Analyses of blood and liver samples from live captured sea otters and liver samples from beachcast sea otter carcasses off the remote Washington coast indicate relatively low exposure to contaminants, but suggest that even at the low levels measured, exposure may be indicated by biomarker response. Evidence of pathogen exposure is noteworthy - infectious disease presents a potential risk to Washington sea otters, particularly due to their small population size and limited distribution. During 2001 and 2002, 32 sea otters were captured, of which 28 were implanted with transmitters to track their movements and liver and blood samples were collected to evaluate contaminant and pathogen exposure. In addition, liver samples from fifteen beachcast animals that washed ashore between 1991 and 2002 were analyzed to provide historical information and a basis of reference for values obtained from live otters. The results indicate low levels of metals, butyltins, and organochlorine compounds in the blood samples, with many of the organochlorines not detected except polychlorinated biphenyls (PCBs), and a few aromatic hydrocarbons detected in the liver of the live captured animals. Aliphatic hydrocarbons were measurable in the liver from the live captured animals; however, some of these are likely from biogenic sources. A significant reduction of vitamin A storage in the liver was observed in relation to PCB, dibutyltin and octacosane concentration. A significant and strong positive correlation in vitamin A storage in the liver was observed for cadmium and several of the aliphatic hydrocarbons. Peripheral blood mononuclear cell (PBMC) cytochrome P450 induction was elevated in two of 16 animals and may be potentially related to aliphatic and aromatic hydrocarbon exposure. Mean concentration of total butyltin in the liver of the Washington beach-cast otters was more than 15 times lower than the mean concentration reported by Kannan et al. (1998) for Southern sea otters in California. Organochlorine compounds were evident in the liver of beach-cast animals, despite the lack of large human population centers and development along the Washington coast. Concentrations of PCBs and chlordanes (e.g., transchlordane, cis-chlordane, trans-nonachlor, cis-nonachlor and oxychlordane) in liver of Washington beach-cast sea otters were similar to those measured in Aleutian and California sea otters, excluding those from Monterey Bay, which were higher. Mean concentrations of 1,1,1,- trichloro-2,2-bis(p-chlorophyenyl)ethanes (DDTs) were lower, and mean concentrations of cyclohexanes (HCH, e.g., alpha BHC, beta BHC, delta BHC and gamma BHC) were slightly higher in Washington beach-cast otters versus those from California and the Aleutians. Epidemiologically, blood tests revealed that 80 percent of the otters tested positive for morbillivirus and 60 percent for Toxoplasma, the latter of which has been a significant cause of mortality in Southern sea otters in California. This is the first finding of positive morbillivirus titers in sea otters from the Northeast Pacific. Individual deaths may occur from these diseases, perhaps more so when animals are otherwise immuno-compromised or infected with multiple diseases, but a population-threatening die-off from these diseases singly is unlikely while population immunity remains high. The high frequency of detection of morbillivirus and Toxoplasma in the live otters corresponds well with the cause of death of stranded Washington sea otters reported herein, which has generally been attributable to infectious disease. Washington’s sea otter population continues to grow, with over 1100 animals currently inhabiting Washington waters; however, the rate of growth has slowed over recent years. The population has a limited distribution and has not yet reached its carrying capacity and as such, is still considered at high risk to catastrophic events. (PDF contains 189 pages)

Cutting Edge: Suppression of GM-CSF Expression in Murine and Human T Cells by IL-27

GM-CSF is a potent proinflammatory cytokine that plays a pathogenic role in the CNS inflammatory disease experimental autoimmune encephalomyelitis. As IL-27 alleviates experimental autoimmune encephalomyelitis, we hypothesized that IL-27 suppresses GM-CSF expression by T cells. We found that IL-27 suppressed GM-CSF expression in CD4+ and CD8+ T cells in splenocyte and purified T cell cultures. IL-27 suppressed GM-CSF in Th1, but not Th17, cells. IL-27 also suppressed GM-CSF expression by human T cells in nonpolarized and Th1- but not Th17-polarized PBMC cultures. In vivo, IL-27p28 deficiency resulted in increased GM-CSF expression by CNS-infiltrating T cells during Toxoplasma gondii infection. Although in vitro suppression of GM-CSF by IL-27 was independent of IL-2 suppression, IL-10 upregulation, or SOCS3 signaling, we observed that IL-27-driven suppression of GM-CSF was STAT1 dependent. Our findings demonstrate that IL-27 is a robust negative regulator of GM-CSF expression in T cells, which likely inhibits T cell pathogenicity in CNS inflammation.

