Twin Attributes of Tyrosyl-tRNA Synthetase of Leishmania donovani

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes essential for protein synthesis. Apart from their parent aminoacylation activity, several aaRSs perform non-canonical functions in diverse biological processes. The present study explores the twin attributes of Leishmania tyrosyl-tRNA synthetase (LdTyrRS) namely, aminoacylation, and as a mimic of host CXC chemokine. Leishmania donovani is a protozoan parasite. Its genome encodes a single copy of tyrosyl-tRNA synthetase. We first tested the canonical aminoacylation role of LdTyrRS. The recombinant protein was expressed, and its kinetic parameters were determined by aminoacylation assay. To study the physiological role of LdTyrRS in Leishmania, gene deletion mutations were attempted via targeted gene replacement. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. LdTyrRS appears to be an essential gene as the chromosomal null mutants did not survive. Our data also highlights the non-canonical function of L. donovani tyrosyl-tRNA synthetase. We show that LdTyrRS protein is present in the cytoplasm and exits from the parasite cytoplasm into the extracellular medium. The released LdTyrRS functions as a neutrophil chemoattractant. We further show that LdTyrRS specifically binds to host macrophages with its ELR (Glu-Leu-Arg) peptide motif. The ELR-CXCR2 receptor interaction mediates this binding. This interaction triggers enhanced secretion of the proinflammatory cytokines TNF-α and IL-6 by host macrophages. Our data indicates a possible immunomodulating role of LdTyrRS in Leishmania infection. This study provides a platform to explore LdTyrRS as a potential target for drug development

Aminoacyl-tRNA synthetases (aaRSs) 3 are the central. enzymes in protein translation, providing charged tRNAs for the appropriate construction of peptide chains. The canonical function of aaRSs is to charge specific tRNAs with their cognate amino acids and thereby contribute to accurate mRNA translation during protein synthesis. Thus, aaRSs are essential components of protein synthesis in every living species.
Apart from their basic function of charging tRNA molecules for protein synthesis, non-canonical functions like ribosomal RNA biogenesis, angiogenesis, apoptosis, transcriptional regulation, and cell signaling have also been reported for several aaRSs (1,2). Novel functions of this group of enzymes depend on the addition of one or more new domains or motifs during the course of evolution (3).
Tyrosyl-tRNA synthetase (TyrRS) is one such aaRS, which belong to a family of class I synthetases, characterized by a structurally well conserved amino-terminal Rossmann-fold domain, which contains the signature sequences "HIGH" and "KMSKS." The mammalian TyrRS contains a closely homologous endothelial monocyte activating polypeptide II (EMA-PII) domain at the C terminus (4). Under specific conditions, human TyrRS is processed by an elastase enzyme into a free carboxyl-terminal EMAPII-like domain and a second aminoterminal part known as mini-TyrRS. Both released proteins are active in distinct immune signaling pathways (4,5). The carboxyl-terminal domain of human TyrRS mimics the cytokine function of EMAPII. On the other hand, human mini-TyrRS, via its ELR (Glu-Leu-Arg) motif, interacts with the CXCchemokine receptor (CXCR1/2), and like IL-8, functions as a chemoattractant for polymorphonuclear leukocytes (4,5). The ELR motif is a signature motif of CXC chemokines such as IL-8 that are active as polymorphonuclear leukocyte chemoattractants (6). This motif is essential for receptor binding and the chemotactic activity of CXC chemokines (7)(8)(9). The ELR motif in mini-TyrRS is also important for its chemokine-like activity (4,5). This data on human TyrRS suggests that the twin attributes of chemokine trigger and aminoacylation coexist in TyrRS.
Not much is known about aaRSs in Leishmania sp. Leishmania genus is the causative agent of leishmaniasis, a group of neglected diseases. The clinical symptoms of the disease depend on the species involved. Leishmania has a digenetic life cycle. Infected sand flies inoculate the mammalian host with promastigotes. Within the mammalian host, promastigotes differentiate into amastigotes that replicate in phagolysosomes. Leishmania parasites have the capability of subverting host function, thereby allowing the parasite to thrive within the organism (10,11). The development of resistance to currently available anti-leishmanial drugs has led to an urgent need to discover novel drug targets (12). In this regard, aaRSs constitute ideal targets for drug development.
The crystal structure of Leishmania major TyrRS has been recently solved, and it is known to exist as an asymmetric pseudodimer (13). In the present study, we report the catalytic promiscuity and moonlighting function of L. donovani TyrRS (LdTyrRS). The twin attributes of L. donovani tyrosyl-tRNA synthetase, namely, aminoacylation and chemokine trigger have been determined. Our earlier comprehensive bioinformatic analysis led to the identification of a total of 26 aaRSs in Leishmania (14). The Leishmania genome encodes a single copy of TyrRS (tritrypDB ID LdBPK_141460.1). The present study characterizes the aminoacylation activity of LdTyrRS. To elucidate the physiological role of LdTyrRS, gene deletion mutations were attempted via targeted gene replacement. Heterozygous knock-out mutants of LdTyrRS showed reduced growth and were attenuated in their infectivity, indicating the essentiality of this protein. Several attempts to generate homozygous null mutants of LdTyrRS were unsuccessful due to the presence of a single copy of the TyrRS gene. Fisetin, a natural flavonoid compound, was found to inhibit parasite growth by inhibiting the aminoacylation activity of LdTyrRS. Apart from its role in translation, we also report the non-canonical function of LdTyrRS. The most notable and intriguing feature of LdTyrRS is the presence of an "ELR" motif, which is the signature motif conserved among IL-8 chemokines (15) and indicates a possible immunomodulating role of this protein.
We explored the significance of this ELR motif in the parasite housekeeping enzyme LdTyrRS. Our comprehensive study involving the characterization, localization, and immunological attributes of LdTyrRS provides a platform to explore LdTyrRS as a potential target for drug development.

