Table 1.
Primers used for the generation of Hyg and Neo specific replacement cassettes.
Table 2.
Primers used for the molecular characterization of genetically modified parasites by PCR based analysis.
Fig 1.
Expression, purification, western blot and enzymatic activity of LdThrRS.
(A) Induction and purification of recombinant LdThrRS (rLdThrRS) by Ni2+-NTA affinity chromatography. M, molecular weight marker; Lane 1, uninduced cell lysate; Lane 2, induced cell lysate; Lane 3 and 4, eluted fractions with 300 mM imidazole showing purified rLdThrRS. (B) Western blot analysis of rLdThrRS with anti-LdThrRS antibody (1:1000), Lane 1, 0.5 μg of rLdThrRS; Lane 2, 1 μg of rLdThrRS; Lane 3, 2 μg of rLdThrRS. (C) Immunoblot analysis of the cell lysate of 30 μg Leishmania with the anti-LdThrRS antibody (1:1000), Lane 1: promastigote; Lane 2: amastigote. (D) Time-dependent aminoacylation assay of rLdThrRS. The aminoacylation reactions were performed with L-threonine and tRNAThr as the substrates. The result shows an average of three different experiments performed in duplicate ± SD. (E) and (F) The aminoacylation kinetics catalyzed by rLdThrRS as a function of L-threonine (E) and tRNAThr (F) concentration. The kinetic parameters were calculated by a Michaelis-Menten algorithm within GraphPad Prism 5.0 for utilization of L-threonine and tRNAThr by the rLdThrRS enzyme. Results are representative data from three separate experiments and are represented as mean ± S.D.
Fig 2.
Localization of LdThrRS in L. donovani.
Immunofluorescence analysis by confocal micrograph of wild-type log phase promastigotes stained with DAPI (C), anti-LdThrRS antibody detected using Alexa 488 (green)-conjugated secondary antibody (B) and mitotracker red CMXRos (A). (E) and (F) merged micrographs and (D) phase contrast image. ‘k’ and ‘n’ indicate kinetoplastid and nuclear DNA respectively. The scale bar represents 5 μm.
Fig 3.
Generation of heterozygous mutants of LdThrRS.
(A) Map of LdThrRS genomic locus and pSP72α-zeo-α-ThrRS episomal construct is shown with the position of the primers used for confirmation of WT and mutant parasites by PCR-based analysis along with the expected band sizes. Primer pairs are shown in Table 2. Primer 4 was designed as a forward primer to match the upstream region of LdThrRS gene, and primers 8, 3 and 6 were designed internal to LdThrRS, HYG and NEO coding regions, respectively. Primer 2 was designed as a reverse primer to match the downstream region of LdThrRS gene and primers 7, 1 and 5 were designed as forward primers, internal to LdThrRS, HYG and NEO coding regions, respectively. Forward primer ZeoFP and reverse primer ZeoRP were designed to match the upstream and downstream region of zeocin resistance gene. Genomic DNA from WT, heterozygous parasites (ThrRS/NEO or ThrRS/HYG) was used as a template for PCR analysis. (B) The specific fusion of the replacement cassette(s) was checked with NEO and gene-specific primers as reported in Table 2 and Fig 3A. Lane 1 (Primers 4 and 6); Lane 2 (Primers 5 and 2); Lane 3 (Primers 4 and 8) and Lane 4 (Primers 7 and 2). (C) The specific fusion of the replacement cassette(s) was checked with HYG and gene-specific primers. Lane 1 (Primers 4 and 3); Lane 2 (Primers 1 and 2); Lane 3 (Primers 4 and 8) and Lane 4 (Primers 7 and 2). (D) WT genomic DNA was used as a negative control. The bands corresponding to the WT gene were obtained in Lane 3 and 4. No bands were observed after using NEO specific primers, Lane 1 (Primers 4 and 6); Lane 2 (Primers 5 and 2) and HYG specific primers, Lane 5 (Primers 4 and 3); Lane 6 (Primers 1 and 2). M indicates the molecular size marker in kb. (E) Southern blot analysis of genomic DNA from wild-type (WT) (Lane 1), ThrRS/NEO (Lane 2) and ThrRS/HYG (Lane 3) parasites. Genomic DNA from WT, ThrRS/NEO and ThrRS/HYG parasites were digested with SphI, separated on a 0.6% agarose gel and probed with 3’UTR of LdThrRS gene. (F) PCR analysis of rescue mutants (ThrRS/NEO[pThrRS+]). The specificity of recombination was checked with Zeor specific primers, Lane 1 (Primers ZeoFP and ZeoRP). M indicates the DNA molecular size marker. (G) Western blot analysis of WT (Lane 1), heterozygous parasites (ThrRS/NEO) (Lane 2), ThrRS overexpressors (WT[pThrRS+]) (Lane 3) and rescue mutant (ThrRS/NEO[pThrRS+]) (Lane 4) parasites. β- tubulin was used as a loading control.
