Localization of Pantoea ananatis inside lesions of maize White Spot Disease using transmission electron microscopy and molecular techniques

The etiological agent of maize white spot (MWS) disease has been a subject of controversy and discussion. Initially the disease was described as Phaeosphaeria leaf spot caused by Phaeosphaeria maydis. Other authors have Suggested the existence of different fungal species causing similar symptoms. Recently, a bacterium, Pantoea ananatis, was described as the causal agent of this disease. The purpose of this Study was to offer additional information on the correct etiology of this disease by providing visual evidence of the presence of the bacterium in the interior of the MWS lesions by using transmission electron microscopy (TEM) and molecular techniques. The TEM allowed Visualization of a large amount of bacteria in the intercellular spaces of lesions collected from both artificially and naturally infected plants. Fungal structures were not visualized in young lesions. Bacterial primers for the 16S rRNA and rpoB genes were used in PCR reactions to amplify DNA extracted from water-soaked (young) and necrotic lesions. The universal fungal oligonucleotide ITS4 was also included to identity the possible presence of fungal structures inside lesions. Positive PCR products from water-soaked lesions, both from naturally and artificially inoculated plants, were produced with bacterial primers, whereas no amplification was observed when ITS4 oligonucleotide was used. On the other hand, DNA amplification with ITS4 primer was observed when DNA was isolated from necrotic (old) lesions. These results reinforced previous report of P. ananatis as the primary pathogen and the hypothesis that fungal species may colonize lesions pre-established by P. ananatis.

Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy

Endophytes are microorganisms that colonize plant tissues internally without causing harm to the host. Despite the increasing number of studies on sweet orange pathogens and endophytes, yeast has not been described as a sweet orange endophyte. In the present study, endophytic yeasts were isolated from sweet orange plants and identified by sequencing of internal transcribed spacer (ITS) rRNA. Plants sampled from four different sites in the state of Sao Paulo, Brazil exhibited different levels of CVC (citrus variegated chlorosis) development. Three citrus endophytic yeasts (CEYs), chosen as representative examples of the isolates observed, were identified as Rhodotorula mucilaginosa, Pichia guilliermondii and Cryptococcus flavescens. These strains were inoculated into axenic Citrus sinensis seedlings. After 45 days, endophytes were reisolated in populations ranging from 10(6) to 10(9) CFU/g of plant tissue, but, in spite of the high concentrations of yeast cells, no disease symptoms were observed. Colonized plant material was examined by scanning electron microscopy (SEM), and yeast cells were found mainly in the stomata and xylem of plants, reinforcing their endophytic nature. P. guilliermondii was isolated primarily from plants colonized by the causal agent of CVC, Xylella fastidiosa. The supernatant from a culture of P. guilliermondii increased the in vitro growth of X. fastidiosa, suggesting that the yeast could assist in the establishment of this pathogen in its host plant and, therefore, contribute to the development of disease symptoms.

Direct effect of Plasmodium vivax recombinant vaccine candidates AMA-1 and MSP-1(19) on the innate immune response

The recombinant apical membrane antigen 1 (AMA-1) and 19-kDa fragment of merozoite surface protein (MSP-1(19)) are the lead candidates for inclusion in a vaccine against blood stages of malaria due to encouraging protective studies in humans and animals. Despite the importance of an efficacious malaria vaccine, vaccine-related research has focused on identifying antigens that result in protective immunity rather than determining the nature of anti-malarial immune effector mechanisms. Moreover, emphasis has been placed on adaptive rather than innate immune responses. In this study, we investigated the effect of Plasmodium vivax vaccine candidates Pv-AMA-1 and Pv-MSP-1(19) on the immune response of malaria-naive donors. Maturation of dendritic cells is altered by Pv-AMA-1 but not Pv-MSP-1(19), as observed by differential expression of cell surface markers. In addition, Pv-AMA-1 induced an increased production of MIP-1 alpha/CCL3 and decreased production of TARC/CCL17 levels in both dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs). Finally, a significant pro-inflammatory response was elicited by Pv-AMA-1-stimulated PBMCs. These results suggest that the recombinant vaccine candidate Pv-AMA-1 may play a direct role on innate immune response and might be involved in parasite destruction. (C) 2007 Elsevier Ltd. All rights reserved.

