Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the proper construction of peptide chains. These enzymes provide raw materials for protein translation and also ensure fidelity of translation. L. donovani is a protozoan parasite that causes visceral leishmaniasis. It is a continuously proliferating parasite that depends heavily on efficient protein translation. Lysyl-tRNA synthetase is one of the aaRSs which charges lysine to its cognate tRNA. Two different coding sequences for lysyl-tRNA synthetases (LdLysRS) are present in this parasite. LdLysRS-1 is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 is closer to bacteria. Here, we have characterized LdLysRS-1 of L. donovani. LdLysRS-1 appears to be an essential gene as the chromosomal null mutants did not survive. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. This study also provides a platform to explore LdLysRS-1 as a potential drug target.

KEYWORDS Leishmania donovani, lysyl-tRNA synthetase, drug targets, genetic validation L eishmaniasis is a vector-borne disease and is caused by the protozoan parasite of the genus Leishmania. The parasite has a dimorphic life cycle alternating between the digestive tract of the female sand fly vector as extracellular flagellated promastigotes and the phagolysosomal compartment of mammalian macrophages as an intracellular amastigote (1). Visceral leishmaniasis (VL) caused by Leishmania donovani is the severe form and is potentially fatal. Due to the lack of an effective vaccine against the disease, VL treatment primarily relies on chemotherapy (2). Moreover, the emergence of resistance to the currently available drugs (3) has worsened the situation. Hence, there is an urgent need to identify novel drug targets to control this disease.
Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein translation, ligating specific amino acids to their cognate tRNAs (4). These enzymes catalyze a two-step process in which the amino acid is activated by formation of an enzymebound aminoacyl-adenylate intermediate followed by the transfer of the activated amino acid to either the 2=-OH or the 3=-OH on the 3=-terminal adenosine of the tRNA (5). The aaRSs can be divided into two classes (class I and class II) based on distinct catalytic domain architectures with exclusive signature motifs for ATP binding (5). Aminoacyl-tRNA synthetases have been a focus of research against the eukaryotic parasites (6). If these enzymes are inhibited, protein translation is halted, which in turn results in the attenuation of parasite growth.
Lysyl-tRNA synthetases (LysRS) are unique as they are found as both class I and class II enzymes (7). Class II LysRS is present in all eukaryotes and most prokaryotes, while class I LysRS has been seen in few bacteria and most archaea (8,9). The class I synthetases contain conserved HIGH and KMSKS residues in the active site. Human LyRS belongs to class II aminoacyl-tRNA synthetases as it lacks both these conserved sequences. The canonical function of LysRS (like that of other aaRSs) is to ligate L-lysine to cognate tRNAs. Besides this, these synthetases can carry out many noncanonical functions like rRNA biogenesis, angiogenesis, apoptosis, transcriptional regulation, and cell signaling in both humans and parasites (10)(11)(12)(13).
LysRS from various organisms like Entamoeba histolytica have been reported to contain a chemokine that imitates the sequence, structure, and role of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte-activating polypeptide II) (14). In Plasmodium falciparum, LysRS have been documented to modulate a variety of cellular functions by synthesizing signaling molecules like diadenosine polyphosphates (15). In Trypanosome brucei, there are two copies of LysRS (TbLysRS-1 and TbLysRS-2). Both the copies are encoded by the nuclear genome. There is a strict functional segregation of the cytosolic and mitochondrial LysRS. The presence of a C-terminal extension in TbLysRS-2 helps the enzyme to remain inactive in the cytosol, but once this enzyme is translocated to mitochondria, the C-terminal sequence is cleaved to produce a mature and active enzyme (16). Crystal structure and functional analysis of human LysRS have revealed that this enzyme can be present in dimeric and tetrameric forms, where the tetrameric form is active during translation and the dimeric form participates in the regulation of transcription (17,18). Previous reports indicate that cladosporin, a fungal secondary metabolite, inhibits LysRS of P. falciparum with high potency (19). Also, LysRS from tropical worm parasites Loa loa (nematode) and Schistosoma mansoni (flatworm) showed 60-fold-better binding with cladosporin than did a human enzyme (20).
