Bioinformatics analysis of <i>L. major</i> LmjF19.0160.

<p>Relationship between human and trypanosomatid members of the M24A subfamily of metallopeptidases was analyzed by multiple alignment and cluster analysis using Clustal X. This subfamily comprises homologs of methionyl aminopeptidases 1 and 2 (METAP1 and 2), and non-peptidases. Alignment was fed into MEGA5.2 software and a Neighbor-Joining tree was computed with 500 bootstrap replicates. Numbers on nodes indicate bootstrap support.</p

2D-DIGE quantitative phosphoproteomics analysis of GFPK7.

<p>An enlarged region of the 2D-DIGE gels showing Cy3-labeled WT stationary promastigotes and Cy5-labeled stationary GFPK7 promastigotes is presented. Spot ID 207 (white arrow) was over-represented in GFPK7. In the lower panel, a graphical representation of the BVA (Biological Variation Analysis) module of Decyder software (GE Healthcare) with statistics for spot ID 207 (2.97 fold and p = 0.00056). The spot was analyzed by mass spectrometry and identified as LmjF19.0160, a putative aminopeptidase.</p

Attenuated virulence in GFP-PA2G4 transgenic parasites.

<p>(A) 10⁵<i>L. major</i> metacyclic mock and transgenic parasites expressing GFP-PA2G4 and cured GFP-PA2G4 grown in the absence of G418 were inoculated into the footpad of female BALB/c mice. Lesion formation was followed by measuring the increase in footpad size with a Vernier caliper. Groups of five mice were analyzed and standard deviation is indicated by the bars. Two independent experiments were performed and one representative experiment is shown. (B) <i>L. donovani</i> transgenic (GFP-PA2G4) lines were established. 2×10⁵ promastigotes were inoculated in low pH medium and 37°C to trigger differentiation to axenic amastigotes. Cells were lysed 24 and 48 h after differentiation, and western blot performed with anti-GFP, amastigote-specific A2 antibody and anti-tubulin as a loading control. The molecular weight of standard proteins is indicated in kDa.</p

Gain-of-function strategy to study LmaPA2G4.

<p>(A) WT or parasites over-expressing an N-terminal GFP-PA2G4 fusion protein were lysed, resolved by SDS–PAGE, electroblotted on a PVDF membrane and analyzed by western blot using monoclonal anti-GFP antibody and anti-tubulin as a loading control. The molecular weight of standard proteins is indicated in kDa. (B) GFP intensity in GFP-PA2G4 transgenic promastigotes was analyzed in a Beckman Coulter FC-500 flow cytometer. 10,000 events were recorded. GFP mock control parasites are clearly distinguished from GFP-PA2G4. (C) Live control and transgenic promastigotes from logarithmic culture were analyzed using spinning disk confocal microscopy. Nuclei were counterstained with 1 µg/mL NucBlue Live Cell stain (Molecular Probes) (red). The bar corresponds to 7 µm. (D) Growth curve of <i>L. major</i> WT, GFP-mock, GFP-PA2G4, pLEXSY and pLEXSY-PA2G4 promastigotes was measured microscopically by counting cells in a Neubauer chamber. The average and standard deviation of triplicate determinations are shown. (E) The infective metacyclic stage in control and transgenic lines was analyzed. Parasites from five day stationary cultures were incubated with peanut agglutinin and numbers of non-agglutinating metacyclics were determined microscopically. Two independent experiments were performed and one representative triplicate experiment with standard deviation denoted by the bars is shown.</p

Electrophoretic Mobility Shift Assay.

<p>Poly (I∶C), a synthetic double stranded RNA was labeled with Cy5 and incubated with 20 ng GFP-PA2G4 fusion protein at room temperature for 45 min. The reaction was resolved in 10% non-denaturing polyacrylamide gels. Lane 1: 40 ng poly (I∶C); lane 2: 20 ng GFP-PA2G4; lane 3: 20 ng GFP-PA2G4 incubated with 40 ng poly (I∶C). Gel was scanned on a Typhoon scanner (GE) using 633/670 nm for Cy5 and 489/508 nm for GFP. Arrows indicate mobility shift of GFP-PA2G4.</p

Establishment of L. major PA2G4 conditional null-mutant parasites.