Inmunología : diagnóstico e interpretación de pruebas de laboratorio

El acelerado avance de la inmunología ha generado el desarrollo de técnicas que permiten resultados más precisos y de métodos de separación, tanto de componentes celulares como humorales, útiles en el diagnóstico. La presente edición tiene como objetivo proveer las herramientas necesarias para entender estos avances, junto con los mecanismos implicados en el desarrollo de algunas técnicas de diagnóstico inmunológico e interpretación clínica. Entre los temas tratados se encuentra el estudio de la estructura y fisiología de Toxoplasma gondii, que sirve de modelo para la elaboración de péptidos sintéticos de proteínas inmunogénicas y, además, puede ser aplicado en el desarrollo de métodos de diagnóstico en otros parásitos de importancia clínica. El presente manual ha sido elaborado con el fin de proporcionar una ayuda en el desarrollo de prácticas de laboratorio en inmunología y está dirigido, principalmente, a estudiantes universitarios y trabajadores de las diferentes áreas de la salud como medicina, bacteriología, biología y química.

Caracterización de la proteína del cuello de las roptrias 5 en plasmodium falciparum (PfRON5)

El conocimiento de las proteínas implicadas en el proceso de invasión de los merozoitos a los eritrocitos por Plasmodium es el punto de partida para el desarrollo de nuevas estrategias para controlar la malaria. Muchas de estas proteínas han sido estudiadas en Toxoplasma gondii, donde se han identificado las proteínas que pertenecen al Tight Junction (TJ), el cual permite una interacción fuerte entre las membranas de la célula huésped y el parásito, necesaria para la invasión parasitaria. En este género, cuatro proteínas del cuello de las roptrias (RON2, RON4, RON5 y RON8) y una proteína de micronemas (TgAMA-1) se han encontrado como parte del TJ. En Plasmodium falciparum, se han caracterizado las proteínas PfRON2 y PfRON4. En el presente estudio se realiza la identificación de la proteína PfRON5, una proteína de ~110 kDa que se expresa en las etapas de merozoitos y esquizontes de la cepa FCB-2 utilizando técnicas de biología molecular, bioinformática e inmuoquímica.

Annotation and characterization of the Plasmodium vivax rhoptry neck protein 4 (PvRON4)

Background: The tight junction (TJ) is one of the most important structures established during merozoite invasion of host cells and a large amount of proteins stored in Toxoplasma and Plasmodium parasites’ apical organelles are involved in forming the TJ. Plasmodium falciparum and Toxoplasma gondii apical membrane antigen 1 (AMA-1) and rhoptry neck proteins (RONs) are the two main TJ components. It has been shown that RON4 plays an essential role during merozoite and sporozoite invasion to target cells. This study has focused on characterizing a novel Plasmodium vivax rhoptry protein, RON4, which is homologous to PfRON4 and PkRON4. Methods: The ron4 gene was re-annotated in the P. vivax genome using various bioinformatics tools and taking PfRON4 and PkRON4 amino acid sequences as templates. Gene synteny, as well as identity and similarity values between open reading frames (ORFs) belonging to the three species were assessed. The gene transcription of pvron4, and the expression and localization of the encoded protein were also determined in the VCG-1 strain by molecular and immunological studies. Nucleotide and amino acid sequences obtained for pvron4 in VCG-1 were compared to those from strains coming from different geographical areas. Results: PvRON4 is a 733 amino acid long protein, which is encoded by three exons, having similar transcription and translation patterns to those reported for its homologue, PfRON4. Sequencing PvRON4 from the VCG-1 strain and comparing it to P. vivax strains from different geographical locations has shown two conserved regions separated by a low complexity variable region, possibly acting as a “smokescreen”. PvRON4 contains a predicted signal sequence, a coiled-coil α-helical motif, two tandem repeats and six conserved cysteines towards the carboxyterminus and is a soluble protein lacking predicted transmembranal domains or a GPI anchor. Indirect immunofluorescence assays have shown that PvRON4 is expressed at the apical end of schizonts and co-localizes at the rhoptry neck with PvRON2.

Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1)

Background: Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections. Methods: The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results: In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions: This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended.

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.

