Adenosine reverses metacyclogenesis <i>in vitro</i> and reduces <i>in vivo</i> development of metacyclic promastigotes.

<p>(A) Metacyclic promastigotes from CGS-treated cultures, obtained by Ficoll gradient centrifugation, were cultivated in “spent” medium (late stationary phase medium from control cultures) or in “spent” medium plus adenosine (Ado). Parasite growth was evaluated by hemocytometer counting. (B) Metacyclic promastigotes from CGS-treated cultures, obtained by Ficoll gradient centrifugation followed by complement mediated lysis (as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001833#pntd-0001833-g002" target="_blank">Figure 2D</a>), were cultured in Grace's insect medium without supplements or in this medium plus adenosine. Parasite growth was evaluated by hemocytometer counting. (C) General aspect of metacyclic promastigotes after double purification (Ficoll + complement) (upper left). Cells after 72 hr of incubation in Grace's insect medium without supplements (upper right), or in the presence of 100 µM (lower left) or 500 µM of adenosine (lower right). Pictures taken with an Axio Cam MR3 attached to a Carl-Zeiss Axio Imager M2 microscope. Adult insects of <i>Lutzomyia longipalpis</i>, majority of females, were allowed to feed in a naturally <i>Leishmania infantum chagasi</i>-infected dog for 30 min. Insects were fed for 8 to10 days with 30% sucrose solution with or without adenosine (5 mM). Each midgut was evaluated by light microscopy for the total amount of parasites (D) and for the percentage of metacyclic promastigotes (E). (F) Image of a metacyclic (upper panel) and non-metacyclic promastigote (lower panel) of <i>L. infantum chagasi</i> from sand fly midgut. Pictures taken with a DFC300FX camera attached to a Leica DM5000B microscope. Means and standard deviations (or medians in graph E) from at least two independent experiments are plotted; *p<0.05, ***p<0.0001 determined by two-tailed Student's t-test indicate significant difference from control group.</p

Presence of CGS 15943 in <i>Leishmania amazonensis</i> culture halts parasite growth and enhances metacyclogenesis.

<p>(A) Evaluation of <i>L. amazonensis</i> growth in Grace's insect medium containing different concentrations of CGS as determined by hemocytometer counting. (B) Viability of parasites was assessed by flow cytometry using propidium iodide incorporation after 72 hr of culture. Phase contrast image of DMSO-treated (C) and CGS-treated (D) cultures of <i>L. amazonensis</i> promastigotes (picture taken with an Axio Cam MR3 attached to a Carl-Zeiss Axio Imager M2 microscope). Percentage of metacyclic promastigotes in control (Medium; DMSO) and CGS-treated cultures was evaluated by differential counting in optical microscopy (E) and Ficoll density gradient purification (F). Means and standard deviations from at least three independent experiments are plotted; *p<0.05, **p<0.01 determined by two-tailed Student's t-test indicate significant difference from control group.</p

Control of metacyclogenesis <i>in vitro</i>.

<p>(A) Uptake of [³H]adenosine by mid-log phase promastigotes of <i>L. amazonensis</i> in presence of CGS or dipyridamole (DIP). Experiments were performed at pH 7.4. # indicates p<0.01 when we compared DIP with CGS groups using the two-tailed Student's t-test. (B) <i>L. amazonensis g</i>rowth curve and metacyclogenesis (C) in the presence of DIP (please see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001833#pntd-0001833-g001" target="_blank">Figure 1</a> for details). (D) Growth evaluation of <i>L. amazonensis</i> in presence of N⁶methyladenine (N⁶-MA) followed by metacyclogenesis assessment (E). (F) Assessment of metacyclogenesis in cultures stimulated with CGS and CGS plus adenosine (Ado), inosine (Ino), adenine (Ade) or hypoxanthine (Hyp). CGS was always used at concentration of 50 µM and for nucleosides/nucleobases, the concentration is indicated in parentheses (µM). Experiments were performed in Grace's culture medium supplemented with fetal bovine serum, glutamine and penicillin, pH 6.5. (G) Compilation of results from experiments evaluating growth curve and metacyclogenesis in different conditions. In all experiments metacyclogenesis was evaluated by Ficoll density gradient. Means and standard deviations from at least three independent experiments are plotted; *p<0.05, **p<0.01 determined by two-tailed Student's t-test indicate significant difference from control group.</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

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

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

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

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

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

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