Luzia Monteiro de Castro Côrtes

Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro

BackgroundLeishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. The Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event.MethodsFlagellar (Ff) and membrane (Mf) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. In addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis.ResultsThe success of promastigotes subcellular fractionation led to the obtainment of Ff and Mf proteins, both of which presented two main protein bands (65.0 and 55.0kDa) with affinity to heparin. The contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17 ± 2.0% of culture cells with adhered parasites): 30% (for HS 20μg/ml) and 16% (for HS 10μg/ml); HBP Mf (35.2% for 10μg/ml and 25.4% for 20μg/ml) and HBP Ff (10.0% for 10μg/ml and 31.4% for 20μg/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. The SPR analysis performed further confirmed the presence of GAGs ligands on L. (V.) braziliensis promastigote surfaces.ConclusionsThe data presented here point to evidences that HBPs present on the surface of L. (V.) braziliensis promastigotes participate in adhesion of these parasites to Lulo cells through HS participation.

Leishmania (Viannia) braziliensis: insights on subcellular distribution and biochemical properties of heparin-binding proteins.

Leishmaniasis is a vector-borne disease and an important public health issue. Glycosaminoglycan ligands in Leishmania parasites are potential targets for new strategies to control this disease. We report the subcellular distribution of heparin-binding proteins (HBPs) in Leishmania (Viannia) braziliensis and specific biochemical characteristics of L. (V.) braziliensis HBPs. Promastigotes were fractionated, and flagella and membrane samples were applied to HiTrap Heparin affinity chromatography columns. Heparin-bound fractions from flagella and membrane samples were designated HBP Ff and HBP Mf, respectively. Fraction HBP Ff presented a higher concentration of HBPs relative to HBP Mf, and SDS-PAGE analyses showed 2 major protein bands in both fractions (65 and 55 kDa). The 65 kDa band showed gelatinolytic activity and was sensitive to inhibition by 1,10-phenanthroline. The localization of HBPs on the promastigote surfaces was confirmed using surface plasmon resonance (SPR) biosensor analysis by binding the parasites to a heparin-coated sensor chip; that was inhibited in a dose-dependent manner by pre-incubating the parasites with variable concentrations of heparin, thus indicating distinct heparin-binding capacities for the two fractions. In conclusion, protein fractions isolated from either the flagella or membranes of L. (V.) braziliensis promastigotes have characteristics of metallo-proteinases and are able to bind to glycosaminoglycans.

Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus.

Heparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

Epoxy-α-lapachone has in vitro and in vivo anti-Leishmania (Leishmania) amazonensis effects and inhibits serine proteinase activity in this parasite.

ABSTRACT Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone on L. (L.) amazonensis were analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm2), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm2) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm2). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore, in silico simulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) of L. (L.) amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compounds effects on the parasite.

Production of MMP-9 and inflammatory cytokines by Trypanosoma cruzi-infected macrophages

Matrix metalloproteinases (MMPs) constitute a large family of Zn(2+) and Ca(2+) dependent endopeptidases implicated in tissue remodeling and chronic inflammation. MMPs also play key roles in the activation of growth factors, chemokines and cytokines produced by many cell types, including lymphocytes, granulocytes, and, in particular, activated macrophages. Their synthesis and secretion appear to be important in a number of physiological processes, including the inflammatory process. Here, we investigated the interaction between human and mouse macrophages with T. cruzi Colombian and Y strains to characterize MMP-9 and cytokine production in this system. Supernatants and total extract of T. cruzi infected human and mouse macrophages were obtained and used to assess MMP-9 profile and inflammatory cytokines. The presence of metalloproteinase activity was determined by zymography, enzyme-linked immunosorbent assay and immunoblotting assays. The effect of cytokines on MMP-9 production in human macrophages was verified by previous incubation of cytokines on these cells in culture, and analyzed by zymography. We detected an increase in MMP-9 production in the culture supernatants of T. cruzi infected human and mouse macrophages. The addition of IL-1β or TNF-α to human macrophage cultures increased MMP-9 production. In contrast, MMP-9 production was down-modulated when human macrophage cultures were treated with IFN-γ or IL-4 before infection. Human macrophages infected with T. cruzi Y or Colombian strains produced increased levels of MMP-9, which was related to the production of cytokines such as IL-1β, TNF-α and IL-6.

