Burkholderia kururiensis is a diazotrophic bacterium originally isolated from a polluted aquifer environment and presents a high level of similarity with the rice endophyte "B. brasilensis" species. This work assessed the ability of B. kururiensis to endophytically colonize rice plantlets by monitoring different tissues of root-inoculated plants for the presence of bacterial growth in different media, electron microscopy and by 16S rDNA analysis. Observations of roots, stems and leaves of inoculated rice plantlets by electron microscopy revealed B. kururiensis colonization predominantly on root hair zones, demonstrating endophytic colonization primarily through the endodermis, followed by spreading into xylem vessels, a possible pathway leading to aerial parts. Although indifferent for the bacterial growth itself, addition of a nitrogen source was a limiting factor for endophytic colonization. As endophytic colonization was directly associated to an enhanced plant development, production of phytohormone auxin/indole-3-acetic acid by B. kururiensis was assayed with transgenic rice plantlets containing an auxin-responsive reporter (DR5-GUS). Our findings suggest the ability of auxin production by plant-associated B. kururiensis which may have a stimulatory effect on plant development, as evidenced by activation of DR5-GUS. We hereby demonstrate, for the first time, the ability of B. kururiensis to endophytically colonize rice, promoting both plant growth and rice grain yield.

Promastigote forms of a trypanosomatid were isolated from the third and fourth ventricles of the midgut and from the hindgut of the phytophagous hemipteran Oncopeltus fasciatus. Some individuals had adhered to its anterior region, close to the flagellar pocket, or to the flagellum up to four rounded aflagellated forms known as straphangers cysts. Scanning electron microscopy revealed that the flagellated forms presented a twisted cell body and a long flagellum, and the cysts, smaller than the parental promastigote, had a nascent flagellum. Transmission electron microscopy showed that promastigotes were typical, while cystic forms were ovoid dense cells devoid of a cyst wall, but presenting a cell coat, a special subpellicular region limited by a membrane unit, and a condensed cytoplasm. The kinetoplast-DNA fibrils appeared as dense spots and the condensed chromatin was arranged in a labyrinthic structure. Desmosome-like structures, observed in the region of adhesion of the precystic forms to the parental promastigote, could explain how cysts remain attached to the mother cell during the encystation process. Release of membranes from the surface of promastigotes and cysts seems to be correlated with the condensation of the cytoplasm during encystment. Morphological and isozyme analyses indicated that this trypanosomatid belongs to the genus Leptomonas. The molecular karyotype of this isolate was compared with that of a strain of Leptomonas oncopelti obtained from Oncopeltus varicolor by contour-clamped homogeneous electric field (CHEF) electrophoresis and revealed similar DNA banding patterns between 2,200-825 Kb, but not in lower bands (825-225 Kb). This suggested that the isolate from O. fasciatus and that from O. varicolor were not identical. Based on our findings we are describing Leptomonas wallacei n. sp. for our isolate from O. fasciatus.

In a previous work, we have investigated the effects of piperine and several of its chemical derivatives on the proliferation of the protozoan parasite Trypanosoma cruzi. It was observed that natural piperine is more active against intracellular amastigotes than axenically grown epimastigotes with IC50 values of 4.91 and 7.36 microM, respectively. Despite its superior trypanocidal activity against the intracellular amastigotes, here, we show that piperine did not enhance microbiocidal characteristics of murine peritoneal macrophages (Mø) based on nitric oxide production. As shown by light and electron microscopy analysis, epimastigotes treated with sublethal concentrations of piperine presented a reversible cell cycle arrestment and become round shaped, with swelling of the mitochondrion matrix and intense intracellular vacuolization with structures displaying complex membrane invaginations. Similar to the effects of exposing epimastigotes to the antitumor and microtubule stabilizer taxol, multiplication of cell organelles such as the flagellum, kinetoplast, and nucleus occurred, but division into daughter cells was impaired. Unlike the effects caused by the anti-microtubular vinca alkaloids vincristine and vinblastine, which also induce cytokinesis arrestment in T. cruzi epimastigotes, piperine did not induce the formation of giant multinucleated cells. The data reinforce the selectivity of the mechanisms of action of piperine against T. cruzi.

Background Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands.Methodology/Principal FindingsHere we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities.Conclusions/SignificanceTaken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.

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