Article Figures & Data
Figures
- FIG 1
Multiple-sequence alignment of kinetoplastid leucyl-tRNA synthetases along with representative enzymes from other eukaryotes and bacteria generated using the T-Coffee tool. The signature motifs of class Ia synthetases are indicated in red bold font. The GTG and T-rich motifs are highlighted in yellow. The catalytic aspartic acid residue is highlighted in blue. The CP1 domain is highlighted in light gray. The zinc-binding cysteine motifs are shaded in cyan. The inhibitor binding residues are highlighted in green. The conserved residues are shaded in black, and the conservative mutations are shaded in dark gray. The GenBank accession numbers of the aligned sequences are as follows: Leishmania donovani, XP_003859311.1; Leishmania major, XP_001681854.1; Trypanosoma brucei, XP_828454.1; Trypanosoma cruzi, XP_805515.1; Candida albicans, EEQ43286.1; Homo sapiens, BAA95667.1; Escherichia coli, EFI86361.1; Thermus thermophilus, WP_014628983.1.
- FIG 2
Sequence-based phylogeny of leucyl-tRNA synthetase (LRS) homologs from kinetoplastids, mammals, plants, insects, plasmodia, fungi, bacteria, and archaea. The neighbor-joining bootstrap tree was constructed using MEGA (v5) software. Bootstrap values of >90 are shown in the phylogenetic tree. For analysis, the following sequences were used. The following accession numbers correspond to the species listed from top to bottom, respectively: for Mammalia. BAA95667.1, XP_018883476.1, XP_014996611.1, XP_014710275.1, XP_007080026.1, XP_011962813.1, NP_001095962.1, XP_010848946.1, NP_598898.2, and NP_001009637.1; for Insecta, XP_004930202.1, XP_013140644.1, XP_395743.2, XP_019551695.1, ETN58342.1, XP_011294281.1, JAC42657.1, XP_017855445.1, and AAM50317.1; for Fungi, AAA33599.1, EEQ43286.1, and AAA34805.1; for Plantae, XP_017630544.1, XP_020404072, AAD36946.1, XP_009118303.1, and XP_018483207.1; for Kinetoplastida, XP_009310821.1, XP_805515.1, XP_828454.1, XP_001563232.2, XP_003873311.1, XP_001681854.1, XP_003859311.1, and XP_001464129.1; for Zoomastigophora, EFO65558.1; for Bacteria, NP_213240.1, WP_014628983.1, EFI86361.1, and AMP26935.1; for Plasmodiidae, XP_001349244.1, XP_012762667.2, XP_001614881.1, SCA48531.1, and CDU17148.1; and for Archaea, WP_018153661.1, WP_064496564.1, WP_007043736.1, WP_055430094.1, WP_010885055.1, and WP_010867994.1.
- FIG 3
(A) Purification of the recombinant LdLRS (rLdLRS) protein on Ni2+-NTA acid affinity resin. Lane M, molecular weight marker; lane 1, uninduced cell lysate; lane 2, induced cell lysate; lane 3, fraction eluted with 100 mM imidazole showing purified LdLRS. (B) Immunoblotting analysis of the WT promastigote cell lysate (lane 1) using anti-LdLRS antibody. Two different concentrations (0.5 and 1.0 μg) of the recombinant LdLRS protein were used as a control (lanes 2 and 3). Lane M, molecular weight marker. (C to E) Michaelis-Menten plots and Lineweaver-Burk plots (insets) for aminoacylation kinetics catalyzed by the LdLRS enzyme. The enzyme assay was carried out as described in the Materials and Methods section. The kinetic constants for the utilization of tRNALeu, l-leucine, and ATP by the LdLRS enzyme were computed using the Michaelis-Menten algorithm within GraphPad Prism (v5.0) software. Results are representative data from three separate experiments and are represented as the mean ± SD. V, velocity.
- FIG 4
Binding of LdLRS with leucine (A), norvaline (B), and isoleucine (C). (Top) The raw calorimetric data, denoting the amount of heat (endothermic peaks) produced following each injection of leucine. Upon each titration, as the active site of protein gets saturated with ligand, the area under the peaks gradually becomes smaller. (Bottom) The amount of heat generated per injection as a function of the molar ratio of leucine to enzyme. The experiment was performed in 10 mM phosphate buffer (pH 7.5) at 25°C; the concentration of protein used was 50 μM, and that of the substrate was 400 μM.