<p>(A) Schematic representation of the null-mutant strategy. Two alleles of LmaPA2G4 were replaced by homologous recombination with hygromycin B (HYG B) and puromycin (PAC) resistance markers. Replacement was performed in the presence of an ectopic copy of PA2G4 (pXNG- PA2G4), carrying a nourseothricin (SAT) marker, a fluorescent protein (GFP) and a <i>Herpes simplex</i> virus thymidine kinase (TK). (B) PCR analysis of total DNA from wild-type (WT) parasites or one independent clone (PA2G4 −/− [pXNG-PA2G4]). F1,R1 primers (expected size 338 bp) confirm the presence of the endogenous copy of PA2G4, while F2,R1 primers (expected size 513 bp) confirm of the episomal PA2G4 ORF. F1, R2 primer pair (expected size 413 bp) and F1, R3 (expected size 440 bp) show the integration of hygromycin b and puromycin genes, respectively. Molecular weight marker (M) is shown. (C) (Upper panel). WT parasites carrying a copy of pXNG-PA2G4 were selected and grown in SAT and the GFP intensity was analyzed by flow cytometry (GCV−). After negative selection with the addition of 50 µg/mL GCV (GCV+) to the culture (3 passages) a reduction in GFP fluorescence is observed (lower panel). In contrast, conditional PA2G4 null-mutant parasites retained the ectopic copy of pXNG-PA2G4 as shown by the minimal reduction in GFP intensity. Histograms plots of one representative analysis are shown. Dotted lines correspond to fluorescence background levels of control parasites.</p

2D-DIGE analysis of the <i>L.major</i> mock and GFP-PA2G4 proteome.

<p>(A) Representative 2D-DIGE gel. Protein extracts from promastigotes of three independent biological experiments were differentially labeled with the CyDye fluors Cy3, Cy5 and Cy2, and separated by two-dimensional electrophoresis on 13 cm pH 4–7 IPG strips and 12.5% polyacrylamide gels. Gels were scanned on a Typhoon FLA 9500 (GE Healthcare) imager. A merged image of Cy5-labeled GFP-mock (red) and Cy3-labeled GFP-PA2G4 (green) is shown. The molecular weight of marker proteins and the pI range of the IEF gradient are indicated. (B) Differences in protein abundance were revealed by analyzing the gel images with Delta2D v4.3 software (Decodon). Histograms are a graphic representation of these significant differences. For normalization purposes, a Cy2-labeled internal standard was included, corresponding to a pool of protein from all extracts used in the analysis (yellow bars). Fold change and p-values of the spots selected are shown, as well as protein identification by mass spectrometry. Raw data of MS data and Mascot searches is presented in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002646#pntd.0002646.s003" target="_blank">Table S1</a>.</p

Identification of point mutations previously identified in MIL-resistant <i>L</i>. <i>donovani</i> (T421N, L856P, W210*, M1) and <i>L</i>. <i>major</i> (G582D, M547del).

<p>Previously identified mutations were sequenced and are indicated with an asterisk (*) and highlighted in bold font, using <i>L</i>. <i>major</i> FVI wild-type as the reference strain. No mutations were detected in any of the resistant lines.</p><p>Identification of point mutations previously identified in MIL-resistant <i>L</i>. <i>donovani</i> (T421N, L856P, W210*, M1) and <i>L</i>. <i>major</i> (G582D, M547del).</p

Susceptibility of MIL-resistant <i>L</i>. <i>major</i> FVI populations generated by step-wise selection and determined by EC₅₀ analysis.

<p>1×10⁶ Log-phase parasites were incubated in the presence of a range of drug concentrations for 48 hours at 27°C, and the surviving cells were quantified with Cell Titer Blue proliferation assay using a Typhoon FLA-9500 laser scanner. Populations of parasites were grown in increasing concentrations of MIL ranging from 10 μM (R10) to 40 μM (R40), showing increased resistance to MIL. Horizontal dashed line represents WT threshold for MIL resistance. “Rno” are resistant lines grown in the absence of MIL for at least 75 passages. Results are the average of triplicate experiments ± SD.</p

Virulence of WT and MIL-resistant <i>L</i>. <i>major</i>.

<p>R40 demonstrate attenuated virulence <i>in vivo</i> compared with WT promastigotes. 1×10⁶ WT (n = 5) and R40 (n = 6) metacyclic promastigotes were injected into the footpads of female BALB/c mice. Lesion size was recorded weekly by taking measurements of footpad thickness with a Vernier caliper, results are averages ± SD.</p