Results
Characterization of Leishmania Tyrosyl-tRNA Synthetase (LdTyrRS)-Multiple sequence alignment of LdTyrRS (Uniprot ID A4HW83; tritrypDB ID LdBPK_141460.1) with representative sequences from other eukaryotes such as human (Uniprot ID P54577), Plasmodium (UniProt ID Q8IAR7), Trypanosoma brucei (Uniprot ID Q57WH7; tritrypDB ID Tb927.7.3620), and L. major (Uniprot ID Q4QFJ7; tritrypDB ID LMJF_14_1370) was generated using CLUSTALW. This multiple sequence alignment showed that LdTyrRS belongs to a family of class I synthetases, characterized by a structurally well conserved amino-terminal Rossmann-fold domain that contains the signature sequences HIGH and KMSKS. Our earlier bioinformatics analysis has revealed the presence of an "ELR (Glu-Leu-Arg) motif," which is the signature motif conserved among CXC chemokines (14). The ELR motif in LdTyrRS was found to be present at the 22nd amino acid position and indicated a possible immunomodulating role of this enzyme (Fig. 1). The alignment also suggested complete conservation of the ELR motif in parasitic tyrosyl-tRNA synthetases (Plasmodium, T. brucei, and L. major). Interestingly, the ELR motif was found to be evolutionarily absent in TyrRSs from lower eukaryotes and prokaryotes (Fig. 2).
To characterize the recombinant LdTyrRS protein, the fulllength gene was cloned into a bacterial expression vector pET30a. A histidine-tagged fusion protein with an estimated molecular mass of ϳ80 kDa was induced. This size correlated with the amino acid composition of the LdTyrRS protein (ϳ74 kDa) with a His 6 tag (ϳ6 kDa) at the N terminus (Fig. 3A). Recombinant LdTyrRS was purified to homogeneity by metal affinity chromatography (Fig. 3A). Purification yielded ϳ2 mg of purified protein/liter of bacterial culture. The recombinant protein (rLdTyrRS) was recognized by an anti-His tag monoclonal antibody (Fig. 3B). To further characterize rLdTyrRS, the purified protein was analyzed by MALDI-TOF/TOF mass spectroscopy (data not shown). The spectrum of the protein analyzed by BioTools version 2.2 showed an intensity coverage of 46.6% for tyrosyl-tRNA synthetase (Leishmania infantum). The expression of the full-length LdTyrRS enzyme was confirmed in Leishmania cell lysates by immunoblot analysis (Fig.  3C). The anti-LdTyrRS antibody detected a ϳ74 kDa band in the cell extracts of both the promastigotes and the amastigotes (Fig. 3C).
Enzymatic Activity and Kinetic Parameters for LdTyrRS-To assess the aminoacylation activity of rLdTyrRS, a coupled enzyme assay was performed. The aminoacylation reaction was carried out with rLdTyrRS in the presence of inorganic pyrophosphatase, and the P i produced was measured with a malachite green solution. Recombinant LdTyrRS acylated tRNA Tyr in a time-dependent manner, demonstrating that the L. donovani TyrRS gene encodes a functional enzyme (Fig. 3D). The kinetic parameters and specificity of rLdTyrRS were determined with L-tyrosine and tRNA Tyr as substrates in vitro (Fig. 3, E and F). Enzyme kinetics was performed with varying concentrations of L-tyrosine (from 0.1 to 20 M), whereas other components were kept constant (Fig. 3E). The results showed that the enzyme reaction was dependent on L-tyrosine concentration (Fig. 3E). The K m value of rLdTyrRS for L-tyrosine was 0.21 Ϯ 0.0245 M, which is closer to that reported in the case of human TyrRS (0.3 M) (16). Because tRNA Tyr is another essential substrate of the aminoacylation reaction, therefore, we also performed tRNA Tyr -dependent enzyme kinetics studies (Fig.  3F). The estimated K m of LdTyrRS, for tRNA Tyr (1.177 Ϯ 0.2271 M), was closer to that of humans (0.9 M) (16) but higher than that of Saccharomyces cerevisiae (0.2 M) (17).
Subcellular Localization of LdTyrRS-The amino acid sequence analysis of LdTyrRS by web-based programs like sig-nalP and pSORT II predicted that LdTyrRS does not contain any detectable signal peptide or cleavage site. Moreover, predictions with MARSPred and LocTree3 also indicated a preferentially cytosolic localization. To ascertain the localization of TyrRS in L. donovani, immunofluorescence analysis of log phase promastigotes stained with anti-LdTyrRS antibody and DAPI was conducted. The kinetoplast and nuclear DNA in these cells were readily identified by their bright staining with DAPI (Fig. 4B). LdTyrRS was found to be localized only in the cytoplasm of the parasite (Fig. 4, C and D). Earlier data from mass spectrometry has demonstrated a predominantly cytoplasmic localization of TyrRS in T. brucei (18). Controls performed with mouse preimmune sera, non-permeabilized cells, and secondary antibody alone showed no detectable signal (data not shown). The key residues present in the aminoacylation and catalytic domains are highlighted in a gray background. The ELR motif is highlighted in bold font.

Gene Deletion Studies of Tyrosyl-tRNA Synthetase-Because
TyrRS is an important component of protein translation, we explored whether its depletion from the cell would affect aminoacylation and impact parasite growth and infection. The essentiality of LdTyrRS was assessed by classical gene replacement experiments where attempts were made to replace both the alleles of LdTyrRS by drug-resistance genes. This was achieved by the generation of inactivation cassettes with hygro-FIGURE 2. Domain organization of TyrRS from eukaryotes and prokaryotes. The catalytic and anticodon binding domains are indicated. The ELR motif is present at the N terminus and is shown in red. The HIGH and KMSKS active site motifs are common to all class I catalytic domains. The AIDQ motif is characteristic of the ATP binding site in TyrRS. The AC1 motif corresponds to the anticodon-binding domain that interacts with the anticodon stem of tRNA Tyr . The AC2 motif specifically recognizes the anticodon bases G34 and U/35. mycin phosphotransferase (HYG) or neomycin phosphotransferase (NEO) as selection markers along with 5Ј-and 3Ј-UTRs of the TyrRS gene, as described under "Experimental Procedures." Linear replacement cassettes made by fusion PCR were electrotransfected into wild-type (WT) L. donovani promastigotes leading to the generation of heterozygous parasites (TyrRS/HYG or TyrRS/NEO) in which one allele of the LdTyrRS gene was replaced with either the hygromycin or neomycin drug resistance gene. The replacement of a single allele of the LdTyrRS gene by the drug resistance gene cassette was confirmed by a PCR-based analysis. After 3-4 passages, DNA from heterozygous mutant parasites (TyrRS/HYG or TyrRS/NEO) was isolated and subjected to a PCR-based analysis using primers external to the inactivation cassette of the LdTyrRS gene (Fig. 5A). The PCR analysis demonstrated the correct integration of HYG and NEO replacement cassettes at the TyrRS locus in heterozygotes (TyrRS/HYG or TyrRS/NEO), as indicated by the appearance of 1. bands corresponding to the WT LdTyrRS gene. This data confirmed that a single allele of the LdTyrRS gene had been replaced in heterozygous mutant parasites (TyrRS/HYG or TyrRS/NEO). Several attempts to replace both alleles of the LdTyrRS gene to generate homozygous gene deletion mutants failed. Although few clones resistant to both drugs were obtained, PCR analyses demonstrated that the LdTyrRS gene was still present in the genome of these parasite lines (data not shown), thus indicating that LdTyrRS is an essential gene.