- FIG 5
(A) Restriction map of the LdLRS genomic locus and the locations of the primers used for confirmation by PCR-based analysis along with the expected band sizes. Primer 4 was designed as a forward primer to match the upstream region of the LdLRS gene, and primers 8, 3, and 6 were designed to be specific for regions internal to the LRS, HYG, and NEO coding regions, respectively. Primer 2 was designed as a reverse primer to match the downstream region of the LdLRS gene, and primers 7, 1, and 5 were designed as forward primers to be specific for regions internal to the LRS, HYG, and NEO coding regions, respectively. (B, C) Genomic DNA from LRS/HYG or LRS/NEO parasites (B) and ΔLRS(pLRS+) parasites (C) was used as a template for PCR analysis. The specific integration of the replacement cassette(s) was checked with HYG, NEO, and LdLRS (WT) gene-specific primers. ZEO, amplification using zeocin resistance cassette-specific primers for detection of the pSP72α-zeo-α-LRS episome. The numbers above the gel refer to the primers. Lane M, molecular size markers. (D and E) Genomic DNA was digested with MluI and separated on a 0.6% agarose gel for Southern blot analysis of wild-type (WT) strain Bob, single transfectant LRS/NEO, and double transfectant LRS/NEO/HYG parasites probed with the 5′ UTR of the LdLRS gene (D) and WT, LRS/NEO(pLRS+), and ΔLRS(pLRS+) parasites probed with the 370-bp zeocin resistance gene (E). The pSP72α-zeo-α-LRS plasmid digested with MluI was used as a control. Molecular weight markers are indicated to the right of the blots.
- FIG 6
(A) Comparison of the growth curve characteristics of WT, LRS/NEO, and LRS/NEO(pLRS+) promastigotes in M199 medium. The experiment was repeated thrice independently in triplicate. Representative data from one experiment are shown. (B) Comparison of the infectivity profile of L. donovani WT, LRS/NEO, and LRS/NEO(pLRS+) parasites in the J774A.1 murine macrophage cell line. The murine macrophage cell line J774A.1 was infected with stationary-phase promastigotes at a multiplicity of infection of 20:1. Cells were stained after 12 h and 24 h, and amastigotes were enumerated visually. (C) Aminoacylation activity of LdLRS in the cell lysates of L. donovani WT and heterozygous (LRS/NEO) and LRS/NEO(pLRS+) parasites. The results represent the mean ± SD (n = 3). **, a statistically significant difference (P < 0.01) from the wild-type control; ns, nonsignificant.
- FIG 7
(A) Structure of AN2690. (B) ITC binding of AN2690 with LRS. The inhibitor (800 μM AN2690) was titrated into 100 μM protein in phosphate buffer (pH 7.5) at 25°C. The data were fitted with a single-site model available in the Origin program. (C) Dose-response inhibition of the aminoacylation activity of LdLRS in the presence of the inhibitor AN2690. Inhibitor concentrations are plotted on the log scale on the x axis. The experiment was performed with 0.1 to 10 μM AN2690.
- FIG 8
(A and C) The shift in the growth inhibition profile of AN2690 and miltefosine according to the EC50 values for L. donovani LRS heterozygous mutants compared to those for WT cells. LRS/NEO(pLRS+) promastigotes served as a control. L. donovani log-phase (WT) and genetically manipulated promastigotes were seeded in a 96-well flat-bottom plate and incubated with the indicated concentration of AN2690 and miltefosine. The graphs show the cell concentrations relative to those for the untreated controls that were reached after 72 h of incubation at 22°C, as determined by the MTT assay. The experiments were done in triplicate; the graphs depict the mean for the relative cell concentrations and include the standard errors. (B and D) The intracellular parasite load was determined using Giemsa staining of infected J774.A1 murine macrophages at 72 h after treatment with the indicated concentration of AN2690 and miltefosine. The graphs depict the parasite loads relative to those for the untreated controls. The results were obtained in duplicate as representatives of two independent experiments.