Our previous in silico analysis led to the identification of a total of 26 aaRSs in Leishmania (21). The Leishmania genome encodes two copies of LdLysRS (TriTrypDB identifiers [IDs] LdBPK_150270.1 and LdBPK_300130.1). The gene present on chromosome 15 encodes 586-amino-acid-long LdLysRS-1, and the gene present on chromosome 30 encodes 536-amino-acid-long LdLysRS-2. LdLysRS-1 belongs to the class II synthetases. In the present study, we for the first time report the molecular and enzymatic characterization of the LysRS-1 enzyme from Leishmania donovani. The physiological role of LdLysRS-1 was elucidated by making gene deletion mutations using targeted gene replacement methodology. Heterozygous knockout mutants of LdLysRS-1 showed reduced growth and were attenuated in their infectivity, indicating the essentiality of this protein. Cladosporin, a fungal secondary metabolite, and 3-epiisocladosporin, an isoform of isocladosporin (22,23), showed antileishmanial activity in both the promastigote and intracellular amastigote stages in vitro. Both drugs were found to be effective in inhibiting the aminoacylation activity of the recombinant LdLysRS-1. In sum, the data show that LdLysRS-1 is essential for the survival of L. donovani and can be used as a drug target.
demonstrating that the L. donovani LysRS gene encodes a functional enzyme. The kinetic parameters of LdLysRS-1 were established utilizing L-lysine and tRNA Lys as the substrates. The effect of different concentrations of L-lysine was examined while other constituents were kept constant (Fig. 3G). The K m value of rLdLysRS-1 for L-lysine was 111 Ϯ 15 M, which is closer to that documented in the case of humans (25). Since tRNA Lys is another essential substrate for the aminoacylation reaction, we, therefore, performed analysis of tRNA Lys -dependent enzyme kinetics (Fig. 3H). The estimated K m of LdLysRS for tRNA Lys was 3.33 Ϯ 0.80 M. Subcellular localization of LdLysRS-1. Our earlier studies using web-based prediction of signal sequences using PSORT-II indicated cytosolic localization of LdLysRS-1 (21). The localization of LysRS-1 in L. donovani was ascertained by immunofluorescence analysis of log-phase promastigotes using an anti-LdLysRS-1 antibody and 4=,6diamidino-2-phenylindole (DAPI). Figure 4B shows the kinetoplast (k) and nuclear DNA (n) as indicated by the bright staining with DAPI. Analysis by confocal microscopy revealed that LdLysRS is localized in the cytosol of the parasites (Fig. 4C). The mouse preimmune sera, nonpermeabilized cells, and secondary antibody were used as controls. No detectable signal was detected with this control (data not shown).
Gene deletion of LdLysRS-1. In order to determine the indispensability of LdLysRS-1 in the parasite, classical gene replacement experiments were employed, where efforts were made to replace both the wild-type (WT) alleles of LdLysRS-1 with cassettes harboring drug resistance marker genes. As elucidated in Materials and Methods, this was done by the generation of inactivation cassettes having hygromycin phosphotransferase (HYG) or neomycin phosphotransferase (NEO) as a selection marker fused with the flanking 5= untranslated region (UTR) and 3= UTR of the LdLysRS-1 gene (Fig. 5A). Linear replacement cassettes were prepared by PCR-based fusion reactions and were electroporated into the wild-type (WT) L. donovani promastigotes. This resulted in the generation of heterozygous parasites (LysRS-1/HYG or LysRS-1/NEO) in which either the hygromycin or neomycin drug resistance gene replaced one allele of the LdLysRS-1 gene. Further, the PCR-based analysis was done to confirm the genotype of the heterozygous parasites (LysRS-1/HYG or LysRS-1/NEO) by utilizing primers (Table 1) external to the linear replacement cassette of the LdLysRS-1 gene (Fig. 5A). The correct integration of HYG and NEO replacement cassettes at the LdLysRS-1 locus was FIG 1 (A) Multiple sequence alignment of representative LysRS sequences from kinetoplastids, humans, yeast, plasmodia, and bacterial species generated using Clustal W (35). The ELR motif is highlighted in yellow. The key residues present in the ATP-binding site are highlighted in blue and red. For analysis, we used Linj. 15   To further establish the essentiality of the LdLysRS-1 gene, the construction of homozygous null mutants was attempted in the presence of a rescuing episome that has the LdLysRS-1 gene (pSP72␣-zeo-␣-LysRS-1). The heterozygous parasites (LysRS-1/ HYG) were transfected with pSP72␣-zeo-␣-LysRS-1 to generate LysRS-1/HYG[pLysRS-1 ϩ ] mutants. After selection of these parasites in double-antibiotic-containing M199 medium, these mutant parasites (LysRS-1/HYG[pLysRS-1 ϩ ]) were transfected with the 5= UTR-NEO-3= UTR construct. After 3 to 4 passages, genomic DNA was isolated and investigated for the presence of the LdLysRS-1 gene in these ⌬LysRS-1[pLysRS-1 ϩ ] triple-drug-resistant parasites. PCR analysis revealed the absence of the LdLysRS-1 gene in these parasites (Fig. 5C, lanes 4-8 and 7-2), and bands corresponding to the The aminoacylation activity of LysRS-1 was measured in genetically modified parasites and compared to that of WT parasites (Fig. 6A). This was done to establish if the deletion of a single allele of LysRS-1 resulted in the decrease in aminoacylation activity of LysRS-1. A significant reduction in the aminoacylation activity of LysRS-1 was observed in the heterozygous parasites (LysRS-1/NEO) (2.8-fold) compared to that of the WT parasites. Comparable LysRS-1 activity levels were exhibited in add-back mutants (LysRS-1/HYG[pLysRS-1 ϩ ]) and the WT strain (Fig. 6A).
