Leishmania chagasi: An ecto-3′-nucleotidase activity modulated by inorganic phosphate and its possible involvement in parasite–macrophage interaction
Graphical abstract
Adenosine and 3′ AMP added during the Leishmania chagasi macrophage interaction increased adhesion at the same rate. Parasites grown at low Pi concentration, expressing higher ecto-3′-nucleotidase activity, infected more marcophages than Parasites grown at high Pi.
Research highlights
►L. chagasi was able to hydrolyze 3’AMP ten times more than 5’AMP. ► Parasites grown in low Pi concentration decreased cell growth compared to those grown in high Pi concentration. ► Ecto-3’nucleotidase activity increased when L. chagasi was grown in a low Pi concentration. ► Altogether, the results describe an ecto-3’nucleotidase activity that could be involved on parasite-macrophage interaction.
Introduction
Leishmaniasis, a disease caused by obligate intra-macrophage protozoa, is endemic in the tropics and subtropics. Leishmaniasis has two main clinical forms: cutaneous and visceral leishmaniasis (Neuber, 2008). Visceral leishmaniasis is a systemic disease that is fatal if left untreated and is caused by species of the Leishmania donovani complex. In Brazil, it is typically caused by Leishmania chagasi. Following an incubation period that generally lasts between 2 and 6 months, patients with visceral leishmaniasis develop symptoms such as enlarged lymph nodes, spleen and liver (Chappuis et al., 2007).
Trypanosomatid protozoans are incapable of synthesising purines de novo and must rely on their hosts for these essential nutrients (Hassan and Coombs, 1988, De Koning et al., 2000, Berrêdo-Pinho et al., 2001, Meyer-Fernandes, 2002, Meyer-Fernandes et al., 2004, Pinheiro et al., 2006, Leite et al., 2007). Leishmania species have membrane bound 3′-nucleotidases on their extracellular surface that hydrolyse extracellular 3′-nucleotides or nucleic acids to form nucleosides (Dwyer and Gottlieb, 1984, Gbenle, 1993, Lakhal-Naouar et al., 2008). As the plasma membrane of Leishmania is permeable to nucleosides rather than nucleotides, this enzyme plays a role in the acquisition of purines for the cell (Gbenle and Dwyer, 1992). It was first identified in Leishmania, and then in other pathogenic and non-pathogenic trypanosomatids such as African trypanosomes, Crithidia and Herpetomonas. No similar enzyme has been reported from any mammalian cell or tissue. L. donovani and Crithidia luciliae grown in limiting conditions of purines and phosphate had higher 3′-nucleotidase/nuclease activity and an increased distribution of the enzyme at the cell surface (Gotllieb, 1985, Gottlieb et al., 1988, Gottlieb and Dwyer, 1983, Sacci et al., 1990, Debrabant et al., 1995).
In mammals and protozoan parasites 5′-nucleotidase enzymes are involved in purine salvage, generating adenosine (Tasca et al., 2003, Tasca et al., 2005, Maioli et al., 2004, Borges et al., 2007, Marques-da-Silva et al., 2008). The production of adenosine by the 5′-nucleotidase activity may be associated with the establishment of the Leishmania infection, in murine model (Maioli et al., 2004, Marques-da-Silva et al., 2008).
Some microbial cells have enzymes that provide Pi by hydrolysing phosphomonoester metabolites (Braibant and Content, 2001, Li et al., 2002, Bozzo et al., 2004, Fonseca-de-Souza et al., 2008, Fonseca-de-Souza et al., 2009, Dick et al., 2010). The concentration of Pi in the growth of Trypanosoma rangeli influences the ability of this parasite to colonise the anterior midgut of insect vector Rhodnius prolixus (Dick et al., 2010). The capacity to survive and proliferate in several adverse environments such as in macrophages and in the midgut of sandflies suggests that Leishmania has probably developed survival strategies, such as the acquisition of extracellular nutrients, release of virulence factors, microbiocidal resistance and evasion of host immune responses (Cunningham, 2002).
This report shows an ecto-3′nucleotidase activity present on the surface of L. chagasi and examines its modulation by exogenous phosphate content. The changes in ecto-3′-nucleotidase activity were correlated with the modulation of macrophage infection by the parasites. These findings suggest that ecto-3′nucleotidase may constitute a new member of the L. chagasi purine scavenging pathway with a possible role in Leishmania–host cell interactions.