- FIG 9
(A) In vivo efficacy of AN2690 against Leishmania donovani in BALB/c mice infected with 1 × 108 stationary-phase L. donovani promastigotes and treated orally after day 7 for 5 days with miltefosine (25 mg/kg) or AN2690 (11 mg/kg) or left untreated. The parasite burden in the spleen, expressed in LDU, was determined 3 days after the completion of the treatment. (B) Real-time PCR of Leishmania donovani in infected mouse spleen tissues showing the fold change in the kDNA level for treated and untreated mice. Ten nanograms of total DNA isolated from the infected and uninfected mouse spleen tissues was used as the template for quantitative PCRs. kDNA levels were quantitated using the 2-ΔΔCT (comparative CT) method. The results for all the infected samples (from vehicular control-, miltefosine-, and AN2690-treated mice) were normalized to those for the uninfected samples. The results are the means from two replicate experiments with five mice in each group. Error bars indicate the standard error. The statistical significance was determined using Student's t test (*, P < 0.05).
Tables
- TABLE 2
Real-time PCR confirmation of Leishmania donovani in infected mouse spleen tissues for treated and untreated groupsa
Group and serial no. Description CT value Treated groups 1 Naive mice 31.859 ± 0.24 2 Vehicular control group 14.799 ± 0.02 3 Miltefosine treatment 23.160 ± 0.08 4 AN2690 treatment 19.621 ± 0.88 Controls 1 Nontemplate control 30.953 ± 0.94 2 Positive control (10 ng DNA from L. donovani parasites) 12.492 ± 0.32 ↵a The data presented are representative of those from two experiments with similar results and represent the mean ± SEM for 5 mice in each group.
- TABLE 3
Primers used for generation of hygromycin- and neomycin-specific linear replacement cassette fragments
Primera Sequence A 5′-TTGAAGGCTCACATGTCGCGTA-3′ BHYG 5′-GGTGAGTTCAGGCTTTTTCATGTTCTTCTCCAGCTGCGCCTTCA-3′ BNEO 5′-CAATCCATCTTGTTCAATCATGTTCTTCTCCAGCTGCGCCTTCA-3′ CHYG 5′-TGAAGGCGCAGCTGGAGAAGAACATGAAAAAGCCTGAACTCACC-3′ CNEO 5′-TGAAGGCGCAGCTGGAGAAGAACATGATTGAACAAGATGGATTG-3′ DHYG 5′-GAAGAGGGTAGACACCCCAACGTCTATTCCTTTGCCCTCGGACGAG-3′ DNEO 5′-GAAGAGGGTAGACACCCCAACGTTCAGAAGAACTCGTCAAGAAG-3′ EHYG 5′-CTCGTCCGAGGGCAAAGGAATAGACGTTGGGGTGTCTACCCTCTTC-3′ ENEO 5′-CTTCTTGACGAGTTCTTCTGAACGTTGGGGTGTCTACCCTCTTC-3′ F 5′-ATGCTTGTCCCGTTCGTGTCGT-3′ G 5′-TTTTTCTAGAATGTCCACGGCACGTCGCGATG-3′ H 5′-TTTTTTCATATGCTACTCGCGCTTCACTACTGG-3′ ↵a HYG, hygromycin; NEO, neomycin.
- TABLE 4
Primers used for molecular characterization of the genetically manipulated parasites by PCR-based analysis
Primer Sequence 1 5′-TGTAGAAGTACTCGCCGATAGTGG-3′ 2 5′-GTTTCAGGTGGCACCAGAAGA-3′ 3 5′-CGCAGCTATTTACCCGCAGGACAT-3′ 4 5′-TGGCCTTTAGCGTCTTCAGCGT-3′ 5 5′-ATAGCGTTGGCTACCCGTGATATTGC-3′ 6 5′-AACACGGCGGCATCAGAGCAGCCGATTG-3′ 7 5′-TCATCGACGATGCCGTGATGTT-3′ 8 5′-TACATGAACGGCAAGCTTCACCT-3′