Plasmodium vivax recombinant vaccine candidate AMA-1 plays an important role in adaptive immune response eliciting differentiation of dendritic cells

The Apical Membrane Antigen-1 (AMA-1) is a well-characterized and functionally important merozoite protein and is currently considered a major candidate antigen for a malaria vaccine. Previously, we showed that AMA-1 has an influence on cellular immune responses of malaria-naive subjects, resulting in an alternative activation of monocyte-derived dendritic cells and induction of a pro-inflammatory response by stimulated PBMCs. Although there is evidence, from human and animal malaria model systems that cell-mediated immunity may contribute to both protection and pathogenesis, the knowledge on cellular immune responses in vivax malaria and the factors that may regulate this immunity are poorly understood. In the current work, we describe the maturation of monocyte-derived dendritic cells of P. vivax naturally infected individuals and the effect of P. vivax vaccine candidate Pv-AMA-1 on the immune responses of the same donors. We show that malaria-infected subjects present modulation of DC maturation, demonstrated by a significant decrease in expression of antigen-presenting molecules (CD1a, HLA-ABC and HLA-DR), accessory molecules (CD40, CD80 and CD86) and Fc gamma RI (CD64) receptor (P <= 0.05). Furthermore, Pv-AMA-1 elicits an upregulation of CD1a and HLA-DR molecules on the surface of monocyte-derived dendritic cells (P=0.0356 and P=0.0196, respectively), and it is presented by AMA-1-stimulated DCs. A significant pro-inflammatory response elicited by Pv-AMA-1-pulsed PBMCs is also demonstrated, as determined by significant production of TNF-alpha, IL-12p40 and IFN-gamma (P <= 0.05). Our results suggest that Pv-AMA-1 may partially revert DC down-modulation observed in infected subjects, and exert an important role in the initiation of pro-inflammatory immunity that might contribute substantially to protection. (c) 2009 Elsevier Ltd. All rights reserved.

Violet ZnSe/ZnS as an Alternative to Green CdSe/ZnS in Nanocrystal-Fluorescent Protein FRET Systems

Fluorescent proteins from the green fluorescent protein family strongly interact with CdSe/ZnS and ZnSe/ZnS nanocrystals at neutral pH. Green emitting CdSe/ZnS nanocrystals and red emitting fluorescent protein dTomato constitute a 72% efficiency FRET system with the largest alteration of the overall photoluminescence profile, following complex formation, observed so far. The substitution of ZnSe/ZnS for CdSe/ZnS nanocrystals as energy donors enabled the use of a green fluorescent protein, GFP5, as energy acceptor. Violet emitting ZnSe/ZnS nanocrystals and green GFP5 constitute a system with 43% FRET efficiency and an unusually strong sensitized emission. ZnSe/ZnS-GFP5 provides a cadmium-free, high-contrast FRET system that covers only the high-energy part of the visible spectrum, leaving room for simultaneous use of the yellow and red color channels. Anisotropic fluorescence measurements confirmed the depolarization of GFP5 sensitized emission.

Plasmodium vivax apical membrane antigen-1: comparative recognition of different domains by antibodies induced during natural human infection

The Apical Membrane Antigen-1 (AMA-1) of Plasmodium sp. has been suggested as a vaccine candidate against malaria. This protein seems to be involved in merozoite invasion and its extra-cellular portion contains three distinct domains: DI, DII, and DIII. Previously, we described that Plasmodium vivax AMA-1 (PvAMA-1) ectodomain is highly immunogenic in natural human infections. Here, we expressed each domain, separately or in combination (DI-II or DII-III), as bacterial recombinant proteins to map immunodominant epitopes within the PvAMA-1 ectodomain. IgG recognition was assessed by ELISA using sera of P. vivax-infected individuals collected from endemic regions of Brazil or antibodies raised in immunized mice. The frequencies of responders to recombinant proteins containing the DII were higher than the others and similar to the ones observed against the PvAMA-1 ectodomain. Moreover, ELISA inhibition assays using the PvAMA-1 ectodomain as substrate revealed the presence of many common epitopes within DI-II that are recognized by human immune antibodies. Finally, immunization of mice with the PvAMA-1 ectodomain induced high levels of antibodies predominantly to DI-II. Together, our results indicate that DII is particularly immunogenic during natural human infections, thus indicating that this region could be used as part of an experimental sub-unit vaccine to prevent vivax malaria. (C) 2008 Elsevier Masson SAS. All rights reserved.