Prevenção da toxoplasmose congénita em Portugal

A toxoplasmose congénita é uma doença infecciosa, causada pelo parasita Toxoplasma gondii e, adquirida por transmissão materno-fetal, a qual pode acarretar sequelas neurológicas e oculares muito graves, no recém-nascido. O presente estudo incide sobre as linhas de prevenção da doença, em Portugal. A base da prevenção define-se como primária, através da determinação do estatuto imunológico da mulher, do aconselhamento e adopção de medidas higiénico-dietéticas das mulheres seronegativas, de forma a evitar a infecção materna. A vigilância serológica, na detecção de uma possível infecção materna, e a instituição da terapêutica de profilaxia, constituem a prevenção secundária, de modo a evitar a infecção fetal. A prevenção terciária recai, sobre o estabelecimento de um novo esquema terapêutico, dotado de alguma teratogenicidade, com o intuito de minimizar as sequelas da infecção. Em Portugal, existem muitas mulheres seronegativas, mal informadas acerca da doença, e que não tomam medidas preventivas correctas, para evitar a infecção. Esta problemática é decrescente, de norte para sul do país. A prevenção da doença pode ser bem-sucedida, através da implementação de directrizes específicas, dirigidas aos diferentes grupos de risco e da orientação correcta, pelos profissionais de saúde. A realização de estudos, em várias áreas de intervenção da doença, optimiza a sua prevenção e a sua relação de custo-benefício.

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H-heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs. (C) 2010 Elsevier B.V. All rights reserved.

Melatonin and IP(3)-induced Ca(2+) Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

In Vitro and In Vivo Antiplasmodial Activities of Risedronate and Its Interference with Protein Prenylation in Plasmodium falciparum

The increasing resistance of malarial parasites to almost all available drugs calls for the identification of new compounds and the detection of novel targets. Here, we establish the antimalarial activities of risedronate, one of the most potent bisphosphonates clinically used to treat bone resorption diseases, against blood stages of Plasmodium falciparum (50% inhibitory concentration [IC(50)] of 20.3 +/- 1.0 mu M). We also suggest a mechanism of action for risedronate against the intraerythrocytic stage of P. falciparum and show that protein prenylation seems to be modulated directly by this drug. Risedronate inhibits the transfer of the farnesyl pyrophosphate group to parasite proteins, an effect not observed for the transfer of geranylgeranyl pyrophosphate. Our in vivo experiments further demonstrate that risedronate leads to an 88.9% inhibition of the rodent parasite Plasmodium berghei in mice on the seventh day of treatment; however, risedronate treatment did not result in a general increase of survival rates.

Sequence-specific reconstruction from fragmentary databases using seed sequences: implementation and validation on SAGE, proteome and generic sequencing data

Motivation: DNA assembly programs classically perform an all-against-all comparison of reads to identify overlaps, followed by a multiple sequence alignment and generation of a consensus sequence. If the aim is to assemble a particular segment, instead of a whole genome or transcriptome, a target-specific assembly is a more sensible approach. GenSeed is a Perl program that implements a seed-driven recursive assembly consisting of cycles comprising a similarity search, read selection and assembly. The iterative process results in a progressive extension of the original seed sequence. GenSeed was tested and validated on many applications, including the reconstruction of nuclear genes or segments, full-length transcripts, and extrachromosomal genomes. The robustness of the method was confirmed through the use of a variety of DNA and protein seeds, including short sequences derived from SAGE and proteome projects.

A Community-based Survey of Human Toxoplasmosis in Rural Amazonia: Seroprevalence, Seroconversion Rate, and Associated Risk Factors

IgG antibodies to Toxoplasma gondii were detected in, March-April 2004, in 65.8% (95% confidence interval, 60.8-70.8%) of 342 systematically sampled subjects 5-90 years of age (87.5% of the eligible) living in a rural settlement in Amazonia, with a seroconversion rate of 9% over I year of follow-up of 99 seronegative subjects. Multiple logistic regression analysis identified age as the only significant independent predictor of seropositivity at the baseline. Each additional year of age increases the odds of being seropositive by 6%, and 76.8% of the subjects are expected to be seropositive at 30 years of age. A single high-prevalence spatial cluster, comprising 11.9% of the seropositive subjects, was detected in the area; households in the cluster were less likely to have dogs as pets and their heads had a lower education level, when compared with households located outside the cluster. The challenges for preventing human toxoplasmosis in tropical rural settings are discussed.