Analysis of the growth kinetics of heterozygous and rescue mutant parasites was undertaken to verify if the reduced expression of LysRS-1 affects the growth of the parasites. The heterozygous parasites (LysRS-1/HYG) showed a consistent growth delay compared to their WT counterparts (Fig. 6B). Add-back mutants (LysRS-1/HYG[pLysRS-1 ϩ ]) rescued the growth of these parasites similar to that of the WT control (Fig. 6B). It is possible that a gene dosage resulted in the lesser synthesis of LysRS-1 protein, thereby leading to suboptimal cell proliferation.

Drug binding and inhibition of recombinant LdLysRS-1.
We also checked the effect of these compounds on the aminoacylation activity of recombinant LdLysRS-1 (Fig. 9A). Cladosporin inhibited the enzymatic activity of rLdLysRS-1 with an IC 50 of 4.07 &micro;M, while 3-epi-isocladosporin inhibited rLdLysRS-1 with an IC 50 of 25.5 &micro;M. A concentration of isocladosporin as high as 1 mM failed to inhibit the enzymatic activity of LdLysRS-1 (Fig. 9A). The binding of cladosporin or 3-epiisocladosporin and LdLysRS-1 was further established by checking the relative binding affinities of cladosporin, 3-epi-isocladosporin, or ATP for LdLysRS-1 by performing thermal shift assays. The thermal melting profile of LdLysRS-1 was only slightly altered by ATP with a shift of~1.5°C (Fig. 9B). In contrast, addition of cladosporin and 3-epi-isocladosporin shifted the melting curve by~8°C and~3°C, respectively (Fig. 9B). These data indicate higher affinity and greater thermal stability of the LdLysRS-1cladosporin complex than the LdLysRS-1-3-epi-isocladosporin complex.

DISCUSSION
Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes of the protein translation machinery that ensure fidelity in the translation of mRNA. These enzymes are the validated targets for the development of new antiparasitic agents with novel mechanisms of action (6). Among all aaRSs, lysyl-tRNA synthetase (LysRS) is unusual because it belongs to either class I or class II enzymes (26). In most organisms, LysRS is present as the class II form. However, in many archaea, in several alphaproteobacteria, and in spirochetes, a very different type of LysRS which is homologous to class I aaRSs is present (27).
While humans possess a single copy of LysRS (28), Leishmania and trypanosomes encode two copies of LysRS (21,29). One of the LdLysRS (LdBPK_150270.1) (LdLysRS-1) has an N-terminal extension of 80 amino acids (DUF972). The N-terminal extension in mammals has been reported to participate in tRNA binding (25), whereas its role in Leishmania is not known. The N-terminal extension of LdLysRS-1 also contains an ELR motif that is known to have chemokine activity in humans. LdLysRS-2 has a C-terminal extension similar to that reported in TbLysRS-2. This C-terminal extension in TbLysRS-2 enables the enzyme to remain inactive in the cytosol, but once the enzyme is translocated to the mitochondria, the C-terminal sequence is cleaved to produce a mature and active enzyme (16). However, experimental verification of the role of N-and C-terminal extensions and the ELR motif in L. donovani is required to address this. We checked the triggering of cytokine secretion by a murine macrophage cell line using recombinant LdLysRS-1. The culture supernatants were analyzed for the presence of proinflammatory cytokines. Time kinetic analysis by enzyme-linked immunosorbent assay (ELISA) revealed no trigger of cytokine release from macrophages (data not shown). These data indicate that LdLyRS-1 is probably not a chemokine.