Trypanosoma cruzi heparin-binding proteins present a flagellar membrane localization and serine proteinase activity.

Heparin-binding proteins (HBPs) play a key role in Trypanosoma cruzi-host cell interactions. HBPs recognize heparan sulfate (HS) at the host cell surface and are able to induce the cytoadherence and invasion of this parasite. Herein, we analysed the biochemical properties of the HBPs and also evaluated the expression and subcellular localization of HBPs in T. cruzi trypomastigotes. A flow cytometry analysis revealed that HBPs are highly expressed at the surface of trypomastigotes, and their peculiar localization mainly at the flagellar membrane, which is known as an important signalling domain, may enhance their binding to HS and elicit the parasite invasion. The plasmon surface resonance results demonstrated the stability of HBPs and their affinity to HS and heparin. Additionally, gelatinolytic activities of 70 kDa, 65·8 kDa and 59 kDa HBPs over a broad pH range (5·5-8·0) were revealed using a zymography assay. These proteolytic activities were sensitive to serine proteinase inhibitors, such as aprotinin and phenylmethylsulfonyl fluoride, suggesting that HBPs have the properties of trypsin-like proteinases.

Leishmania (Viannia) braziliensis: Influence of successive in vitro cultivation on the expression of promastigote proteinases

Cysteine proteinases are an important virulence factor in Leishmania parasites. In this study we analyzed the cysteine proteinase expression of infective Leishmania (Viannia) braziliensis promastigotes, examining the expression induced by successive in vitro passages in culture. We observed that this parasite presents a decrease in its virulence over BALB/c macrophages, after successive passages in culture, but still they present proteinase activity, being capable of hydrolyzing the substrate pGlu-Phe-Leu-p Nitroanilide at pH 7.0. This proteinase activity also decreases in the course of the successive passages. Additionally, the decrease in the amount of CPB proteins following successive passages of promastigotes was verified by immunoblotting assays, using an anti-CPB antiserum. Real-time PCR assays were performed to assess the relative cpb expression when compared to a housekeeping gene in promastigote cDNA preparations from the first, fourth and seventh passages. Interestingly, the data indicate a relative increase in cpb gene transcripts as the promastigotes were maintained under in vitro culture: 2.2 times higher for fourth and 2.7 times higher for seventh passages when compared to the first passage. Thus, the information gathered here shows that the expression of cysteine proteinases is modified during in vitro cultivation of L. (V.) braziliensis promastigotes.

Ultrastructural aspects of the replication of dengue virus type 2 isolated in Brazil

Our purpose here is to describe and compare ultrastructural aspects of DEN-2 virus replication in the C6/36 mosquito cell line of the two otbreaks in the states of Ceara and Bahia. Sera from four patients from Fortaleza (Ceara) and two from Eunapolis (Bahia) were isolated and typed previously as DEN-2 at the Laboratory of Flaviviruses, Department of Virology, Instituto Oswaldo Cruz (Nogueira et al. 1995 Rev Inst Med Trop Sao Paulo submitted) by indirect immmunofluorescence test with type specific monoclonal antibodies. Cells were inoculated with 50 μl of the original human sera containing virus and maintained at 28°C in Leibovitz-15 medium. Showing 30-50% of cytopathic effect, the cell cultures were fixed in glutaraldehyde and processed as described previously (Barth et al. 1994 loc. cit.). Ultrathin sections were observed with a Zeiss EM-900 electron microscope. At least 50 cells were analyzed for virus replication in each sample. The Brazilian DEN-2 virus replication in mosquito cell cultures of all examined sera present some peculiar morphological aspects, when compared with the reference New Guinea C strain. Typical featured virus particles, that occur in great number inside the rough endoplasmatic reticulum (RER) and derived vesicles after about three days of inoculation, are observed at high magnification in ultrathin sections and present always a smooth surface (Fig. 1); small granules on the virus particle surfaces can be observed by the negative staining technique at higher magnifications, corresponding to the virus membrane and envelope proteins (Fig. 2 arrow). Beside these classical structured DEN-2 virus particles, virus-like particles occur in the RER and derived vesicles, also with the isolates of patient sera from State of Rio de Janeiro, but not in the cells infected with the reference New Guinea C strain. These particles, characterized by a fuzzy coated surface, are less numerous but persistent just to the 5th passage level in cells (Barth et al. 1994 loc. cit.). Cell controls were always free from these particles, as well as the same cell line infected by DEN-1 viruses. No antibodies, that may be engulfed by the cells during endocytosis, were found inside the infected cells; this means that the last described particles are not virus particles covered by antibodies. Human sera from Fortaleza and Eunapolis cities, show the same morphological aspects of the virus particles as described for DEN-2 from Rio de Janeiro (Fig. 3). Classical structured virus particles occur beside the fuzzy coated ones in the same RER vesicles. Smooth membrane structures, RESEARCH NOTE

