Aedes fluviatilis cell lines as new tools to study metabolic and immune interactions in mosquito‑Wolbachia symbiosis

In the present work, we established two novel embryonic cell lines from the mosquito Aedes fluviatilis containing or not the naturally occurring symbiont bacteria Wolbachia, which were called wAflu1 and Aflu2, respectively. We also obtained wAflu1 without Wolbachia after tetracycline treatment, named wAflu1.tet. Morphofunctional characterization was performed to help elucidate the symbiont-host interaction in the context of energy metabolism regulation and molecular mechanisms of the immune responses involved. The presence of Wolbachia pipientis improves energy performance in A. fluviatilis cells; it affects the regulation of key energy sources such as lipids, proteins, and carbohydrates, making the distribution of actin more peripheral and with extensions that come into contact with neighboring cells. Additionally, innate immunity mechanisms were activated, showing that the wAflu1 and wAflu1.tet cells are responsive after the stimulus using Gram negative bacteria. Therefore, this work confirms the natural, mutually co-regulating symbiotic relationship between W. pipientis and A. fluviatilis, modulating the host metabolism and immune pathway activation. The results presented here add important resources to the current knowledge of Wolbachia-arthropod interactions

The roles of haemolymphatic lipoproteins in the oogenesis of Rhodnius prolixus

The fates of purified 32P-vitellin and 32P-lipophorin were followed in vitellogenic females of Rhodnius prolixus. While the radioactivity from 32P-vitellin 6 hours after injection was found almost exclusively in the ovary, the radioactivity from injected 32P-lipophorin was found distributed among several organs. In the ovary, the radioactivity from 32P-vitellin was associated with the contents of the yolk granules. 32P-lipophorin delivered a great amount of radioactive phospholipids to the ovary with no accumulation of its protein moiety, as observed after its iodination with 131I. The delivery of phospholipids was inhibited at 0ºC and by the metabolic inhibitors, sodium azide and sodium fluoride. Comparison of the radioactivity incorporation from 32P-lipophorin with that of 14C-inulin suggests that the 32P-phospholipids from lipophorin are not taken up by fluid phase endocytosis. The data presented here are compatible with the concept of lipophorin as a carrier of lipids in insects and provide evidence that lipophorin transports phospholipids as shown previously for other classes of lipids. The utilization by the oocytes of the phospholipids transported by lipophorin is discussed

Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp (125I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when 125I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a Kdof 7.1 ± 0.9 x 10-8M and a maximal binding capacity of 430 ± 40 ng 125I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma

Localization and function of Rhipicephalus (Boophilus) microplus vitellin-degrading cysteine endopeptidase

The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite’s physiology. The vitellin-degrading cysteine endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate cysteine endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development

Localization and function of Rhipicephalus (Boophilus) microplus vitellin-degrading cysteine endopeptidase

The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite’s physiology. The vitellin-degrading cysteine endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate cysteine endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development

Leishmania amazonensis: characterization of an ecto-3´-nucleotidase activity and its possible role in virulence

Submitted by Martha Martínez Silveira ([email protected]) on 2015-03-31T16:09:19Z No. of bitstreams: 1 Palleta-Silva R Leishmania amazonienses.....pdf: 603227 bytes, checksum: 83ce2fdf0647e6b62e497920ee4e59cc (MD5)Approved for entry into archive by Martha Martínez Silveira ([email protected]) on 2015-03-31T16:27:40Z (GMT) No. of bitstreams: 1 Palleta-Silva R Leishmania amazonienses.....pdf: 603227 bytes, checksum: 83ce2fdf0647e6b62e497920ee4e59cc (MD5)Made available in DSpace on 2015-03-31T16:27:40Z (GMT). No. of bitstreams: 1 Palleta-Silva R Leishmania amazonienses.....pdf: 603227 bytes, checksum: 83ce2fdf0647e6b62e497920ee4e59cc (MD5) Previous issue date: 2011Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem. Rio de Janeiro, RJ, BrasilUniversidade Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Prof. Paulo de Góes. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem. Rio de Janeiro, RJ, BrasilEcto-30-nucleotidase/nuclease (30NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 30mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-30-nucleotidase activity (La30-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La30-nucleotidase inhibitor and approach the possible involvement of ecto-30-nucleotidase in cellular adhesion. La30-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La30-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu2+ ions. Interestingly, ecto-30-nucleotidase activity is 60-fold higher than that of ecto-50-nucleotidase in L. amazonensis. Additionally, ecto-30-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage–parasite attachment/invasion was increased by 400% in the presence of adenosine 30-monophosphate (30AMP); however, this effect was reverted by TTM treatment. We believe that La30-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infectio