In the present study, we for the first time report the molecular characterization of LdLysRS-1. This study provides genetic validation of LdLysRS-1 as an essential enzyme in Leishmania. The open reading frame (ORF) of LdLysRS-1 encodes a 586-amino-acidlong polypeptide. Kinetic analysis of the recombinant LdLysRS-1 showed that it exhibited catalytic efficiency similar to that reported for other mammalian LysRS (25). Our results indicate that the LdLysRS-1 gene encodes an aaRS that is present in the cytosol. Gene deletion studies stated that LysRS-1 is essential for L. donovani viability and may be explored as a possible antileishmanial drug target. Earlier reports show that the knockdown of expression of the gene encoding TbLysRS-1 in T. brucei resulted in parasite growth arrest, indicating the essentiality of this gene for parasite growth (29).
Cladosporin is a fungal secondary metabolite, and its efficacy as a lysyl-tRNA synthetase inhibitor has been reported in the case of P. falciparum (19). Isocladosporin, isolated from the fungus Cladosporium cladosporioides, is also composed of a THP ring similar to cladosporin. 3-Epi-isocladosporin is an isomer of isocladosporin (23). Cladosporin mimics the adenosine part of ATP and hence interacts with the catalytic site of the LysRS (30). The basis of cladosporin selectivity has been reported earlier (19). The majority of the amino acid residues in the ATP-binding pocket are highly conserved across different species. However, a clear variation has been reported at 2 amino acid positions corresponding to Saccharomyces cerevisiae residue Gln324 and Thr340 (19) (Fig. 1A). In LdLysRS-1 of Leishmania spp., these positions are occupied by Gln308 and Ser324 residues (Fig. 1A). However, in the case of LdLysRS-2, these positions are occupied by Val189 and Thr205, respectively. A clear correlation has been predicted between cladosporin activity and the identity of these amino acids at these two key positions in the ATP-binding pocket (19). Reduced cladosporin potency is predicted whenever a bulkier residue, e.g., in replacement of serine with threonine, is present at position 340, as in the case of Saccharomyces cerevisiae. However, P. falciparum, which has Val328 and Ser344, has much higher potency toward cladosporin than L. donovani LdLysRS-2, which has Val189 and Thr205 (P. falciparum, IC 50 of 0.04 to 0.08 &micro;M [19]; L. donovani, IC 50 of 2.56 &micro;M [19]).
We analyzed the effects of these three compounds on parasite survival and aminoacylation activity of LdLysRS-1. Cladosporin, 3-epi-isocladosporin, and isocladosporin were found to inhibit parasite growth. Cladosporin was the most efficient with the lowest IC 50 s (4.2 &micro;M in promastigotes and 1.1 &micro;M in amastigotes) in comparison to the other two analogues. Our data show that LdLysRS-1 has glutamine at position 308 and serine at position 324, and in humans, these positions are occupied by Gln321 and Thr337, respectively (Fig. 1A). Since a bulkier amino acid (Thr) is replaced in the case of humans, this possibly results in reduced cladosporin potency (19). Our data show that cladosporin has relatively higher IC 50 s in THP-1-derived macrophages (CC 50 , 113 &micro;M) than in amastigotes (1.1 &micro;M).
The aminoacylation activity of LdLysRS-1 was inhibited by cladosporin (IC 50 , 4.0 &micro;M) and 3-epi-isocladosporin (IC 50 ,~25.5 &micro;M). A concentration of isocladosporin as high as 1 mM failed to inhibit the enzymatic activity of LdLysRS-1. Cladosporin possessed a 50% inhibitory concentration of 4.0 &micro;M against LdLysRS-1 (Fig. 9A), which is comparable to its activity in cellular screens (IC 50 s, 4.2 &micro;M in promastigotes and 1.1 &micro;M in amastigotes). These data indicate that lysyl-tRNA synthetase is the primary target within the cell. The specificity of inhibition of lysyl-tRNA synthetase by cladosporin is also supported by using LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. In conclusion, we have characterized Leishmania LysRS-1 and show that it is essential for parasite growth or infectivity in vitro. Further studies are ongoing in the laboratory to check the efficacy of these inhibitors in the in vivo mouse model. The inhibitors studied here may provide a framework for the development of a new class of drugs against Leishmania parasites.