The effect of disruption of a single allele of the TyrRS gene at the protein level was studied by Western blotting analysis. Densitometric analysis was performed to evaluate the levels of TyrRS protein across different parasite lines. Comparative densitometry of the bands revealed a ϳ1.8-fold decreased expression of TyrRS protein in heterozygous mutants (TyrRS/HYG) (Fig. 5C, lane 4) as compared with that in WT parasites. Complementation of the heterozygous mutant parasites (TyrRS/ HYG) with an episomal copy of the TyrRS gene (TyrRS/HYG-[pTyrRS ϩ ]) restored protein expression to levels comparable with that of WT parasites (Fig. 5C, lane 3). The overexpression of LdTyrRS protein in overexpressing mutants (WT[pTyrRS ϩ ]) was also confirmed by Western blotting analysis. A ϳ1.5-fold increase in the TyrRS protein level was observed in TyrRS overexpressors (WT[pTyrRS ϩ ]) (Fig. 5C, lane 1) as compared with that in WT parasites.
To further establish that the elimination of a single allele of the TyrRS gene in L. donovani conferred TyrRS enzyme deficiency, the aminoacylation activity of TyrRS was determined in genetically manipulated parasites and compared with that in WT L. donovani parasites. A ϳ2-fold decrease in the aminoacylation activity of TyrRS was observed in heterozygous parasites (TyrRS/HYG) as compared with that in WT parasites (Fig. 5D). Similar results were obtained with TyrRS/NEO parasites (data not shown). The WT and the "add-back" lines (TyrRS/HYG[p-TyrRS ϩ ]) exhibited comparable TyrRS activity levels (Fig. 5D).
To assess if the reduced expression of TyrRS compromised the cellular growth of heterozygous mutant parasites, growth kinetic studies were undertaken. Heterozygous parasites (TyrRS/HYG) consistently showed growth delay as compared with WT parasites (Fig. 5E). The complementation of TyrRS/ HYG mutants with an episomal copy (pTyrRS ϩ ) rescued the growth of these parasites similar to that of the WT control. Thus, it is reasonable to assume that a gene dosage effect resulted in the production of lesser TyrRS protein, and such a conjecture would suggest that TyrRS is involved in optimal cell proliferation.
We also examined the survival of heterozygous mutant parasites (TyrRS/HYG) inside murine macrophages in vitro. Virulence studies in a mouse macrophage cell line were carried out to determine the effects of genetic deficiency of TyrRS and to characterize the LdTyrRS enzyme further as a potential therapeutic target. To this end, a murine macrophage cell line was infected with WT, TyrRS heterozygous mutant (TyrRS/HYG), and add-back (TyrRS/HYG[pTyrRSϩ]) parasites at an multiplicity of infection of 20:1. WT parasites were capable of infecting and sustaining robust infection in murine macrophages, whereas the parasitemia of the heterozygous mutants was reduced by ϳ50% relative to WT parasites 24 h post-infection (Fig. 5F). Similar results were obtained with TyrRS/NEO parasites (data not shown). Complementation with an episomal copy of the TyrRS gene restored the infectivity of the heterozygous (TyrRS/HYG) mutants similar to that of the WT parasites. Taken together, our data suggests that the LdTyrRS gene has a significant role in the growth and intramacrophage survival of amastigotes.
Leishmanicidal Activity of TyrRS Inhibitors-Resveratrol is a natural phenolic compound that has recently been shown to inhibit the activity of human TyrRS (19). A co-crystal structure of resveratrol bound to the active site of human TyrRS has been reported (19). Another flavanoid compound fisetin has also been identified to bind TyrRS of L. major (13). To test the effi- cacy of these compounds on L. donovani, log phase promastigotes were cultured in the presence of increasing concentrations of fisetin and resveratrol. Both compounds were found to inhibit the growth of promastigotes in a dose-dependent manner (Fig. 6A). The effective concentration that caused 50% inhibition of growth (IC 50 ) after 72 h of drug addition was 31.07 M for fisetin and 47.21 M for resveratrol (Fig. 6A). The sensitivities of amastigotes were also tested in an intracellular amastigote-macrophage model. The IC 50 of fisetin and resveratrol for amastigotes after 3 days of drug treatment was 1.53 and 9.3 M, respectively ( Fig. 6B). At these concentrations, both fisetin and resveratrol did not affect the viability of the macrophage cell line J774A.1, with the IC 50 being Ͼ400 M after 48 h of drug treatment.
The effect of these compounds on the aminoacylation activity of LdTyrRS (Fig. 6C) was also tested. Fisetin inhibited the enzymatic activity of recombinant LdTyrRS with an IC 50 of ϳ14.23 M (Fig. 6C), whereas a concentration of resveratrol as high as 1 mM failed to inhibit the enzymatic activity of LdTyrRS ( Fig. 6C). Thus, our results suggest that the anti-leishmanial effect of resveratrol is not due to the inhibition of LdTyrRS.