Biophysical characterization of the recombinant merozoite surface protein-3 of Plasmodium vivax

Plasmodium vivax Merozoite Surface Protein-3 alpha and 3 beta are members of a family of related merozoite surface proteins that contain a central alanine-rich domain with heptad repeats that is predicted to form alpha-helical secondary and coiled-coil tertiary structures. Seven recombinant proteins representing different regions of MSP-3 alpha and MSP-3 beta of P. vivax were generated to investigate their structure. Circular dichroism spectra analysis revealed that some proteins are folded with a high degree of alpha-helices as secondary structure, whereas other products contain a high content of random coil. Using size exclusion chromatography, we found that the two smaller fragments of the MSP-3 alpha, named CC4 and CC5, predicted to form coiled-coil (CC) structures, eluted at volumes corresponding to molecular weights larger than their monomeric masses. This result suggests that both proteins are oligomeric molecules. Analytical ultracentrifugation experiments showed that the CC5 oligomers are elongated molecules. Together, these data may help to understand important aspects of P. vivax biology. (C) 2008 Elsevier B.V. All rights reserved.

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund`s Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria. (C) 2010 Elsevier Ltd. All rights reserved.

On the Cytoadhesion of Plasmodium vivax-Infected Erythrocytes

Background. Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. Methods. Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. Results. Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. Conclusions. These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

Molecular characterization of the Aspergillus fumigatus NCS-1 homologue, NcsA

Here, we characterize the Aspergillus homologue ncsA Neuronal Calcium Sensor. We that ncsA is not an essential gene and Delta ncsA growth decreased in the presence of EGTA and SDS. the Delta ncsA mutant is more resistant to calcium NcsA: mRFP localizes to the cytoplasm and its localization is not affected by the cellular response to calcium chloride or EGTA. The Delta ncsA mutant strain more sensitive to voriconazole, itraconazole, and Polar growth in the Delta ncsA mutant was also more aVected by lovastatin than in the wild type The Spitzenkorper can be visualized in both strains although the vacuolar system does not seem to be very different, there is an increase in the staining intensity on the germling surface of the Delta ncsA strain. NcsA promotes pmcA and pmcB expression and therefore there is a reduced expression of these ion pumps in the Delta ncsA mutant background, and also of other genes involved in the response to calcium in A. fumigatus. The ncsA inactivation mutation is not causing loss of virulence in a low dose murine infection when compared to the corresponding wild type strain.

Role of phospholipase C and protein kinase C in Aspergillus nidulans during growth on pectin or glucose: Effects on germination and duplication cycle

The effects of PLC and Pkc inhibitors on Aspergillus nidulans depend on the carbon source. PLC inhibitors Spm and C48/80 delayed the first nuclear division in cultures growing on glucose, but stimulated it in media supplemented with pectin. Less intense were these effects on the mutant transformed with PLC-A gene rupture (AP27). Neomycin also delayed the germination in cultures growing on glucose or pectin; however, on glucose, the nuclear division was inhibited whereas in pectin it was stimulated. These effects were minor in AP27. The effects of Ro-31-8425 and BIM (both Pkc inhibitors) were also opposite for cultures growing on glucose or pectin. On glucose cultures of both strains BIM delayed germination and the first nuclear division, whereas on pectin both parameters were stimulated. Opposite effects were also detected when the cultures were growing on glucose or pectin in the presence of Ro-31-8425.

