Identification of novel rhoptry proteins in Neospora caninum by LC/MS-MS analysis of subcellular fractions

Apicomplexan parasites possess an apical complex that is composed of two secretory organelles recognized as micronemes and rhoptries. Rhoptry contents are secreted into the parasitophorous vacuole during the host cell invasion process. Several rhoptry proteins have been identified in Toxoplasma gondii and seem to be involved in host-pathogen interactions and some of them are considered to be important virulence factors. Only one rhoptry protein, NcROP2, has been identified and extensively characterized in the closely related parasite Neospora caninum, and this has showed immunoprotective properties. Thus, with the aim of increasing knowledge of the rhoptry protein repertoire in N. caninum, a subcellular fractionation of tachyzoites was performed to obtain fractions enriched for this secretory organelle. 2-D SDS-PAGE followed by MS and LC/MS-MS were applied for fraction analysis and 8 potential novel rhoptry components (NcROP1, 5, 8, 30 and NcRON2, 3, 4, 8) and several kinases, proteases and phosphatases proteins were identified with a high homology to those previously found in T. gondii. Their existence in N. caninum tachyzoites suggests their involvement in similar events or pathways that occur in T. gondii. These novel proteins may be considered as targets that could be useful in the future development of immunoprophylactic measures.

The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences

Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC50 (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.

Manipulação gênica do protozoário Neospora caninum como ferramenta para o estudo das interações parasito-hospedeiro

Neospora caninum is an obligate intracellular parasite classified in the phylum Apicomplexa, characterized by the presence of the apical complex composed by micronemes proteins, rhoptries and dense granules, used by parasite during the adhesion and invasion process of the host cell. This is the mean event in infection pathogenesis generated by N. caninum and other parasites from the phylum Apicomplexa, promoting influence in the parasite biology and the interface between the parasite and its host. Therefore, molecular tools have been developed in order to identify and characterize these possible virulence factors. Thus, the present study sought to establish a specific system of genetic manipulation of N. caninum, searching for the improvement of the genetics manipulation of this parasite. So, we developed genetically depleted N. caninum to Rop9 rhoptry using the pU6-Universal CRISPR-Cas9 plasmid of T. gondii modified by the insertion of Ku80. The Rop9 depleted parasite showed important during initial phase of invasion and replication of the parasite, however it was not characterized as a potential virulence fator for N. caninum. Furthermore, T. gondii proteins were expressed in N. caninum by the use of specific vectors for this parasite, showing an heterologous system for the study of Toxoplasma proteins, due to the fact that Gra15 or Gra24 of type II T. gondii and Rop16 of type I T. gondii were expressed in N. caninum tachyzoites in a stable way and keept its biological phenotype, as already presented the former parasite, that naturaly expresses these proteins. In addition, it was observed that N. caninum induced an inflammasome activation through NLRP3, ASC and Caspase-1. IL-1R/MyD88 demonstrated an indirect pathway in the control of parasite replication. Furthermore, it was observed that this activation is dependent of the potassium efflux and that different strains of N. caninum keep this activation profile. However, T. gondii strains block this activation, making necessary a prior signal in order to active the inflamosome pathway. Type I T. gondii Rop16 was identified as responsible for blocking this activation, in a dependent way to the STAT3 activation. Therefore, the development of molecular tools and their application in N. caninum may prove to be useful to identify and characterize virulent factors involved in the pathogenesis by these two protozoans.

Prior and concomitant dehydroepiandrosterone treatment affects immunologic response of cultured macrophages infected with Trypanosoma cruzi in vitro?

DHEA, a steroid hormone synthesized from cholesterol by cells of the adrenal cortex, plays an essential role in enhancing the host`s resistance to different experimental infections. Receptors for this hormone can be found in distinct immune cells (especially macrophages) that are known to be the first line defense against Trypanosoma cruzi infection. These cells operate through an indirect pathway releasing nitric oxide (NO) and cytokines such TNF-alpha and IL-12 which in turn trigger an enhancement of natural killer cells and lymphocytes which finally secrete pro and anti-inflammatory cytokines. The effects of pre- and post-infection DHEA treatment on production of IL-12, TNF alpha and NO were evaluated. T. cruzi infected macrophages post treated with DHEA displayed enhanced concentrations of TNF-alpha, IL-12 and NO. Probably, the mechanisms that induced the production of cytokines by infected cells are more efficient when the immune system has been stimulated first by parasite invasion, suggesting that the protective role of DHEA is greater when administered post infection. (C) 2011 Elsevier B.V. All rights reserved.