To ascertain whether the anti-leishmanial activity of fisetin is mediated through the inhibition of LdTyrRS, we also evaluated the effect of fisetin on the growth of genetically manipulated parasites (Fig. 6D). WT, overexpressors (WT[pTyrRS ϩ ]), heterozygous mutants (TyrRS/HYG), and add-back (TyrRS/ HYG[pTyrRS ϩ ]) parasites were treated with fisetin at a concentration of 35 M. In the absence of drug treatment (untreated), the growth of each parasitic line was normalized to a value of 1.0. After 72 h of treatment with fisetin, the growth rate of each parasitic line was calculated relative to the untreated control. Parasites overexpressing LdTyrRS (WT[pTyrRS ϩ ]) were found to be more resistant to growth inhibition by fisetin as compared with WT parasites (Fig. 6D). In contrast, heterozygous mutant parasites (TyrRS/HYG) were found to be more susceptible to inhibition by fisetin when compared with WT parasites (Fig.  6D). Complementation of TyrRS/HYG parasites with an episomal TyrRS gene (TyrRS/HYG[pTyrRS ϩ ]) decreased the sen- sitivity of heterozygous mutant parasites (Fig. 6D). The increased susceptibility of the heterozygous mutants to fisetin may be explained by the reduced levels of LdTyrRS expression in heterozygous knock-out parasites. The above data further indicates that fisetin exerts its antileishmanial effect through the inhibition of LdTyrRS.
Localization of TyrRS during Macrophage Infection with Leishmania-To determine the localization of LdTyrRS during macrophage infection, confocal microscopy of infected macrophages was performed. Infected and uninfected macrophages were fixed, permeabilized, and processed for immunofluorescence using either the anti-LdTyrRS antibody or preimmune sera (Fig. 7, B and C). DAPI staining was utilized for visualizing both the host and parasite nuclei. The anti-LdTyrRS-specific fluorescence observed in infected macrophages was not only associated with parasite nuclei staining but was also present throughout the cytosol of the infected macrophages (Fig. 7C). Thus, the confocal data demonstrates the presence of LdTyrRS in the cytosol of infected macrophages. Uninfected macrophages served as a negative control (Fig. 7A). No signal could be detected with the preimmune sera in infected macrophages (Fig. 7B). The immunofluorescence data thus indicates the possible secretion of TyrRS from Leishmania amastigotes (Fig. 7, The presence of LdTyrRS in the host cytosol also hinted toward its possible secretion into the extracellular milieu. Therefore, culture supernatants from uninfected or L. donovani-infected macrophages were analyzed for the presence of LdTyrRS by immunoprecipitation and immunoblotting analysis (Fig. 7D). As expected, no TyrRS was detected in the culture supernatant of uninfected macrophages (Fig. 7D). In contrast, a single band corresponding to the wild-type LdTyrRS (ϳ74 kDa) was observed in the culture supernatant of infected macrophages (Fig. 7D).
We also analyzed the secretion of TyrRS by Leishmania promastigotes (Fig. 7E). A ϳ74-kDa band corresponding to wildtype LdTyrRS was observed in the parasite culture supernatants (Fig. 7E). To exclude the possibility that the apparent secretion of LdTyrRS was due to cell lysis, we measured the amount of the cytosolic marker glucose-6-phosphate dehydrogenase in the parasite (promastigote) culture supernatants by an enzymatic assay (20). The glucose-6-phosphate dehydrogenase activity in culture supernatants was found to be negligible (data not shown). As a control, parasite culture supernatants were also analyzed for the presence of mitochondrial tryparedoxin peroxidase, which has been reported to be a parasitic non-secretory protein (21) (Fig. 7F). No secretion of mitochondrial tryparedoxin peroxidase was observed in promastigote culture supernatants (Fig. 7F). Taken together, our immunofluorescence, immunoprecipitation, and Western blotting data provide compelling evidence indicating the secretion of LdTyrRS from both promastigotes and amastigotes.
Tyrosyl-tRNA Synthetase of Leishmania Possesses an Immunologically Active ELR Motif -Previously, human "mini"-TyrRS has been shown to display potent neutrophil chemotaxis activity because of the presence of an immunologically active ELR motif (4). A transwell migration assay was performed to evaluate the chemotactic activity of LdTyrRS toward mouse neutrophils (Fig. 8A). The neutrophils that migrated into the lower chamber were quantitated by measuring their myeloperoxidase activity (Fig. 8B) as described under "Experimental Procedures." N-Formyl-Met-Leu-Phe (fMLP) is a chemotactic peptide that was used as a positive control. The addition of both fMLP and rLdTyrRS stimulated the chemotactic migration of neutrophils (Fig. 8, B and C). Recombinant LdTyrRS stimulated the chemotactic activity of neutrophils at a concentration of 0.1 nM (6 ng), which is equivalent to the physiological amount of protein present in ϳ2 ϫ 10 5 parasites. Because the ELR motif is critical for chemotactic activity, we also prepared LdTyrRS mutant in which the ELR motif was mutated to "AAA." The mutant LdTyrRS (ELR/AAA) did not show any chemotactic activity toward neutrophils (Fig. 8, B and C). Also, under these experimental conditions, spontaneous migration of neutrophils was negligible as indicated by the media control (Fig. 8, B and C).
Overall, these results suggest that because of its ELR motif, LdTyrRS functions as a chemoattractant for neutrophils. It is also known that the Leishmania parasite exploits neutrophils as Trojan horses before they enter their definitive host cells, i.e. macrophages (22). In light of the role of neutrophils as host cells for Leishmania, it may be hypothesized that secreted LdTyrRS functions as a virulence factor to induce an inflammatory recruitment of neutrophils at the site of infection.
LdTyrRS Interaction with Host Macrophages-In addition to attracting neutrophils, another important function of the ELR motif is to mediate the interaction of CXC chemokines with host immune cells like macrophages. This interaction further triggers the release of proinflammatory cytokines from host White arrows indicate intracellular amastigotes. Parasite and macrophage nuclei were visualized with DAPI (blue). Anti-LdTyrRS antibody (green) was used to visualize the parasitic LdTyrRS. Staining of infected macrophages with rabbit preimmune sera was used as a negative control (B). Scale bar corresponds to a size of 10 m. D, Western blotting analysis of the parasite culture supernatants using antibodies against LdTyrRS. The culture supernatant of the infected (lane 1) and uninfected (lane 2) macrophages was electroblotted onto nitrocellulose membrane and probed with the anti-LdTyrRS antibody and HRP-conjugated anti-rabbit antibody. The blot was then developed with commercially available ECL reagent. Western blotting analysis of the promastigote culture supernatants was used to check for the presence of LdTyrRS (E) and mitochondrial tryparedoxin peroxidase (mTXNPx) (F). Lane 1, L. donovani promastigote cell lysate (20 g); lane 2, promastigote culture supernatant. The parasites were suspended at a concentration of ϳ10 8 parasites/ml in RPMI 1640 media without FBS and incubated for 8 h under normal culture conditions. The LdTyrRS protein was immunoprecipitated from a minimum of ϳ80 ml of culture supernatants containing a total of ϳ8 ϫ 10 9 promastigotes and detected by a Western blotting analysis as described under "Experimental Procedures." immune cells (23)(24)(25). Direct binding of rLdTyrRS to host macrophages was visualized by immunofluorescence analysis. The specific interaction of rLdTyrRS with mouse macrophages was observed by using anti-LdTyrRS (Fig. 9A) and anti-His antibodies (Fig. 9E). On the other hand, the mutant LdTyrRS (ELR/AAA) did not show any binding to host macrophages (Fig. 9, B and F). Also, no binding could be detected in macrophages that were not incubated with any protein and preimmune sera controls (Fig. 9, C and D). This data indicated an ELR motif-mediated interaction of LdTyrRS with host macrophages.
