Effect of sphingosine and phorbol-12-myristate-13-acetate on the growth and dimethylsulfoxide-induced differentiation in the insect trypanosomatid Herpetomonas samuelpessoai

Abstract

We investigated the effect of two modulators of protein kinase C, Herpetomonas samuelpessoai is a non-pathogenic trypanosomatid isolated from the predatory insect Zelus leucogrammus that shares important antigens with Trypanosoma cruzi, the etiologic agent of Chagas disease (Souza et al. 1974), and with Leishmania spp. (reviewed by Santos et al. 2006). Several Brazilian researchers have used this parasite as a very interesting model to study cellular differentiation, since H. samuelpessoai possesses three distinct morphological stages (promastigote, paramastigote, and opisthomastigote) during its life cycle either in culture or in the invertebrate vector (Angluster et al. 1977, Castellanos et al. 1980, Souza et al. 1980, Thomas et al. 1981a,b, Santos et al. 2001, 2002a,b, 2003a.In protozoa as in higher eukaryotes, cellular differentiation occurs as result of selective gene expression, which is controlled in various ways. Differentiation is often initiated in response to external stimuli such as the binding of ligands to the cell surface (Parsons & Ruben 2000). The H. samuelpessoai differentiation mechanism is triggered by chemical and physical changes in the growth conditions. Previous studies have shown that the binding and/or interaction to the plasma membrane of drugs such as 2-deoxy-D-glucose (Angluster et al. 1977), Concanavalin A (Souza et al. 1980, cholinergic drugs (Thomas et al. 1981a), local anesthetic (Thomas et al. 1981b, and dimethylsulfoxide (DMSO) (Castellanos et al. 1980) induce the differentiation process in H. samuelpessoai, which is associated with several changes in the parasite biochemical machinery (Santos et al. 2001(Santos et al. , 2002a(Santos et al. ,b, 2003a. Nevertheless, very little is known about the signal transduction pathways involved in the differentiation process of this monoxenous flagellate.Protein phosphorylation-dephosphorylation in eukaryotes is involved in the regulation of signal transduction, metabolism, differentiation, proliferation, death, and other cellular processes (Nishizuka 2003). Protein kinase inhibitors have been used extensively to study the role of these enzymes in higher eukaryotes, but few similar studies on parasites have been carried out. In this context, we have tested the effects of two distinct modulators of protein kinase C (PKC) activity, sphingosine and phorbol-12-myristate-13-acetate (PMA), throughout the cellular growth and DMSO-induced differentiation process of H. samuelpessoai.H. samuelpessoai (CT-IOC-067) was maintained by weekly transfers in chemically defined conditions as previously reported (Santos et al. 2001). Experiments were made in 18 × 150 mm glass tubes containing 5 ml of medium and the inoculum consisted of 2% of a 48 h culture containing about 1.0 × 10 6 cells. Drugs were dissolved as follows: sphingosine was dissolved in water, at 500 ng/ml, PMA in ethanol, at 200 ng/ml, and DMSO (Sigma Chemical Co.) in the culture medium, at 4% (final concentration). The parasites were grown at 26°C, for 48 h (exponential growth phase), in ...

Cysteine peptidases inHerpetomonas samuelpessoaiare modulated by temperature and dimethylsulfoxide-triggered differentiation

“…DMSO has also been used as a powerful cell-differentiating agent in numerous biological systems (reviewed by Santos et al 2003 b ), including the insect trypanosomatid H. samuelpessoai (Castellanos et al 1981). Santos et al (2007 b ) demonstrated that the reduction of parasite proliferation was accompanied by an increase in the differentiation rate in H. samuelpessoai . It has been shown that H. samuelpessoai grown in the absence of DMSO presented a preponderance of promastigote forms during 24 to 72 h (Castellanos et al 1981; Santos et al 2001, 2002 a , b , 2003 a ).…”

“…Nevertheless, no T. cruzi cruzipain homologues were detected by means of the employed methodologies. Since DMSO is able to interfere with several signalling cascades, including some in which Ca 2+ participates as second messenger (reviewed by Santos et al 2003 b), as well as it causes modification in the cellular architecture during the transformation of promastigote into paramastigote and opisthomastigote observed in H. samuelpessoai (Santos et al 2007 a), we have investigated the possible participation of calpain-like molecules in the DMSO-stimulated differentiation of H. samuelpessoai. Our results showed for the first time that DMSO-untreated H. samuelpessoai expresses a molecule of 80 kDa that presents homology to D. melanogaster calpain-like protein.…”

“…Cysteine peptidases of parasitic protozoa have been implicated in a variety of biological events including differentiation, nutrition, host cell infection, evasion of the host immune response, pathogenesis and virulence, and some have also been validated as drug targets of high promise, especially providing an alternative to traditional therapy against drug-resistant parasites (reviewed by Sajid and McKerrow, 2002 ;Vermelho et al 2007). Recently, our research group has reported that some insect and plant trypanosomatids presented immunological similarities with cruzipain, the major cysteine peptidase produced by the human pathogen Trypanosoma cruzi (d'Avila-Levy et al 2005 ;Santos et al 2006Santos et al a, 2007. In addition, previous studies showed that H. samuelpessoai displays close antigenic similarities to T. cruzi (Souza et al 1974) and Leishmania spp.…”

Effects of direct electric current on Herpetomonas samuelpessoai: An ultrastructural study

“…Non‐pathogenic trypanosomatids have emerged as important models for the study of basic biological processes performed by an archetypal eukaryotic cell. In this context, several Brazilian researchers have used Herpetomonas samuelpessoai , a monoxenous trypanosomatid, as a very interesting model to study physiological, genetic, biochemical, and ultrastructural aspects of flagellate trypanosomatids [Angluster et al, 1977; McGhee and Cosgrove, 1980; Soares et al, 1984; Santos and Soares, 2007]. H. samuelpessoai was originally isolated from the predatory insect Zelus leucogramus (Hemiptera: Reduviidae) [Galvao et al, 1970], which can be easily cultivated either in complex or chemically defined medium at 26 °C (insect vector temperature) or 37 °C (mammalian temperature) [Roitman et al, 1972].…”