Background

Trypanosomatid protozoan parasites are a diverse group of primitive parasitic organisms (Kaufer et al., 2017). They are both of medical and veterinary importance as many Trypanosomatid species cause serious or fatal diseases in humans and agricultural animals in tropical and subtropical regions worldwide (Reference 16). Virulence factors of these parasites include molecules expressed on their cell surface as well as those secreted or shed into the extracellular medium (Gottlieb and Dwyer, 1981; Fonseca-de-Souza et al., 2008 and 2009). Trypanosomatidae are exposed to diverse environmental stimuli throughout their life cycles in all biological systems they reside. Central events in recognition of the external and internal signals in these cells, which lead to specific responses for adaptation and survival in all these environments, are protein phosphorylation and dephosphorylation events carried out by a multitude of kinases and phosphatases. The trypanosomatid kinome and phosphatome have been recently deciphered (Brenchley et al., 2007; Sadatomi et al., 2013).

Phosphatase activities modulate different aspects of the infection by members of the trypanosomatid family (Neves et al., 2014). Ecto-phosphatases, the enzymes localized in parasite’s surface membranes, are of special interest as they are candidate molecules for the interaction between the parasite and its host and thereby they could be intimately involved in many cellular events implicated in parasite adhesion to the host, protection from the host’s immune response and establishment of infection. Moreover, several alternative functions have been suggested for ecto-phosphatases, such as participation in proliferation, differentiation, adhesion, virulence and infection (Katakura and Kobayashi, 1988; Singla et al., 1992). Little is known about their physiological roles in protozoa parasites (Cosentino-Gomes et al., 2011; Gomes et al., 2011). In particular, acid ecto-phosphatases that hydrolyze phosphomonoesters at acidic pHs, have been associated with the survival of intracellular parasites within phagocytes through inhibition of the respiratory burst (Saha et al., 1985; Remaley et al., 1985; Baca et al., 1993; Reilly et al., 1996; Jungnitz et al., 1998; Saleh and Belisle, 2000; Burtnick et al., 2001; Steenkamp, 2002; Cosentino-Gomes et al., 2009; Aragon et al., 2011; Cosentino-Gomes and Meyer-Fernandes, 2011), suggesting that they may act as virulence factors (Katakura and Kobayashi, 1988; Singla et al., 1992; Papadaki et al., 2015). They may also be related to the ability of parasites to survive and multiply in the insect host (Remaley et al., 1985; Saha et al., 1985; Singla et al., 1992). The field of the acid ecto-phosphatases was reopened with the characterization of the L. donovani acid ecto-phosphatase LdMAcP (Papadaki et al., 2015) and the identification of at least two more open reading frames in the L. donovani genome that could be potentially expressed at the surface membrane of the parasites (Papadaki and Boleti. data not published).

Therefore assaying acid phosphate activity at the surface of live cells is a putative diagnostic parameter for virulence of Leishmania parasite strains as well as for the evaluation of the expression of the ecto-phosphatases under study at the surface of transgenic parasite strains overexpressing them.Acid phosphatase enzymatic activity can be measured in crude membrane fractions and in membrane fractions enriched in plasma membrane (Glew et al., 1982; Shakarian et al., 2002), however, in these cases, the intracellular acid phosphatases, mainly localized in lysosomes, contribute to the final result.

The protocol provided herein was used to measure acid ecto-phosphatase activity in live Leishmania promastigotes but it could be used to detect and quantitate acid ecto-phosphatase activity in all suspension cells of prokaryotic or eukaryotic origin.

Our method was adapted from previously described methodologies (Remaley et al., 1985; Saha et al., 1985; Katakura and Kobayashi, 1988; Singla et al., 1992) for assaying ecto-phosphatases in other organisms.