Lipid microspheres loaded with antigenic membrane proteins of the Leishmania amazonensis as a potential biotechnology application

Lipid microspheres (LM) are excellent drug delivery or vaccines adjuvant systems and are relatively stable. The aim of this work is to develop and characterize a system that is able to encapsulate and present antigenic membrane proteins from Leishmania amazonensis. Membrane proteins are important for vaccine`s formulation because these proteins come in contact with the host cell first, triggering the cell mediated immune response. This is a useful tool to avoid or inactivate the parasite invasion. The LM are constituted by soybean oil (SO), dipalmitoylphosphatidilcholine (DPPC), cholesterol and solubilized protein extract (SPE). The particles formed presented an average diameter of 200 run, low polydispersion and good stability for a period of 30 days, according to dynamic light scattering assays. Isopycnic density gradient centrifugation of LM-protein showed that proteins and lipids floated in the sucrose gradient (5-50%w/v) suggesting that the LM-protein preparation was homogeneous and that the proteins are interacting with the system. The results show that 85% of SPE proteins were encapsulated in the LM. Studies of cellular viability of murine peritoneal macrophages show that our system does not present cytotoxic effect for the macrophages and still stimulates their NO production (which makes its application as a vaccine adjuvant possible). LM-protein loaded with antigenic membrane proteins from L. amazonensis seems to be a promising vaccine system for immunization against leishmaniasis. (C) 2009 Elsevier Inc. All rights reserved.

Involvement and interactions of different immune cells and their cytokines in human visceral leishmaniasis

Visceral leishmaniasis (VL) or kala-azar, a disseminated infection of the lymphoreticular system of the body, is marked by severe defect in immune system of the host. Successful cure of VL depends on the immune status of the host in combination with the effects of the antileishmanial drugs. The rationale approach towards eradication of this disease would be to potentiate the immune functioning of the host in addition to parasite killing. This review deals with different aspects of adaptive and innate immune responses and explores their role in protection or pathogenesis of VL. IL-10 has emerged as the principal cytokine responsible for disease pathogenesis, although evidences regarding its source during active VL remain inconclusive. On the other hand, IFNγ, under the influence of IL-12, is mostly correlated with healing of the disease. Chemokines are important in mounting cell-mediated immune response as they can prevent parasite invasion in association with cytokines. Different types of T cells like CD4, CD8 and NK T cells also contribute to the immunology of this disease. In spite of conflicting reports, the role of regulatory T cells in VL pathogenesis is important. Recently discovered Th17 subset and its different members have been reported to perform diverse functions in the course of VL and leishmaniasis as a whole. Innate immune responses, depending on the cell types, are essential in early parasite detection and subsequent development of an efficient NK cell response. Immunotherapy targeting IL-10 could be looked upon as an interesting option for the treatment of VL.

Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 µM) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.

Triatoma rubrovaria (Blanchard, 1843) (Hemiptera - Reduviidae - Triatominae) IV: bionomic aspects on the vector capacity of nymphs

Triatoma rubrovaria has become the most frequently captured triatomine species since the control of T. infestans in the state of Rio Grande do Sul (RS), Brazil. The aim of this study was to evaluate aspects of the vectorial competence of T. rubrovaria using nymphs raised in laboratory under environmental conditions of temperature and humidity and fed on mice. The average developmental period of T. rubrovaria was 180.1 days. The percentage of defecation shortly after feeding was still higher than previous studies in which samples of T. rubrovaria subjected to a slight starvation period before the blood meal were used. The obtained results support former indication that T. rubrovaria presents bionomic characteristics propitious to be a good vector of Trypanosoma cruzi to man. Therefore its domiciliary invasion process must be continuously monitored.

Antigenicity and immunogenicity of a novel chimeric peptide antigen based on the P. vivax circumsporozoite protein.