MATERIALS AND METHODS
Chemicals. All DNA-modifying enzymes and restriction enzymes were obtained from New England Biolabs. Hygromycin, zeocin, and paromomycin were attained from Sigma. pET-30a plasmid was acquired from Novagen. Protein markers and DNA ladders were obtained from New England Biolabs. Escherichia coli DH10␤ and BL21(DE3) were utilized as hosts for plasmid cloning and protein expression, respectively. Ni 2ϩ -nitrilotriacetic acid (NTA) agarose was purchased from Qiagen. Cladosporin, 3-epiisocladosporin, and isocladosporin were synthesized by Debendra K. Mohapatra, CSIR-Indian Institute of Chemical Technology, Hyderabad, India (22,23). Sypro orange dye was obtained from Sigma. The rabbit antitubulin antibody was acquired from Neomarker (Fremont, CA). Other materials utilized as part of this study were of analytical grade and were commercially available.
The axenic amastigotes were obtained by the standard protocol as described earlier (31). THP-1, an acute monocytic leukemia-derived human cell line obtained from ATCC, was grown in RPMI 1640 medium (Sigma) supplemented with 10% FBS and antibiotics (100 units/ml penicillin and 100 g/ml streptomycin) at 37°C with 5% CO 2 .
Sequence and phylogenetic analysis. LysRS sequences retrieved from TriTrypDB (32), Swiss-Prot/ UniProtKB (33), and PlasmoDB (34) were used for multiple sequence alignment. Multiple sequence alignment of these sequences was done using ClustalW (35) using default parameters and utilized as seed alignment for phylogenetic tree generation utilizing the Jones-Taylor-Thornton (JTT) model. MEGA version 5.0 (36) was utilized for both analysis and visualization of the phylogenetic tree.
Expression and purification of the recombinant LdLyRS-1 protein.
In order to express the LdLysRS-1 gene (TriTrypDB ID LdBPK_150270.1), the coding region was PCR amplified from L. donovani genomic DNA using a sense primer with an adjacent BamHI site (5= AAAGGATCCATGTCGTCCCTCGAAG AGCTCCGTA 3=) and an antisense primer with an adjoining HindIII site (5= AAAAAGCTTCTACAGCAGGG GAACACCCTGACCAT 3=). The digested 1,761-bp PCR product covering the LdLysRS open reading frame (ORF) was cloned in frame into BamHI and HindIII restriction sites of pET-30a vector (Novagen). The resulting construct (LdLysRS-1-pET-30a) with a His 6 tag at the N-terminal end was transformed into the E. coli BL21(DE3) strain (Novagen). The protein expression of recombinant LdLysRS (rLdLysRS) was induced at an optical density at 600 nm (OD 600 ) of 0.6 with 0.3 mM IPTG (isopropyl-␤-Dthiogalactopyranoside) at 16°C for 16 h. The protein was purified by affinity chromatography using Ni 2ϩ -nitrilotriacetic acid agarose resin (Qiagen) by eluting with increasing concentrations of imidazole. The protein was further purified by gel permeation chromatography on a Superdex 200 16/60 GL column (GE Healthcare). Eluted fractions were checked by SDS-PAGE, and fractions were pooled and concentrated.
