An evaluation of lipid metabolism in the insect trypanosomatid Herpetomonas muscarum uncovers a pathway for the uptake of extracellular insect lipoproteins.

Lipid uptake and metabolism by trypanosomatid parasites from vertebrate host blood have been well established in the literature. However, there is a lack of knowledge regarding the same aspects concerning the parasites that cross the hemolymph of their invertebrate hosts. We have investigated the lipid composition and metabolism of the insect trypanosomatid Herpetomonas muscarum by 3H- palmitic acid and phosphate (32Pi) and the parasite interaction with Lipophorin (Lp) the main lipid carrying protein of insect hemolymph. Gas chromatography-mass spectrometry (GC-MS) analyses were used to identify the fatty acids and sterols composition of H.muscarum. Furthermore, we investigated the Lp binding site in the plasma membrane of parasite by Immunolocalization. We showed that H. muscarum incorporated 3H-palmitic acid and inorganic phosphate (32Pi) which were readily used as precursor molecules of lipid biosynthetic pathways. Furthermore, H. muscarum was able to take up both protein and lipid moieties of Lp which could be used as nutrient sources. Moreover, we have also demonstrated for the first time the presence of a Lp binding site in the membrane of a parasite. Such results point out the role of describing the metabolic pathways of trypanosomatids in order to provide a better understanding of parasite-host interaction peculiarities. Such studies may enhance the potential form the identification of novel chemotherapeutic targets in harmful parasites.

Transovum transmission of trypanosomatid cysts in the Milkweed bug, Oncopeltus fasciatus.

Leptomonas wallacei is a trypanosomatid that develops promastigotes and cystic forms in the gut of the hemipteran insect Oncopeltus fasciatus. Insect trypanosomatids are thought to be solely transmitted from one host to another through the ingestion of parasite-contaminated feces. However, here we show that L. wallacei cysts present on the eggshells of eggs laid by O. fasciatus can also act as infective forms that are transmitted to the insect offspring. Newly hatched O. faciatus nymphs are parasite-free, but some of them become contaminated with L. wallacei after feeding on eggshell remnants. The present study is the first report of transovum transmission of a trypanosomatid, a process that may have a relevant role in parasite's within-host population dynamics.

Glycoinositolphospholipids from Trypanosomatids subvert nitric oxide production in Rhodnius prolixus salivary glands.

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 FINDINGS: Here 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/SIGNIFICANCE: Taken 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.

Endophytic colonization of rice (Oryza sativa L.) by the diazotrophic bacterium Burkholderia kururiensis and its ability to enhance plant growth.

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.

Endophytic colonization of rice (Oryza sativa L.) by the diazotrophic bacterium Burkholderia kururiensis and its ability to enhance plant growth

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.

Burkholderia kururiensis é uma bactéria diazotrófica, originalmente isolada de um ambiente aquático poluído e apresenta alto nível de similaridade com a espécie endofítica "B. brasilensis" encontrada na planta de arroz. Este artigo demonstrou a habilidade de B. kururiensis colonizar endofiticamente plântulas de arroz, após esta bactéria ter sido inoculada na raiz das plantas. Esta capacidade foi confirmada pelo crescimento bacteriano em diferentes tecidos da planta, por microscopia eletrônica e pela análise do 16S rADN. Observação por microscopia eletrônica das raízes, caule e folhas das plântulas de arroz inoculadas, revelou predominância da colonização de B. kururiensis na zona pilífera da raiz, demonstrando que a colonização endofítica inicia-se na endoderme, espalha-se pelo xilema, sendo esta a possível via para a bactéria alcançar as partes aéreas. A adição de uma fonte de nitrogênio, embora não tenha influenciado no crescimento bacteriano, foi um fator limitante para a colonização endofítica. Como a colonização endofítica mostrou-se diretamente associada ao aumento no desenvolvimento da planta, a produção do fitohormônio auxina/ácido 3-indolacético pela B. kururiensis foi verificada utilizando uma plântula de arroz transgênica, contendo o repórter responsivo para auxina (DR5-GUS). Nossos resultados sugerem que a produção de auxina pela B. kururiensis é responsável pelo estímulo no desenvolvimento da planta verificado pela ativação do DR5-GUS. Neste trabalho demonstramos, pela primeira vez, a habilidade de B. kururiensis colonizar endofiticamente a planta de arroz, promovendo tanto o aumento no crescimento da planta como a produção de sementes de arroz.

The toxic effects of piperine against Trypanosoma cruzi: ultrastructural alterations and reversible blockage of cytokinesis in epimastigote forms.

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.

Development of a ligand blot assay using biotinylated live cells.