BACKGROUND: Plasmodium vivax circumsporozoite (PvCS) protein is a major sporozoite surface antigen involved in parasite invasion of hepatocytes and is currently being considered as vaccine candidate. PvCS contains a dimorphic central repetitive fragment flanked by conserved regions that contain functional domains. METHODS: We have developed a chimeric 137-mer synthetic polypeptide (PvCS-NRC) that includes the conserved region I and region II-plus and the two natural repeat variants known as VK210 and VK247. The antigenicity of PvCS-NRC was tested using human sera from PNG and Colombia endemic areas and its immunogenicity was confirmed in mice with different genetic backgrounds, the polypeptide formulated either in Alum or GLA-SE adjuvants was assessed in inbred C3H, CB6F1 and outbred ICR mice, whereas a formulation in Montanide ISA51 was tested in C3H mice. RESULTS: Antigenicity studies indicated that the chimeric peptide is recognized by a high proportion (60-70%) of residents of malaria-endemic areas. Peptides formulated with either GLA-SE or Montanide ISA51 adjuvants induced stronger antibody responses as compared with the Alum formulation. Sera from immunized mice as well as antigen-specific affinity purified human IgG antibodies reacted with sporozoite preparations in immunofluorescence and Western blot assays, and displayed strong in vitro inhibition of sporozoite invasion (ISI) into hepatoma cells. CONCLUSIONS: The polypeptide was recognized at high prevalence when tested against naturally induced human antibodies and was able to induce significant immunogenicity in mice. Additionally, specific antibodies were able to recognize sporozoites and were able to block sporozoite invasion in vitro. Further evaluation of this chimeric protein construct in preclinical phase e.g. in Aotus monkeys in order to assess the humoral and cellular immune responses as well as protective efficacy against parasite challenge of the vaccine candidate must be conducted.

Antigenicity and immunogenicity of a novel Plasmodium vivax circumsporozoite derived synthetic vaccine construct.

BACKGROUND: The circumsporozoite (CS) protein is a major malaria sporozoite surface antigen currently being considered as vaccine candidate. Plasmodium vivax CS (PvCS) protein comprises a dimorphic central repeat fragment flanked by conserved regions that contain functional domains involved in parasite invasion of host cells. The protein amino (N-terminal) flank has a cleavage region (region I), essential for proteolytic processing prior to parasite invasion of liver cells. METHODS: We have developed a 131-mer long synthetic polypeptide (LSP) named PvNR1R2 that includes the N-terminal flank and the two natural repeat variant regions known as VK210 and VK247. We studied the natural immune response to this region in human sera from different malaria-endemic areas and its immunogenicity in mice. RESULTS: PvNR1R2 was more frequently recognized by sera from Papua New Guinea (PNG) (83%) than by samples from Colombia (24%) when tested by ELISA. The polypeptide formulated in Montanide ISA51 adjuvant elicited strong antibody responses in both C3H and CB6F1 mice strains. Antibodies from immunized mice as well as affinity-purified human IgG reacted with native protein by IFA test. Moreover, mouse immune sera induced strong (90%) in vitro inhibition of sporozoite invasion (ISI) of hepatoma cell lines. CONCLUSIONS: These results encourage further studies in non-human primates to confirm the elicitation of sporozoite invasion blocking antibodies, to assess cell mediated immune responses and the protective efficacy of this polypeptide.

Exopolysaccharides produced by the symbiotic nitrogen-fixing bacteria of leguminosae

The process of biological nitrogen fixation (BNF), performed by symbiotic nitrogen fixing bacteria with legume species, commonly known as α and β rhizobia, provides high sustainability for the ecosystems. Its management as a biotechnology is well succeeded for improving crop yields. A remarkable example of this success is the inoculation of Brazilian soybeans with Bradyrhizobium strains. Rhizobia produce a wide diversity of chemical structures of exopolysaccharides (EPS). Although the role of EPS is relatively well studied in the process of BNF, their economic and environmental potential is not yet explored. These EPS are mostly species-specific heteropolysaccharides, which can vary according to the composition of sugars, their linkages in a single subunit, the repeating unit size and the degree of polymerization. Studies have showed that the EPS produced by rhizobia play an important role in the invasion process, infection threads formation, bacteroid and nodule development and plant defense response. These EPS also confer protection to these bacteria when exposed to environmental stresses. In general, strains of rhizobia that produce greater amounts of EPS are more tolerant to adverse conditions when compared with strains that produce less. Moreover, it is known that the EPS produced by microorganisms are widely used in various industrial activities. These compounds, also called biopolymers, provide a valid alternative for the commonly used in food industry through the development of products with identical properties or with better rheological characteristics, which can be used for new applications. The microbial EPS are also able to increase the adhesion of soil particles favoring the mechanical stability of aggregates, increasing levels of water retention and air flows in this environment. Due to the importance of EPS, in this review we discuss the role of these compounds in the process of BNF, in the adaptation of rhizobia to environmental stresses and in the process of soil aggregation. The possible applications of these biopolymers in industry are also discussed.