Aminoacylation assays. The L. donovani tRNA Lys was synthesized by in vitro transcription from a PCR product template, having a T7 RNA polymerase promoter followed by a gene encoding the L. donovani tRNA Lys sequence (TriTrypDB ID LinJ.10.tRNA1) and the terminal CCA sequence. The in vitro transcription reaction was carried out with the MEGAscript T7 polymerase kit (Ambion; Life Technologies) at 37°C for 16 h according to the manufacturer's guidelines. Transcripts were extracted using acid phenolchloroform (5:1) solution, pH 4.5 (Ambion; Life Technologies), and were precipitated with isopropanol (Sigma). The tRNA was folded prior to the aminoacylation reactions by heating at 70°C for 10 min, followed by the addition of 10 mM MgCl 2 and slow cooling at room temperature (RT). The aminoacylation reaction was done in 30 mM HEPES (pH 7.5), 150 mM NaCl, 30 mM KCl, 50 mM MgCl 2 , 1 mM dithiothreitol (DTT), 200 M ATP, 10 mM L-lysine, 8 M tRNA Lys , 2 units/ml inorganic pyrophosphatase (PPiase) (Sigma), and 0.2 M rLdLysRS-1 protein at 37°C (37). The aminoacylation reaction was stopped at different time points by the addition of 10 mM EDTA and developed by addition of malachite green (Echelon Bioscience). Absorbance was measured at 620 nm with a SpectraMax M2 reader (Molecular Devices). The K m and V max for L-lysine and tRNA Lys were determined by varying the concentration of L-lysine or tRNA Lys in the reaction mixture while the other components were maintained in excess. For rLdLyRS-1 inhibition, a reaction mixture containing rLdLysRS-1 (0.2 M) was incubated with different concentrations of cladosporin, 3-epi-isocladosporin, and isocladosporin (0.1 nM to 1 mM) for 30 min at 37°C. Reactions were stopped and quantitated as described above. The 50% inhibitory concentration (IC 50 ) was determined. Using GraphPad Prism, the dose-response data were fitted to the log (inhibitor)versus-response equation.
Generation of molecular constructs for the substitution of LdLysRS-1 alleles. A targeted gene replacement strategy based on PCR fusion was employed (38) for the inactivation of the LdLysRS-1 gene. Briefly, flanking regions of LdLysRS-1 were PCR amplified from genomic DNA of L. donovani and were linked to the hygromycin phosphotransferase gene (HYG) or the neomycin phosphotransferase gene (NEO). The 5= UTR (783 bp) and 3= UTR (925 bp) of the LdLysRS-1 gene were PCR amplified using primers A and B Hyg or A and B Neo and primers E Hyg and F or E Neo and F (Table 2), respectively. NEO and HYG genes were amplified from pX63-NEO and pX63-HYG templates using primers C Neo and D Neo and primers C Hyg and D Hyg (Table 2), respectively. The 5= UTR of the L. donovani LysRS-1 gene was then fused to either of the antibiotic resistance marker genes (HYG/NEO) by PCR utilizing primers A and D Hyg or primers A and D Neo . 5= UTR-marker gene-3= UTR constructs were obtained using primers A and F by utilizing 5= UTR-marker gene and 3= UTR as the templates. An episomal copy of the LdLysRS-1 gene was generated by amplification of the LdLysRS-1 coding sequence with a sense primer possessing the XbaI site (primer 7) and antisense primer with the HindIII site (primer 8) (Table 1). After amplification of LdLysRS-1, the gene was cloned into the pSP72␣-zeo-␣ vector to get the pSP72␣-zeo-␣-LysRS-1 construct. All the synthesized fragments and constructs were sequenced before transfection.
Creation of genetically modified parasites. After the generation of linear replacement fragments, 2 g of the fragment (5= UTR-Hyg 3= UTR or 5= UTR-Neo 3= UTR) was separately transfected into wild-type L. donovani promastigotes (38). Drug selection was carried out depending on the marker gene. In order to check for the correct integration of inactivation cassettes, the parasites resistant to antibiotic selection were further subjected to PCR-based analysis using primers shown in Table 1. To knock out the other allele of the LysRS-1 gene, the second round of transfection was initiated. In order to check the genotype of mutants, Southern analysis was done utilizing a standard protocol (39).
Growth and infectivity assays. Growth rate experiments were done by seeding stationary-phase parasites at a density of 1 ϫ 10 6 cells/ml in drug-free M199 medium with 5% FBS in 25-cm 2 flasks at 22°C. The growth rate of cultures was monitored microscopically at 24-h intervals for 7 days with a Neubauer hemocytometer. The experiments were repeated at least three times. For the infectivity assay, the THP-1 cell line was plated at a cell density of 5 ϫ 10 5 cells/well in a 6-well flat-bottom plate. THP-1 cells were treated with 0.1 &micro;M phorbol myristate acetate (PMA; Sigma) at 37°C for 48 h to achieve differentiation into adherent, nondividing macrophages. After activation, adherent cells were infected with stationary-phase promastigotes, at an MOI of 20:1 for 6 h. Extra nonadherent promastigotes were then removed by incubating the cells for 30 s in phosphate-buffered saline (PBS). These were then maintained in RPMI 1640 medium containing 10% FBS at 37°C with 5% CO 2 . Propidium iodide staining was done to visualize the intracellular parasite load.
