Analyses of expression and purification of rLicNTPDase-2.

<p>(A) Protein extract from <i>E. coli</i> carrying the empty vector pET21b. (B) Protein extract from <i>E. coli</i> carrying the vector pET21b plus rLicNTPDase-2. (C) Purified rLicNTPDase-2 (3 µg) stained by the silver method. SDS lanes indicate samples analyzed after SDS (10%)-PAGE stained with Coomassie blue. WB lanes indicate the same SDS-PAGE samples analyzed by Western blot using anti-His produced in rabbit as primary antibody (1∶4000) and anti-rabbit-IgG conjugated with FITC as secondary antibody (1∶6000). The nitrocellulose membrane was analyzed using an FLA 5100 (Fujifilm) instrument at 475 nm, with a blue filter.</p

Phylogenetic tree using representatives of the ENTPDases from mammals and Trypanosomatids (<i>Leishmania</i> and <i>T. cruzi</i>).

<p>ENTPDase sequences were aligned by the CLC workbench program and used to construct the phylogenetic tree using the Neighbor Joining method with bootstrap analysis (number in the branches). <i>Mus musculus</i> (Mm); <i>Homo sapiens</i> (Hs). Trypanosomatids have two ENTPDases with exception of <i>T. cruzi</i>, which has only one ENTPDase in databank. Trypanosomatid ENTPDases are more similar to mammalian ENTPDases isoforms 5 and 6 and are grouped at the upper branch of the tree.</p

Sub-cellular localization of ENTPDases in <i>L. infantum chagasi</i> promastigotes.

<p>Electron micrographs using polyclonal antibodies to rLicNTPDase-2 anti-IgG conjugated to 10 nm colloidal gold (A–H). Letters and symbols indicate different localizations: nucleus (N) (white arrow), mitochondria and kinetoplasts (K) (white arrowhead), internal vesicles (black arrowhead), flagellum (F) and flagellar pocket (black arrow) and cell surface (dashed black arrow). No staining was observed in the control (H). Bars: A, C, F, G = 0.1 µm and B = 0.3 µm, C, D and E, H 0.2 µm.</p

rLic-NTPDase-2: refolding, substrate preference and pH dependence.

<p>(A) Refolding assay- the enzymatic activity was measured just after the refolding (zero point) and up to six days later using ADP as the substrate. (B) The preference for different substrates was assessed in the presence of the cofactors calcium (grey bars) or magnesium (black bars). Inset shows the hydrolysis values with SD. (C) ATP (open circles) and ADP (black circles) were used to evaluate enzymatic activity as a function of pH. The pH-dependence test was performed in buffer containing 50 mM MES, 50 mM Tris, 50 mM HEPES, 3 mM MgCl₂, 116 mm NaCl, 5.4 mM KCl and 2.5 mM nucleotide. The SDs represent the those from the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method.</p

Evaluation of role of LicNTPDases in the infection and adhesion of <i>L. infantum chagasi</i> to macrophages.

<p>(A) Adhesion of <i>Leishmania</i> promastigotes to macrophages. (B) Infection of macrophages by <i>Leishmania</i> promastigotes. (C) Treatment did not affect the amount of parasites in macrophages. In all assays (A, B and C): Macrophages treated with polyclonal antiserum anti-rLicNTPDase-2 prior to the infection or with the purified rLicNTPDase-2 before the contact with parasites are compared with macrophages treated with the parasites in the absence of intervention. Control  =  adhesion and infection assay without any intervention. Control with enzyme buffer  =  adhesion and infection assay in the presence of the buffer used to suspend rLicNTPDase-2. BSA was used as a non-related protein. The data reflect the mean + SE from three analyzed slides from each of three independent assays. The asterisks indicate significant differences (<i>p</i><0.05) between the control and other samples.</p

Immunohistochemistry using anti-rLicNTPDase-2 in the lymph nodes of naturally infected dogs.

<p>(A) Lymph nodes from 48 <i>Leishmania</i>-positive dogs were evaluated by immunohistochemistry (IHC) using anti-rLicNTPDase-2. The results of the IHC are compared with ELISA data of the same samples using a Biomanguinhos Kit. (B) An example of IHC result using polyclonal anti-rLicNTPDase-2. (C) The zoom of section C is from image B.</p

<i>L. infantum chagasi</i> promastigote ecto-nucleotidase activity and surface ENTPDase localization using anti-rLicNTPDase-2.

<p>(A) The promastigote ecto-nucleotidase activity assays were performed in live total promastigotes from log phase growth using different substrates and Mg²⁺ as cofactor. The activities of recombinant enzyme (rLicNTPDase-2) are shown in the inset box. The SDs represent those of the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method. (B) Western blotting using anti-rLicNTPDase-2 recognizes both recombinant isoforms rLicNTPDase-1 and rLicNTPDase-2. Purified rLicNTPDase-1 and rLicNTPDase-2 were run in 10% SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was incubated with purified rabbit polyclonal antibodies to anti-rLicNTPDase-2 (1∶100) as the primary antibody and with anti-rabbit IgG conjugated with FITC (1∶10,000) as the secondary antibody. (C) Distribution of ENTPDases at the surface of <i>L. infantum chagasi</i> promastigotes. Non-permeabilized cells fixed with paraformaldehyde were incubated with anti- rLicNTPDase-2 (1∶50) as primary antibody and with Alexa 488-conjugated goat anti-rabbit IgG (1∶400) as secondary antibody. The glass slides were mounted with Prolong Gold Antifade Reagent (Molecular Probes) and examined by confocal microscopy (Leica, SP5). Bar scale  = 2 µm.</p

rLicNTPDase-2 ATPase activity in the presence of known partial inhibitors of ENTPDases and a 5′nucleotidase inhibitor.

<p>Purified rLicNTPDase-2 was assayed in the absence of inhibitors (control column) or in the presence of inhibitors: ARL 67156 (ARL) 300 µM, gadolinium chloride 300 µM, suramin 100 µM, sodium azide 2 mM and ammonium molybdate 3 µM (a 5′nucleotidase inhibitor). The SDs represent those from the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method. Statistical analyses were performed using ANOVA, and the significant differences between the control and inhibitors assays are shown with asterisks (p<0.05).</p

Alignment of representatives of TpNTPDases-1 and TpNTPDases-2.

<p>TpNTPDases from <i>L. infantum</i> (Li), <i>L. braziliensis</i> (Lb), <i>L. major</i> (Lm) and <i>T. cruzi</i> (Tc) were aligned using the CLC main workbench and manually inspected. The ACRs are shown in dashed line rectangles. The prevalent amino acids are shown in the consensus line and the level of conservation of each position at the primary sequences of proteins is shown in the conservation box.</p

Genome alignments.

<p>The figure shows the alignment of the <i>A. deanei</i> endosymbiont (Endo-<i>A. deanei</i>) and the <i>S. culicis</i> endosymbiont (Endo-<i>S. culicis</i>) (A); between Endo-<i>A. deanei</i> and <i>T. asinigenitalis</i> (B), <i>T. equigenitalis</i> (C), or <i>Wolbachia</i> (D); and between <i>Wolbachia</i> and <i>T. asinigenitalis</i> (E). Alignments were performed with the ACT program based on tblastx analyses. Red (direct similarity) and blue lines (indirect similarity) connect similar regions with at least 700 bp and a score cutoff of 700. The numbers on the right indicate the size of the entire sequence for each organism.</p