Alexandre Romeiro

Ultrastructural and Biochemical Characterization of Promastigote and Cystic Forms of Leptomonas wallacei N. Sp. Isolated from the Intestine of its Natural Host Oncopeltus fasciatus (Hemiptera: Lygaeidae)

Abstract 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.

Secreted Phosphatase Activities in Trypanosomatid Parasites of Plants Modulated by Platelet-Activating Factor

ABSTRACT The secreted phosphatase activities of two trypanosomatid parasites were characterized and compared with supernatants of living cells. The plant parasite Phytomonas françai and the phytophagous hemipteran parasite Herpetomonas sp. hydrolyzed p-nitrophenylphosphate at a rate of 15.54 and 6.51 nmol Pi/mg of protein per min, respectively. Sodium orthovanadate (N(a)VO(3)) and sodium fluoride (NaF) decreased the phosphatase activities. The phosphatase activity of P. françai was drastically diminished (73% inhibition) in the presence of sodium tartrate, whereas the phosphatase activity of Herpetomonas sp. was inhibited by 23%. Cytochemical analysis showed the localization of these enzymes on the external surface and in the flagellar pocket of the two trypanosomatids. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor modulated the phosphatase activities, inhibiting P. françai activity and stimulating Herpetomonas sp. phosphatase activity.

Characterization of ectophosphatase activities in trypanosomatid parasites of plants.

ABSTRACT In the present work ectophosphatase activities of three trypanosomatid parasites of plants were characterized using intact cells. Phytomonas françai, Phytomonas mcgheei, and Herpetomonas sp. hydrolyzed p-nitro-phenylphosphate at a rate of 5.40, 7.28, and 25.58 nmol Pi/mg of protein per min, respectively. Experiments using classical inhibitors of acid phosphatases such as sodium orthovanadate (NaVO(3)) and sodium fluoride (NaF) showed a decrease in phosphatase activities. Lithium fluoride (LiF) and aluminum chloride (AlCl(3)) were also used. Although AlCl3 had no effect, LiF was able to promote a decrease in the phosphatase activities. Interestingly, the inhibition caused by LiF was enhanced by the addition of AlCl3 during the reaction, probably due to the formation of fluoroaluminate complexes. This effect was confirmed by cytochemical analysis. In this assay, electron-dense cerium phosphate deposits were visualized on the external surface of the three parasites.

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.

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.

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

Abstract 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 (α-galactose, α-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 (β-galactose(1-3) N-acetyl-galactosamine) from Arachis hypogaea.

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. (Journal of Biomolecular Screening 2007:1006-1010)

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.

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.