Triggering of Cytokine Secretion by LdTyrRS-To check if LdTyrRS triggers cytokine activity, a murine macrophage cell line was incubated with rLdTyrRS, mutant ELR/AAA-Ld-TyrRS, lipopolysaccharide (LPS) (as a positive control), and rLdLeuRS (as a negative control). The culture supernatants were analyzed for the presence of proinflammatory cytokines such as TNF-␣, IL-6, IL-12, and IFN-␥. Time kinetic analysis by ELISA revealed maximal production of the inflammatory cytokine TNF-␣ (Fig. 10A) during 12 h of culture and IL-6 within 24 h of culture (Fig. 10B). Interestingly, the mutant ELR/AAA-LdTyrRS did not trigger cytokine release from macrophages and behaved similarly to the control protein rLdLeuRS (Fig. 10,  A and B). Moreover, the release of TNF-␣ and IL-6 by rLdTyrRS was mediated in a concentration-dependent manner (Fig. 10, C  and D). Other proinflammatory cytokines like IL-12 and IFN-␥ were not induced by rLdTyrRS (Fig. 10E). We also checked the induction of an important anti-inflammatory cytokine, IL-10, which is involved in disease progression in leishmaniasis (26). Recombinant LdTyrRS failed to trigger IL-10 secretion (Fig.   10E) by host macrophages. This data indicates that LdTyrRS specifically triggers the release of IL-6 and TNF-␣ in a time-and dose-dependent manner.
We also corroborated our findings with the native LdTyrRS that was immunoprecipitated from promastigote culture supernatants (PDTyrRS). Native LdTyrRS was equally capable of triggering TNF-␣ and IL-6 release from murine macrophages (Fig. 11, A and B). It was also observed that preincubation of native LdTyrRS (PDTyrRS) with anti-LdTyrRS antibodies (PDTyrRS ϩ ␣TyrRS) substantially blocked TNF-␣ (p Ͻ 0.001) and IL-6 production (p Ͻ 0.001) from macrophages (Fig.  11, A and B), thereby suggesting a specific motif-based LdTyrRS interaction with host macrophages.
Potential Receptor(s) of LdTyrRS on Host Macrophages-ELR motif-based binding of CXC chemokines to a CXCR2 receptor on immune cells has been shown to activate NF-B and induce proinflammatory cytokine expression (23). To determine whether the binding of LdTyrRS is also mediated through ELR-CXCR2 receptor interaction, we preincubated human macrophages with anti-CXCR2 and anti-LdTyrRS antibodies and tested whether antibody-mediated receptor or protein blockades inhibited rLdTyrRS-macrophage interaction. The interaction was analyzed by FACS analysis. Recombinant LdTyrRS bound to mouse macrophages was detected by an anti-Ld-TyrRS antibody (Fig. 12A). On the other hand, specific antibody-mediated blocking of the CXCR2 receptor on murine macrophages reduced rLdTyrRS binding (Fig. 12B). The blocking of rLdTyrRS protein with anti-LdTyrRS antibodies also reduced binding of the protein to macrophages (Fig. 12C). As expected, mutant LdTyrRS (ELR/AAA) protein did not show any binding to host macrophages (Fig. 12D). Furthermore, blockage of rLdTyrRS-macrophage interactions using either anti-CXCR2 or anti-LdTyrRS antibodies significantly reduced the release of TNF-␣ (p Ͻ 0.001) and IL-6 (p Ͻ 0.001) from murine macrophages (Fig. 12, E and F). Our data strongly highlights the role of ELR motif in mediating LdTyrRS interactions with host macrophages. We, therefore, conclude that ELR motif-based LdTyrRS binding in vitro likely occurs using specific receptors on host macrophages and that this binding might lead to very specific host immune system activation.

Discussion
aaRSs are key enzymes that drive the protein translational machinery. Apart from their translational functions, aaRSs are implicated in various non-canonical functions such as gene transcription, mRNA translation, inflammation, and immune response (1). Therefore, aaRSs constitute a significant subset of proteins, and inhibition of their enzymatic activity can be deleterious to the organism. Hence, experimental dissection of critical translation components like aaRSs is on high priority as one of the avenues of novel target discovery in pathogen biology.
TyrRS are yet to be experimentally investigated as drug targets in parasites. The few parasite-specific studies available on TyrRS have majorly focused on the structural aspects of the enzyme. A recent comprehensive study highlighted the structural and functional aspect of TyrRS from Plasmodium falciparum (24). The structural analysis of the TyrRS orthologue from L. major reveals several crucial differences between the host and pathogen tyrosyl-tRNA synthetase active sites (13). These differences could potentially be exploited for the design of structure-based inhibitors of parasite TyrRSs. The present study highlights the functional attributes of tyrosyl-tRNA synthetase of Leishmania as a housekeeping protein translation enzyme and also as a mimic of host CXC chemokine.