Adhesive interactions between cells are critical to a variety of processes, including host-pathogen relationships. The authors have developed a new technique for the observation of binding interactions in which molecules obtained from excised tissues are resolved by gel electrophoresis and transferred to a membrane. Biotinylated live cells are then kept in contact with that membrane, and their interactions with proteins of interest are detected by peroxidase-labeled streptavidin, followed by a biotin-streptavidin detection system. The adhesion proteins can eventually be identified by cutting the relevant band(s) and performing mass spectrometry or other amino acid-sequencing methods. The technique described here allows for the identification of both known and novel adhesion molecules capable of binding to live cells, among a complex mixture and without previous isolation or purification. This is especially important for the analysis of host-parasite interactions and may be extended to other types of cell-cell interactions.

Effect of Urtica dioica agglutinin and Arabidopsis thaliana Chia4 chitinase on the protozoan Phytomonas françai.

The genus Phytomonas is responsible for many diseases in different crop plant species. The finding that chitin is an exposed cell surface polysaccharide in Phytomonas françai and the observation that chitinases can inhibit fungal growth raises expectations about the potential effect of plant chitinases on the P. françai cell membrane surface. The plant chitinases Urtica dioica agglutinin (UDA) and Arabidopsis thaliana Chia4 (ATCHIT4) proteins were over-expressed in bacteria and the interaction between these proteins and P. françai surface was analyzed by immunocytochemistry. We showed that UDA and ATCHIT4 proteins can interact with surface-exposed chitin from P. françai.

Interaction of Leptomonas wallacei with the intestinal tract of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae).

While investigating the distribution of Leptomonas wallacei in the intestine of the insect host Oncopeltus fasciatus, promastigotes and cyst-like forms of L. wallacei were observed only in the midgut ventricles V(3) and V(4) and the hindgut. In video-microscopy, once contact had occurred, the parasites remained attached to the midgut epithelium. Scanning electron microscopy revealed the adhesion of flagellates and cyst-like forms to the midgut wall and to the rectal pads of the hindgut. Using transmission electron microscopy, we observed that adhesion occurred mainly between the flagellum and the perimicrovillar membranes secreted by the midgut epithelium. No modifications were observed either in the parasite or in the epithelial cells. In the hindgut, adhesion to the superficial wax layer of the epithelial cells of the rectal pads was via flagellum. Host cell morphology appeared unaffected by L. wallacei.

Leptomonas wallacei shows distinct morphology and surface carbohydrates composition along the intestinal tract of its host Oncopeltus fasciatus (Hemiptera: Lygaeidae) and in axenic culture.

Leptomonas wallacei is a monoxenic trypanosomatid that colonizes the digestive tract of the phytophagous hemipteran Oncopeltus fasciatus. This infection was specific and took place exclusively in midgut intestinal ventricles V3 and V4, and in the hindgut. Abundances of parasites in the hindgut were 54% less than those in the hindgut. Parasites in the hindgut were more slender and had a longer flagellum than those from the hindgut, which were rounded, with a shorter flagellum. Moreover, hindgut forms expressed sugar residues on the cell surface, recognized by the lectins from Griffonia simplicifolia-I (alpha-galactose, alpha-N-acetyl-galactosamine) and Helix pomatia (N-acetyl-galactosamine); those sugar residues were not present in protozoa from the midgut. In culture, parasites were morphologically similar to midgut forms, but differed from them because they did not express sugar residues that bind to lectin (beta-galactose(1-3) N-acetyl-galactosamine) from Arachis hypogaea.

The role of eicosanoids on Rhodnius heme-binding protein (RHBP) endocytosis by Rhodnius prolixus ovaries.

The participation of eicosanoids and second messengers on the regulation of RHBP endocytosis by the ovaries was investigated, using [(125)I]RHBP in experiments in vivo and in vitro. Addition of PGE(2) (one of the products of the cyclooxygenase pathway) decreased in vitro the uptake of RHBP by 35%. The rate of RHBP endocytosis increased in the presence of indomethacin, a potent cyclooxigenase inhibitor, up to 50% in vitro and up to 55% in vivo, thus giving support to the role of cyclooxygenase derivatives on endocytosis regulation. The amount of PGE(2) secreted to the culture medium by the cells of Rhodnius prolixus ovaries was 1.1 ng/ovary following RHBP uptake assay. The amount of PGE(2) decreases approximately 25% in the presence of 5 microM indomethacin. Using a scanning electron microscope we have observed that neither the surface area nor the patencies of follicle cells were affected by treatment with indomethacin, thus suggesting that, its effect is elicited in the oocyte. Finally, we have identified two ovarian peptides that were dephosphorylated after the indomethacin treatment (18 and 25 kDa). Taken together these data show that local mediators such as eicosanoids act upon the oocytes controlling RHBP endocytosis, perhaps using the protein phosphorylation signal transduction pathway.