Section snippets
Chemicals
All reagents were purchased from E. Merck (D-6100 Darmstadt, Germany) or Sigma Chemical Co. (St. Louis, MO).
Parasites
Leishmania chagasi (MHOM/BR/1974/PP75), L. braziliensis (MHOM/BR/75/M2903), L. major (MHOM/TM/1973/5-ASKH) and L. tropica (MHOM/SU/1958/STRAIN OD) were kindly supplied by Dr. E. Cupollilo (Leishmania Type Culture Collection of the Oswaldo Cruz Institute, Fiocruz, Brazil).
L. chagasi parasites were maintained by weekly transfers in BHI modified medium (2 g/l glucose, 2 g/l peptone, 2 g/l BHI,
Results and discussion
The specific induction of various enzymatic activities by nutrient deprivation has been shown to be a general phenomenon in many microorganisms (Braibant and Content, 2001, Bernard et al., 2002, Kneipp et al., 2004). We showed recently that T. rangeli depends strongly of the presence of inorganic phosphate (Pi) in the culture medium to proliferate (Fonseca-de-Souza et al., 2008, Fonseca-de-Souza et al., 2009). In T. rangeli and L. donovani, cellular proliferation decreased by 50% when the cells
Acknowledgments
We thank Mr. Fabiano Ferreira Esteves, Ms. Rosangela Rosa de Araújo and Mr. Paulo Coleto for excellent technical assistance. This work was supported by grants from the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
References (44)
- et al.
A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence
Archives Biochemistry Biophysics
(2001) - et al.
Characterization of an ecto-5′nucleotidase (EC 3.1.3.5) activity in intact trophozoites of Trichomonas gallinae
Veterinary Parasitology
(2007) - et al.
Phosphate or phosphate addition promotes the proteolytic turnover of phosphate-starvation inducible tomato purple acid phosphatase isozymes
FEMS Letters
(2004) - et al.
The cell surface associated phosphatase activity of Mycobacterium bovis BCG is not regulated by environmental Pi
FEMS Microbiology Letters
(2001) - et al.
Purification and characterization of 3′nucleotidase/nuclease from promastigotes of Leishmania donovani
Molecular and Biochemical Parasitology
(1991) Parasite adaptive mechanisms in infection by Leishmania
Experimental Molecular Pathology
(2002)- et al.
Isolation and characterization of the gene encoding the surface membrane 3′-nucleotidase/nuclease of Leishmania donovani
Molecular and Biochemical Parasitology
(1995) - et al.
Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei
Molecular Biochemical Parasitology
(2000) - et al.
Trypanosoma rangeli: differential expression of ecto-phosphatase activities in response to inorganic phosphate starvation
Experimental Parasitology
(2010) - et al.
Surface membrane localization of 3′- and 5′-nucleotidase activities in Leishmania donovani promastigotes
Molecular Biochemical Parasitology
(1984)
A Mg2+-dependent ecto-phosphatase activity on the external surface of Trypanosoma rangeli modulated by exogenous Pi
Acta Tropica
Trypanosoma rangeli: a possible role for ecto-phosphatase activity on cell proliferation
Experimental Parasitology
Characterization of a filtrable 3′-nucleotidase of Leishmania donovani
Biochimica et Biophysica Acta
Evidence for distinct 5′- and 3′-nucleotidase activities in the surface membrane fraction of Leishmania donovani promastigotes
Molecular and Biochemical Parasitology
Crithidia luciliae: factors affecting the expression of 3′-nucleotidase/nuclease activity
Experimental Parasitology
Adenosine: a potential mediator of immunosuppression in multiple organ failure
Current Opinion in Pharmacology
Identification and characterization of a new Leishmania major specific 3′-nucleotidase/nuclease protein
Biochemical and Biophysical Research Communications
Purple acid phosphatases of Arabdopsis thaliana: comparative analysis and differential regulation by phosphate deprivation
Journal Biological Chemistry
Leishmania amazonensis: effects of heat shock on ecto-ATPase activity
Experimental Parasitology
Leishmania amazonensis: biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities
Experimental Parasitology
Leishmania donovani: regulated changes in the level of expression of the surface 3′-nucleotidase/nuclease
Experimental Parasitology
Developmentally regulated expression of a cell surface class I nuclease in Leishmania mexicana
International Journal for Parasitology
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