In the present study, we, for the first time, report the characterization of tyrosyl-tRNA synthetase from L. donovani (LdTyrRS). The overall K m values deduced by kinetic analysis for LdTyrRS appear to be closer to the K m values reported for other mammalian TyrRS. TyrRS appears to be an essential gene as attempts to delete both copies of the TyrRS gene from the parasite genome failed. Heterozygous parasites (TyrRS/HYG) were found to have slower growth kinetics and exhibited attenuated virulence. Because L. donovani TyrRS appears to be an essential gene for parasite survival, we also analyzed the efficacy of known tyrosyl-tRNA synthetase inhibitors, resveratrol and fisetin, on parasite survival and the aminoacylation activity of LdTyrRS. Fisetin was found to inhibit the parasite growth by inhibiting the LdTyrRS aminoacylation activity. This data are in agreement with a previous report that suggests binding of fisetin to the active site of trypanosomal TyrRS (13). Several subtle yet crucial differences between Leishmania and human TyrRS (13) could potentially be exploited for the design of structurebased inhibitors for LdTyrRS.
Leishmania has evolved sophisticated mechanisms to evade or subvert host immune responses and establish chronic infection. Leishmania parasites have the capacity to subvert phagocytosis (27) and modulate cytokine secretion (11,28), thus allowing the parasite to thrive within phagocytic cells and within the host organism as a whole. From previous studies, it has become clear that a range of effector molecules secreted by the Leishmania parasite play a vital role in this process (20,29). In this context, the expression of a mimic of a human cytokine could contribute to the modulation of host signaling pathways to the advantage of the parasite. This report highlights the moonlighting activity of a parasitic tyrosyl-tRNA synthetase (LdTyrRS) to function as a mimic of host CXC chemokine. Aminoacyl-tRNA synthetases have a vital role in translating the genetic code. Numerous studies have shown that members of this enzyme family are quite adept at "moonlighting" (1, 3, 30). Non-canonical roles have been suggested for several parasite tRNA synthetases. Studies have indicated that human tyrosyl-, tryptophanyl-, and lysyl-tRNA synthetases can be secreted extracellularly and can mimic cytokines (4,31,32). TyrRS of Plasmodium and LysRS of Entamoeba also mimic the functions of human cytokines (24,33).
Our immunofluorescence and immunoblotting data reveal that both promastigotes and amastigotes secrete LdTyrRS. Interestingly, the secreted tyrosyl-tRNA synthetase from L. donovani was found to lack a classical N-terminal secretion signal peptide. Previous reports have identified secretion by exosomes as a major mechanism by which Leishmania exports secreted virulence factors that help in communication with the host (29,34). Due to the absence of any defined secretory signal, it may be hypothesized that the release of LdTyrRS also occurs by a non-classical secretion mechanism. Understanding the exact mechanism by which the secretion of LdTyrRS occurs remains to be explored and is the focus of ongoing work.
We further investigated the capacity of LdTyrRS to modulate neutrophil recruitment. Our data shows that by using its ELR motif, secreted LdTyrRS can induce direct chemotaxis of neutrophils. A recent study has highlighted the role of neutrophils as host cells for Leishmania (35)(36)(37). Although parasite culture supernatants have been found to have chemotactic activity toward neutrophils (35), parasite-secreted protein(s) that specifically trigger the initial recruitment of neutrophils are poorly understood. The present study identifies LdTyrRS present in the parasite culture supernatant, as a parasite secretory protein having the capacity to modulate neutrophil recruitment. Thus, FIGURE 10. Effect of rLdTyrRS on mouse macrophages. A and B, the secretion profile of TNF-␣ (A) and IL-6 (B) was measured at different time points using ELISA. C and D, dose-dependent increase in the secretion of cytokines like TNF-␣ (C) and IL-6 (D) from mouse macrophages was also measured. Media alone, rLdLeuRS enzyme, and ELR-AAA mutant of LdTyrRS were used as negative controls. LPS was used as a positive control in all the experiments. E, the secretion profile of other cytokines like IFN-␥, IL-10, and IL-12 that are secreted from mouse macrophages on exposure to rLdTyrRS was also analyzed. The results represent mean Ϯ S.D. with n ϭ 3. Tukey's test was performed, and p values are indicated.
it may be hypothesized that secretion of LdTyrRS is a pro-parasitic response that is likely to bring about the recruitment of neutrophils to the site of infection.
Because ELR motif is also involved in a ligand-based interaction with CXCR2 receptors on immune cells, we also analyzed the binding of LdTyrRS to host macrophages. Our confocal and  FACS data show that via its ELR motif, released LdTyrRS interacts with a specific receptor on host macrophages. Our data also indicates that the LdTyrRS-macrophage interaction triggers the release of the proinflammatory cytokines TNF-␣ and IL-6. Previous studies have shown that Leishmania infection triggers the release of TNF-␣ and IL-6 (38 -41). A central function of these two proinflammatory cytokines is to help in the recruitment of phagocytes like neutrophils and monocytes (42)(43)(44). Both cell types are infection targets and essential for the establishment of Leishmania infection (45)(46)(47). In the case of Leishmania infections, the release of TNF-␣ and IL-6 by the parasite secretory protein GP63 has been directly associated with inflammatory phagocyte (neutrophil and monocyte) recruitment (40). Thus, it may be hypothesized that the secretion of TNF-␣ and IL-6 by LdTyrRS also contributes toward the accrual of neutrophils at the site of Leishmania infection. These results clearly indicate that the secreted LdTyrRS enables Leishmania to elicit proinflammatory cytokine release and neutrophil recruitment, both of which contribute to the establishment of infection. In our present set of experiments, we observed that 0.1 nM (6 ng) LdTyrRS was sufficient to initiate neutrophil chemotaxis and cytokine release from host cells. This concentration was found to correlate with the amount of protein present in ϳ2 ϫ 10 5 parasites.
During Leishmania infection, most sand flies transmit 10 3 -10 5 parasites per blood meal (48). Earlier reports indicate that tissue damage to the skin caused by the infected sand fly bites is important to induce neutrophil recruitment within the first 90 min, further indicating that the initial neutrophil recruitment in the case of a natural infection is parasite-independent (46). However, in the case of an experimental infection in mice, a needle injection of 10 5 Leishmania parasites is required to observe parasite-dependent neutrophil recruitment at later time points (6 -24 h post-infection) (49,50). Thus, in a natural Leishmania infection, apart from parasitic proteins other factors derived from the sand fly also contribute to neutrophil chemotaxis in the infected dermis. Because all these chemotactic factors (both parasite and sandfly derived) work in a synergistic manner, it would be interesting to observe the cooperation between the LdTyrRS and other chemoattractants. Although in the present study, the amount of LdTyrRS used was within the physiological range, the exact amount of protein secreted during natural infection, and the minimal effective concentration required for its chemotactic activity needs to be evaluated by further experimentation.