Drug inhibition assays. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay was performed with L. donovani promastigotes in order to determine susceptibility profile of parasites against cladosporin, 3-epi-isocladosporin, and isocladosporin. Log-phase promastigote parasites (5 ϫ 10 4 cells/well) were seeded in a 96-well flat-bottom plate (Nunc) and incubated with different drug concentrations in M199 medium with 5% FBS at 22°C. After 72 h of incubation, 10 l of MTT (5 mg ml Ϫ1 ) was added to each well, and the plates were further incubated at 37°C for 4 h. The reaction was stopped by the addition of 50 l of 50% isopropanol and 20% SDS followed by gentle shaking at 37°C for 30 min. The absorbance was measured at 570 nm in a microplate reader (SpectraMax M2 from Molecular Devices). The percentage of parasite growth relative to the untreated cells at different drug concentrations was determined, and the 50% inhibitory concentration for each drug was calculated.
The sensitivities of intracellular amastigotes to cladosporin, 3-epi-isocladosporin, and isocladosporin were determined by visualization of the intracellular parasite load using propidium iodide staining of the infected THP-1 differentiated macrophages, 72 h after treatment with different concentrations of the drug.
Thermal shift assay. The thermal shift assay (40) was performed with rLdLysRS-1. LdLysRS-1 (15 &micro;g) diluted in 30 &micro;l buffer containing 50 mM Tris (pH 7.5), 300 mM NaCl, 5 mM MgCl 2 , 1 mM L-lysine, and 2ϫ Sypro orange dye along with different ligands (5 mM ATP [Sigma] and 5 mM drugs) was incubated at room temperature for 10 min. The samples were then heated from 25 to 99°C at a rate of 1°C min Ϫ1 . Fluorescence signals were monitored by the CFX96 real-time system (Bio-Rad). The assays were repeated three times independently. Antibody generation and Western blot analysis. Polyclonal antibodies against highly purified recombinant LdLysRS-1 were raised commercially (Merck) in rabbits. The late-log-phase promastigotes and axenic amastigotes were harvested, and the resultant cell pellets were resuspended in lysis buffer (10 mM Tris-Cl, pH 8.0, 5 mM DTT, 10 mM NaCl, 1.5 mM MgCl 2 , 0.1 mM EDTA, 0.3 mM phenylmethylsulfonyl fluoride [PMSF], and 0.5% Triton X-100). The cells were lysed by freeze-thaw cycles followed by sonication on ice. Lysates were centrifuged at 13,000 rpm, and supernatants were fractionated on a 10% SDS-PAGE gel. Proteins were then transferred onto a nitrocellulose membrane (Bio-Rad). After blocking with 5% bovine serum albumin, the membrane was probed with primary antibodies (1:3,000 dilution) and secondary horseradish peroxidase (HRP)-conjugated antibodies (1:5,000 dilution). The blot was developed using the enhanced chemiluminescence (ECL; Amersham Biosciences) kit according to the manufacturer's protocol.
Immunofluorescence microscopy. For the intracellular localization of LdLysRS-1 promastigotes, the cells were washed with 1ϫ PBS and immobilized on poly-L-lysine-coated coverslips. The cells were then fixed with 4% paraformaldehyde and permeabilized in 0.5% Triton X-100, followed by incubation with the anti-LdLysRS-1 antibody (1:500) for 1 h at room temperature. Cells were washed and incubated for 45 min at room temperature (RT) with Alexa 488-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific). The nuclear and the kinetoplastid DNA were then stained with 1 &micro;g/ml of DAPI (Sigma) for 15 min. The fluorescence of the stained parasites was visualized by a confocal laser scanning microscope (Olympus FluoView FV1000 with PLAPON 60ϫ O objective lenses; numerical aperture [NA], 1.42).
Statistical analysis. Results for aminoacylation activity in cell lysate and in the infectivity assay were shown as column data in GraphPad Prism and were analyzed using Student's t test. Data are represented as means Ϯ standard deviations (SD). A P value of Ͻ0.05 was accepted as an indication of statistical significance.