Synthesis and mobilization of glycogen and trehalose in adult male

15 p. : il.The vector of Chagas’ disease, Rhodnius prolixus, feeds exclusively on blood. The blood meals are slowly digested, and these insects wait some weeks before the next meal. During the life of an insect, energy-requiring processes such as moulting, adult gonadal and reproductive growth, vitellogenesis, muscular activity, and fasting, lead to increased metabolism. Carbohydrates are a major source of energy and their mobilization is important. We determined the amounts of glycogen, trehalose, and glucose present in the fat body and/or hemolymph of adult males of R. prolixus and recorded the processes of accumulation and mobilization of these carbohydrates. We also tested our hypothesis that these processes are under endocrine control. The amount of glycogen in the fat body progressively increased until the fourth day after feeding (from 9.372.2 to 77.377.5 mg/fat body), then declined to values around 36.374.9 mg/fat body on the fifteenth day after the blood meal. Glycogen synthesis was eliminated in decapitated insects and headtransplanted insects synthesized glycogen. The amount of trehalose in the fat body increased until the sixth day after feeding (from 16. 671.7 to 40. 675.3 nmol/fat body), decreased abruptly, and stabilized between days 7 and 15 at values ranging around 15–19 nmol/fat body. Decapitated insects did not synthesize trehalose after feeding, and this effect was reversed in head-transplanted insects. The concentration of trehalose in the hemolymph increased after the blood meal until the third day (from 0.0770.01 to 0.7570.05 mM) and at the fourth day it decreased until the ninth day (0.2170.01 mM), when it increased again until the fourteenth day (0.7970.06 mM) after the blood meal, and then declined again. In decapitated insects, trehalose concentrations did not increase soon after the blood meal and at the third day it was very low, but on the fourteenth day it was close to the control values. The concentration of glucose in the hemolymph of untreated insects remained low and constant (0.1870.01 mM) during the 15 days after feeding, but in decapitated insects it progressively increased until the fifteenth day (2.0070.10 mM). We recorded the highest trehalase activity in midgut, which was maximal at the eighth day after feeding (2,8307320 nmol of glucose/organ/h). We infer that in Rhodnius prolixus, the metabolism of glycogen, glucose, and trehalose are controlled by factors from the brain, according to physiological demands at different days after the blood mea

Identification and Characterization of an Ecto-Pyrophosphatase Activity in Intact Epimastigotes of <i>Trypanosoma rangeli</i>

<div><p>In this study, we performed the molecular and biochemical characterization of an ecto-enzyme present in <i>Trypanosoma rangeli</i> that is involved with the hydrolysis of extracellular inorganic pyrophosphate. PCR analysis identified a putative proton-pyrophosphatase (H⁺-PPase) in the epimastigote forms of <i>T. rangeli</i>. This protein was recognized with Western blot and flow cytometry analysis using an antibody against the H⁺-PPase of <i>Arabidopsis thaliana</i>. Immunofluorescence microscopy confirmed that this protein is located in the plasma membrane of <i>T. rangeli</i>. Biochemical assays revealed that the optimum pH for the ecto-PPase activity was 7.5, as previously demonstrated for other organisms. Sodium fluoride (NaF) and aminomethylenediphosphonate (AMDP) were able to inhibit approximately 75% and 90% of the ecto-PPase activity, respectively. This ecto-PPase activity was stimulated in a dose-dependent manner by MgCl₂. In the presence of MgCl₂, this activity was inhibited by millimolar concentrations of CaCl₂. The ecto-PPase activity of <i>T. rangeli</i> decreased with increasing cell proliferation <i>in vitro</i>, thereby suggesting a role for this enzyme in the acquisition of inorganic phosphate (Pi). Moreover, this activity was modulated by the extracellular concentration of Pi and increased approximately two-fold when the cells were maintained in culture medium depleted of Pi. All of these results confirmed the occurrence of an ecto-PPase located in the plasma membrane of <i>T. rangeli</i> that possibly plays an important role in phosphate metabolism of this protozoan.</p></div

Western blot and flow cytometry analysis of <i>T. rangeli</i> incubated with the anti-326 peptide sequence of the AVP2 antibody.

<p>Panel A: <i>T. rangeli</i> and <i>T. cruzi</i> Dm 28c epimastigote proteins (50 µg/lane) were separated using 8% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. <i>Lane 1</i>, immunoblot probed with antiserum against H⁺PPase (AVP2) in the total extract of <i>T. rangeli</i>. The H⁺PPase antibody recognized a polypeptide with an apparent molecular mass of 64 kDa. <i>Lane 2</i>, immunoblot probed against the total extract of <i>T. cruzi</i>. Panel B: The intact cells of <i>T. rangeli</i> (5×10⁶ cells) were fixed in paraformaldehyde and sodium cacodylate buffer and stained with the anti-326 peptide sequence of <i>A. thaliana</i> vacuolar H⁺PPase (AVP2) produced in rabbit and an Alexa 488-conjugated anti-rabbit secondary antibody produced in mouse, with an emission in the range of 488 nm (red). Abscissa: fluorescence intensity; Ordinate: Events/Positive cells. The gray color represents the autofluorescence of the cells (without antibody).</p