A stabilized demethoxyviridin derivative inhibits PI3 kinase

The viridins like demethoxyviridin (Dmv) and wortmannin (Wm) are nanomolar inhibitors of the PI3 kinases, a family of enzymes that play key roles in a host of regulatory processes. Central to the use of these compounds to investigate the role of PI3 kinase in biological systems, or as scaffolds for drug development, are the interrelated issues of stability, chemical reactivity, and bioactivity as inhibitors of PI3 kinase. We found that Dmv was an even more potent inhibitor of PI3 kinase than Wm. However, Dmv was notably less stable than Wm in PBS, with a half-life of 26 min versus Wm`s half-life of 3470 min. Dmv, like Wm, disappeared in culture media with a half-life of less than 1 min. To overcome Dmv`s instability, it was esterified at the C1 position, and then reacted with glycine at the C20 position. The resulting Dmv derivative, termed SA-DmvC20-Gly had a half-life of 218 min in PBS and 64 min in culture media. SA-DmvC20-Gly underwent an exchange reaction at the C20 position with N-acetyl lysine in a manner similar to a WmC20 derivative, WmC20-Proline. SA-DmvC20-Gly inhibited PI3 kinase with an IC(50) of 44 nM, compared to Wm`s IC(50) of 12 nM. These results indicate that the stability of Dmv can be manipulated by reactions at the C1 and C20 positions, while substantially maintaining its ability to inhibit PI3 kinase. Our results indicate it may be possible to obtain stabilized Dmv derivatives for use as PI3 kinase inhibitors in biological systems. (C) 2009 Elsevier Ltd. All rights reserved.

A new thrombospondin-related anonymous protein homologue in Neospora caninum (NcMIC2-like1)

Neospora caninum is an Apicomplexan protozoan that has the dog as a definitive host and cattle (among other animals) as intermediate hosts. It causes encephalopathy in dogs and abortion in cows, with significant loss in worldwide livestock. As any Apicomplexan, the parasite invades the cells using proteins contained in the phylum-specific organelles, like the micronemes, rhoptries and dense granules. The aim of this study was the characterization of a homologue (denominated NcMIC2-like1) of N. caninum thrombospondin-related anonymous protein (NcMIC2), a micronemal protein previously shown to be involved in the attachment and connection with the intracellular motor responsible for the active process of invasion. A polyclonal antiserum raised against the recombinant NcMIC2-like1 functional core (thrombospondin and integrin domains) recognized the native form of NcMIC2-like1, inhibited the in vitro invasion process and localized NcMIC2-like1 at the apical complex of the parasite by confocal immunofluorescence, indicating its micronemal localization. The new molecule, NcMIC2-like1, has features that differentiates it from NcMIC2 in a substantial way to be considered a homologue dagger.

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.

Identification of Plasmodium relictum causing mortality in penguins (Spheniscus magellanicus) from Sao Paulo Zoo, Brazil

This study reports avian malaria caused by Plasmodium relictum in Magellanic Penguins (Spheniscus magellanicus) from Sao Paulo Zoo. The disease was highly infective among the birds and was clinically characterized by its acute course and high mortality. The penguins of Sao Paulo Zoo were housed for at least 2 years without malaria; however, they had always been maintained in an enclosure protected from mosquito exposure during the night period. When they presented pododermatitis, they were freed at night for a short period. sao Paulo Zoo is located in one of the last forest remnants of the city, an area of original Atlantic forest. In the winter, the space destined for Zoo birds is shared with migratory species. Hence the possibility exists that the disease was transmitted to the penguins by mosquitoes that had previously bitten infected wild birds. Avian malaria parasites are transmitted mainly by mosquitoes of the genera Aedes and Culex, common vectors in the Atlantic forest. In this study, one Culex (Cux.) sp. was found, infected with P. relictum. There are diverse problems in housing distinct species of animals in captivity, principally when occupying the same enclosure, since it facilitates the transmission of diseases with indirect cycles, as is the case of Plasmodium spp., because certain species that cause discrete infections in some bird species can become a serious danger for others, especially penguins, which do not possess natural resistance. Thus, serious implications exist for periodically testing and administrating malaria therapy in captive penguins potentially exposed to mosquitoes during the night period, as well as other captive birds from Sao Paulo Zoo. (C) 2010 Elsevier B.V. All rights reserved.