Reduced genetic diversity, increased isolation and multiple introductions of invasive giant hogweed in the western Swiss Alps.

Abstract The giant hogweed (Heracleum mantegazzianum) has successfully invaded 19 European countries as well as parts of North America. It has become a problematic species due to its ability to displace native flora and to cause public health hazards. Applying population genetics to species invasion can help reconstruct invasion history and may promote more efficient management practice. We thus analysed levels of genetic variation and population genetic structure of H. mantegazzianum in an invaded area of the western Swiss Alps as well as in its native range (the Caucasus), using eight nuclear microsatellite loci together with plastid DNA markers and sequences. On both nuclear and plastid genomes, native populations exhibited significantly higher levels of genetic diversity compared to invasive populations, confirming an important founder event during the invasion process. Invasive populations were also significantly more differentiated than native populations. Bayesian clustering analysis identified five clusters in the native range that corresponded to geographically and ecologically separated groups. In the invaded range, 10 clusters occurred. Unlike native populations, invasive clusters were characterized by a mosaic pattern in the landscape, possibly caused by anthropogenic dispersal of the species via roads and direct collection for ornamental purposes. Lastly, our analyses revealed four main divergent groups in the western Swiss Alps, likely as a consequence of multiple independent establishments of H. mantegazzianum.

What is the potential of spread in invasive bryophytes?

Although the number of invasive bryophytes is much lower than that of higher plants, they threaten habitats that are often species rich and of high conservation relevance. Their potential of spread has, however, never been determined. Here, we assess whether the three most invasive bryophyte species shifted their niche during the invasion process and whether the extent of the study area defined to calibrate the model (geographic background, GB) affects model transferability. We then determine whether ecological niche models (ENMs) developed in their native range can be projected in other areas to assess their invasive potential. The macroclimatic niches of Campylopus introflexus, Orthodontium lineare and Lophocolea semiteres were compared in their native range (Southern Hemisphere) and in their invasion range (Northern Hemisphere) using ordination techniques. ENMs from an ensemble model were calibrated in the native range and projected onto the Northern Hemisphere using different GBs. No evidence for niche expansion in the invaded range was found and the species occur in the invaded range under climate conditions that are similar to those in the native range. The performance of the models to predict occurrences in the invaded range increased with the extent of the GB. The potential range of all species included entire regions on continents where they are still absent. The expansion of the investigated species appears to be constrained by climate conditions that are similar to those currently prevailing in their native range, which is consistent with our failure to demonstrate macroclimatic niche shift in the invaded range. The use of large GBs is recommended in such vagile organisms with large, disjunct distributions. The models indicated that invasive bryophyte species might become a threat in central and eastern Europe, North America and eastern Asia if accidentally introduced or naturally dispersed.

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.

Localization of Cathepsins D and B at the Maternal-Fetal Interface and the Invasiveness of the Trophoblast during the Postimplantation Period in the Mouse

A survey of existing data suggests that trophoblast cells produce factors involved in extracellular matrix degradation. In this study, we correlated the expression of cathepsins D and B in the murine ectoplacental cone with the ultrastructural progress of decidual invasion by trophoblast cells. Both proteases were immunolocalized at implantation sites in lysosome-endosome-like compartments of trophoblast giant cells. Cathepsin D, but not cathepsin B, was also detected ultrastructurally in extracellular compartments surrounded by processes of the invading trophoblast containing extracellular matrix components and endometrial cell debris. The expression of cathepsins D and B by trophoblast cells was confirmed by RT-PCR in ectoplacental cones isolated from implantation chambers at gestation day 7.5. Our data addressed a positive relationship between the expression and presence of cathepsin D at the extracellular compartment of the maternal-fetal interface and the invasiveness of the trophoblast during the postimplantation period, suggesting a participation of invading trophoblast cells in the cathepsin D release. Such findings indicate that mouse trophoblast cells might exhibit a proteolytic ability to partake in the decidual invasion process at the maternal-fetal interface. Copyright (C) 2010 S. Karger AG, Basel