In summary, our data indicates a possible immunomodulating role of LdTyrRS in Leishmania infection. LdTyrRS functions as a direct chemoattractant for neutrophils. LdTyrRS was also found to induce TNF-␣ and IL-6 release, which is known to be associated with inflammatory phagocyte recruitment in Leishmania infections. Improved knowledge of Leishmania-induced inflammation will further our understanding of how the parasite establishes infection, modulates the immune response, metastasizes, and causes pathology.
Considering that drug resistance is a major concern in antiparasitic chemotherapy, there is continuous pressure to identify new drug targets. Our comprehensive analyses on the twin abilities of Leishmania parasite tyrosyl-tRNA synthetase pro-vide a platform to explore LdTyrRS as a potential target for drug development.

Experimental Procedures
Materials-All restriction enzymes and DNA modifying enzymes were obtained from New England Biolabs. Paromomycin and hygromycin were obtained from Sigma. Plasmid pET-30a was obtained from Novagen. Escherichia coli DH10␤ and Rosetta were used as hosts for plasmid cloning and protein expression, respectively. Nickel-nitrilotriacetic acid-agarose was purchased from Qiagen. DNA and protein markers were acquired from New England Biolabs. Resveratrol and fisetin were obtained from Sigma. ELISA kits and antibodies were obtained from BD Bioscience. The rabbit anti-tubulin-␣ antibody was obtained from Neomarker (Fremont, CA). Other materials used in this study were of analytical grade and were commercially available.
Axenic amastigotes were generated by using the standard protocol as described earlier (51,52). Briefly, late-log phase promastigote cultures (ϳ2 ϫ 10 6 /ml) were adapted to grow at 26°C in an acidic amastigote media (RPMI 1640, 25 mM MES, pH 5.5). Once established, these parasites were subsequently grown in acidic medium (RPMI 1640/MES, pH 5.5) at 37°C in a humidified atmosphere containing 5% CO 2 . Throughout this in vitro adaptation process, the cellular morphology of the parasites was examined by both phase-contrast light microscopy and Giemsa-stained preparations. The mouse macrophage-like cell line J774.A1 obtained from ATCC was cultured in RPMI 1640 media (Sigma) supplemented with 10% FBS and antibiotics (100 units/ml of penicillin and 100 g/ml of streptomycin) at 37°C with 5% CO 2 .
Ethics Statement-All animal experiments were performed according to the guidelines approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. The protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Jawaharlal Nehru University (JNU), New Delhi (IAEC code number 11/2013). All mice used for the experiments were ethically sacrificed by asphyxiation with carbon dioxide according to the institutional and the CPC-SEA (Government of India) regulations.

Cloning, Expression, and Purification of Recombinant
LdTyrRS Protein-The gene for LdTyrRS (tritrypDB ID LdBPK_141460.1) was amplified by PCR using a forward primer with a flanking BamHI site (5Ј-TTTTGGATCCAT-GAACACGGACGACCGCTAC-3Ј) and reverse primer with a flanking HindIII site (5Ј-TTTTAAGCTTTACCTCTTCTTT-GCCATCTTCTTC-3Ј) from L. donovani genomic DNA. The 2049-bp amplification product encompassing the LdTyrRS open reading frame (ORF) was cloned into a pET30a vector (Novagen) using BamHI and HindIII restriction sites. This construct (LdTyrRS-pET30a) containing a His 6 tag at the N terminus was transformed into the E. coli Rosetta strain (Novagen). The expression of recombinant LdTyrRS (rLdTyrRS) was induced with 0.5 mM isopropyl ␤-D-thiogalactopyranoside at 18°C for 16 h. The protein was purified by a Ni 2ϩ -nitrilotriacetic acid-agarose resin (Qiagen) by eluting with increasing concentrations of imidazole. The purified protein was found to be Ͼ95% pure as judged by SDS-PAGE.
Site-directed Mutagenesis of the ELR Peptide Motif of LdTyrRS-The ELR motif of LdTyrRS was replaced with AAA by site-directed mutagenesis. The mutation in the LdTyrRS gene was performed using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, USA) following the manufacturer's instructions. Subsequently, a DNA sequence analysis was performed to confirm the mutations. The mutated recombinant protein (ELR/AAA) was expressed and purified using the same protocol as specified above for the recombinant WT LdTyrRS.
Aminoacylation Spectrophotometric Assay-The aminoacylation assays were performed as previously described (53). Briefly, the substrate L. donovani tRNA Tyr was synthesized by in vitro transcription from a PCR product template that contained a T7 promoter followed by the L. donovani tRNA Tyr sequence (tritrypDB ID LinJ.34.tRNA7) and the terminal CCA sequence. The in vitro transcription reaction was performed with the MEGAScript T7 polymerase kit (Ambion; Life Technologies) according to the manufacturer's instructions. The reaction mixtures were then extracted with phenol/chloroform/isoamyl alcohol (25:24:1 (v/v), Sigma), and the tRNAs were precipitated with isopropyl alcohol (Sigma). The tRNA was refolded by heating at 70°C for 10 min, followed by the addition of 10 mM MgCl 2 and slow cooling to room temperature. The aminoacylation reaction was performed as described earlier (53)  The absorbance was then measured at 620 nm by a Spectramax M2 reader (Molecular Devices). The determination of K m and V max for L-tyrosine and tRNA Tyr was achieved by varying the concentration of L-tyrosine or tRNA Tyr in the reaction mixture while maintaining the other components in excess. To determine the effect of the inhibitors on the aminoacylation activity of rLd-TyrRS, reactions were performed in the presence of inhibitors as described previously (53). Briefly, a reaction mixture containing rLdTyrRS (0.2 M) was incubated with different concentrations of fisetin/resveratrol (0.1 nM to 1000 M) for 30 min at 37°C. The reactions were stopped and quantitated as described above. To determine the 50% inhibitory concentration (IC 50 ), the dose-response data were fitted to the log (inhibitor) versus response equation using GraphPad Prism.