Ultrastructural aspects of virus replication in one fatal case and several other isolates from a dengue type 2 outbreak in Rio de Janeiro

Dengue virus replication in mosquito cell cultures was observed by electron microscopy in one fatal and 40 classical isolates from a dengue type 2 outbreak in Rio de Janeiro and compared with the prototype New Guinea C strain. All the Brazilian isolates presented, beside the classical structured dengue virus particles, fuzzy coated virus-like particles, never observed in the referencial New Guinea C virus strain. More numerous DEN-2 virus particles, fuzzy coated virus-like particles, defective virus particles and smooth membrane structures inside the rough endoplasmic reticulum characterized the unique fatal isolate examined.

The Vectorial Potential of Lutzomyia (Nyssomyia) intermedia and Lutzomyia (N.) whitmani in the Transmission of Leishmania (V.) braziliensis Can Also Be Related to Proteins Attaching

We read with great interest the report by Soares et al. [1] on the potential of Leishmania (Viannia) braziliensis to attach to the midgut of the sand flies Lutzomyia (N.) whitmani and Lutzomyia (N.) intermedia. This manuscript assesses relevant information concerning the biomolecular phenomena between Leishmania promastigotes and the midgut of Lutzomyia species that act as vectors of American Cutaneous Leishmaniasis (ACL). However, it is necessary to comment that the basis of these physiological processes is not directly driven by glycolipid lipophosphoglycan (LPG) only. Other L. (V.) braziliensis promastigotes surface components, as proteins, can also be implicated in many steps of the midgut attachment. Since 2007 we have been investigating the potential of heparin binding proteins (HPBs) from L. (V.) braziliensis promastigotes in the attachment of parasites to gut proteins from L. (N.) intermedia and L. (N.) whitmani [2]. We have indicated the existence of physicochemical conditions for the binding between the gut proteins from Lutzomyia spp. and the HPBs—a new macromolecule class involved in the recognition of the sand fly gut epithelium by L. (V.) braziliensis. We proposed that the five HPB ligands (67.0, 62.1, 59.5, 56.0, and 47.5 kDa) observed in both L. (N.) intermedia and L. (N.) whitmani are involved with the promastigote attachments to sand fly gut epithelium. Also, we suggested that the physicochemical conditions for the interaction between HBP and their ligands are more favourable in the midgut of L. (N.) whitmani than in L. (N.) intermedia. Furthermore, heparin similar molecules, synthesized by cells of midgut epithelium seem to act as anchoring sites for L. (V.) braziliensis promastigotes. The ability of promastigotes to adhere to epithelial microvillii of the Phlebotominae digestorium tube is an essential stage for the maintenance of the parasite life cycle, being a factor of distinction between infective and noninfective stains. Similarly to LPG, the HBPs are related to the infective forms of the parasite [3, 4]. In such a way, its presence can be an essential factor for the setting of promastigotes in the digestorium tube and for the continuity of the life cycle, since parasites unable to adhere to the intestinal epithelium would be rejected together with the “feces” of the insect vector [5]. In addition, L. (N.) intermedia and L. (N.) whitmani are related to L. (V.) braziliensis transmission in the same endemic area [6]. The detection of ligands with similar molecular weights in the digestorium tube of both insect species is a biochemical indicative of vectorial homogeneity of these species in the transmission of ACL. The mapping of the interactions between molecules from both parasite and vector molecules can help in the understanding of adhesion to epithelial cells through the parasite surface. Thus, our results considered together with the recent findings by Soares et al. [1] present biochemical indicatives of the epidemiological relevance of L. (N.) whitmani as a primary vector of ACL in Brazil.