Fig 4.
Characterization of genetically modified parasites.
(A) Aminoacylation activity of LdThrRS in the cell lysates of L. donovani WT, heterozygous (ThrRS/NEO) and rescue mutant (ThrRS/NEO[pThrRS+]) parasites. (B) Comparison of the growth curve characteristics of WT, heterozygous (ThrRS/NEO) and rescue mutant (ThrRS/NEO[pThrRS+]) promastigotes in M199 media. The experiment was repeated thrice in triplicate. The data shown here is from one experiment. (C) and (D) Comparison of the infectivity (C) and parasite load (D) of L. donovani WT, ThrRS/NEO and ThrRS/NEO[pThrRS+] parasites in J774A.1 murine macrophage cell line. The stationary phase promastigotes were used to infect murine macrophage cell line J774A.1 at an MOI of 20:1. After 48 h of infection, cells were stained, and amastigotes were counted visually. The results signify mean ± S.D with n = 3, *P < 0.05, **P < 0.01 statistical difference from the wild-type control.
Fig 5.
Cell cycle and morphological study of heterozygous parasites.
Cell cycle analysis of (A) WT, (B) heterozygous parasites (ThrRS/NEO) and (C) Rescue mutants (ThrRS/NEO[pThrRS+]). PI fluorescence (FL2-Area) is plotted versus cell count. The first peak (red color) indicates cells in G0/G1 phase, S phase is indicated in blue color, and third peak (red color) represents cells in G2/M phase. (D) A bar graph is representing the percentage of cells in the G0/G1 phase of WT, ThrRS/NEO and ThrRS/NEO[pThrRS+] parasites. The results represent mean ± S.D with n = 3, ***P < 0.005 statistical difference from the wild-type control. (E) Confocal imaging of WT, ThrRS/NEO and ThrRS/NEO[pThrRS+] parasites.
Fig 6.
Effect of borrelidin on the rLdThrRS enzyme.
(A) 2D structure of borrelidin (B) Dose-response inhibition of the aminoacylation activity of rLdThrRS in the presence of known ThrRS inhibitor, borrelidin. Inhibitor concentrations are plotted in the log scale on X-axis. The experiment was performed with 0.001–10 μM borrelidin. (C) Top Panel: ITC profile for binding of rLdThrRS to borrelidin indicating the sequential injection of the drug into rLdThrRS after correction of the heat of dilution of the drug. Bottom panel: Plot of integrated heat data fitted to a one-site model at 25°C. (D) Bar diagram is describing the variation of the magnitude of thermodynamic parameters of binding of borrelidin to rLdThrRS.
Fig 7.
Effect of borrelidin on the growth of WT, ThrRS/NEO and ThrRS/NEO[pThrRS+] parasites.
(A) and (B) Inhibition profile of borrelidin for the promastigote (A) and intracellular amastigote (B) growth of WT parasites. Percentage parasite survival was plotted against different concentrations of borrelidin. (C) and (D) The leishmanicidal effect of borrelidin was checked on promastigotes (C) and amastigotes (D) of WT, ThrRS/NEO and ThrRS/NEO[pThrRS+] parasites. The mean IC50 values were calculated for borrelidin and plotted as bar graphs. (E) The effect of miltefosine on WT, ThrRS/NEO and ThrRS/NEO[pThrRS+] promastigotes. The bar graphs represent mean ± SD with n = 3. * p < 0.05.
Fig 8.
Structural modelling of LdThrRS.
(A) Structural superposition of LdThrRS model with human ThrRS with an RMSD of 0.21Å. The LdThrRS protein is shown in brown color, and human ThrRS is displayed as green. (B) Active site residues are labelled as sticks.