Molecular Constructs for Replacement of TyrRS Alleles-For inactivation of the LdTyrRS gene, a targeted gene replacement strategy based on PCR fusion was employed (54). Briefly, the flanking regions of the TyrRS gene (5Ј-UTR and 3Ј-UTR) were amplified and fused by PCR to the hygromycin phosphotransferase gene (HYG) or neomycin phosphotransferase gene (NEO). The 5Ј-UTR (599 bp) of the LdTyrRS gene was obtained from WT L. donovani genomic DNA by PCR amplification with either primer A and B Hyg or primers A and B Neo ( Table 1). The NEO gene was amplified from pX63-NEO with primers C Neo and D Neo . The HYG gene was amplified from pX63-HYG with primers C Hyg and D Hyg ( Table 1). The 3Ј-UTR (580 bp) of the LdTyrRS gene was obtained from L. donovani WT genomic DNA by PCR amplification using primers E Hyg /E Neo and reverse primer F ( Table 1). The 5Ј-UTR of L. donovani TyrRS gene was then ligated to either of the antibiotic resistance marker genes (HYG/NEO) by PCR using primers A and D Hyg or A and D Neo . This fragment (5Ј-UTR marker gene) was then fused with the 3Ј-UTR using primers A and F, yielding the linear replacement cassette, 5ЈUTR-Hyg-3ЈUTR or 5ЈUTR-Neo-3ЈUTR.
To generate the episomal construct, the full-length LdTyRS coding sequence was amplified with a forward primer harboring the XbaI site (primer G) and a reverse primer with the Hin-dIII site (primer H) ( Table 1). The amplified LdTyRS gene was then cloned into the pSP72␣-zeo-␣ vector to get the pSP72␣zeo-␣-TyRS episomal construct. All the fragments and constructs were sequenced for confirmation. Generation of Genetically Manipulated Parasites-After PCR amplification and purification, ϳ2 g of the linear replacement cassette (5ЈUTR-Hyg-3ЈUTR or 5Ј-UTR-Neo-3ЈUTR) was individually transfected by electroporation in WT L. donovani promastigotes (55). The transfectants were subjected to antibiotic selection depending on the marker gene. The cells resistant to antibiotic selection were further subjected to PCRbased analysis to check for the correct integration of replacement cassettes using primers as shown in Table 2. Thereafter, the second round of transfection was initiated to knockout the other allele of the TyrRS gene.
Growth and Infectivity Assays-Growth rate experiments were conducted by inoculating stationary phase parasites at a density of 1 ϫ 10 6 cells/ml in M199 medium with 5% FBS in 25-cm 2 flasks without respective selection drug at 22°C. The growth rate of each culture was determined at 24-h intervals with a Neubauer hemocytometer. Growth studies with individual cell lines were done at least three times, and similar results were consistently obtained.
For the infectivity assay, the J774.A1 murine macrophage cell line was plated at a cell density of 5 ϫ 10 5 cell/well in a 6-well flat bottom plate. The adherent cells were infected with stationary phase promastigotes, at a ratio of 20:1 for 6 h. Excess nonadherent promastigotes were then removed by incubating the cells for 30 s in phosphate-buffered saline (PBS). These were then subsequently maintained in RPMI 1640 media containing 10% FBS at 37°C with 5% CO 2 . Giemsa staining was performed to visualize the intracellular parasite load.
Because the inhibitors were dissolved in DMSO, a sample without inhibitors but with an equal volume of DMSO served as an additional control. After 72 h of incubation, 10 l of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/ml) 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% isopropyl alcohol and 20% SDS followed by gentle shaking at 37°C for 30 min to 1 h. The absorbance was measured at 570 nm in a microplate reader (Spectra-Max M2 from Molecular Devices). The percentage of parasite growth at different inhibitor concentrations was determined relative to untreated control cells and 50% inhibitory concentration (IC 50 ) was calculated.
The sensitivity of amastigotes to fisetin and resveratrol was tested in an intracellular amastigote-macrophage model as described earlier (57). Briefly, the J774A.1 cell line (1 ϫ 10 5 cells/well) was cultured in eight-chamber Lab-Tek tissue culture slides (Nunc, USA) and infected with stationary phase promastigotes at an multiplicity of infection of 20:1 as described above. The infected macrophages were then incubated for 72 h with different concentrations of inhibitors (0.1 nM to 10,000 M). The slides were fixed and stained with Giemsa. The number of amastigotes per cell was counted in 100 macrophages at different drug concentrations. The 50% inhibitory concentration (IC 50 ) was obtained by determining the reduction in parasite burden relative to the untreated infected controls.
Antibody Generation and Immunofluorescence Microscopy-Antibodies against the rLdTyrRS were raised commercially in rabbits (Merck). Briefly, the purified rLdTyrRS protein (50 g) was subcutaneously injected in rabbit using Freund's complete adjuvant (Sigma), followed by three booster doses of the recombinant protein (20 g) in Freund's incomplete adjuvant (Sigma), at a 2-week interval. The sera were then collected after the last booster. Protein A-purified IgG fractions (anti-Ld-TyrRS antibody) were utilized for further immunological assays.
For the intracellular localization of LdTyrRS, L. donovani promastigotes were immobilized on poly-L-lysine-coated coverslips. The cells were fixed and permeabilized followed by incubation with the anti-LdTyrRS antibody (1:1000) for 1 h at room temperature. Subsequently, the cells were washed and then incubated for 45 min with the Alexa 546-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific catalogue number A-11071). The nuclear and the kinetoplastid DNA was stained with DAPI (Invitrogen). The fluorescence of the parasites stained with the anti-LdTyrRS antibody was visualized by a confocal laser scanning microscope (Olympus FluoView TM FV1000 with objective lenses PLAPON ϫ60 O, NA-1.42) at an excitation wavelength of 556 nm. The cellular DNA stained with DAPI was visualized at an excitation wavelength of 405 nm.
Immunofluorescence analysis of the infected macrophages was performed as previously described (58). Briefly, J774 cells (1 ϫ 10 5 ) were grown on coverslips in RPMI 1640 supplemented with 10% FBS. The adherent cells were then infected with L. donovani stationary-phase promastigotes at an multiplicity of infection of 20:1 or left uninfected. After 24 h of infection, the cells were fixed and permeabilized followed by incu- bation with the primary (anti-LdTyrRS and secondary (Alexa 488-conjugated goat anti-rabbit IgG) antibodies. All antibody incubations were of 50 min duration and were followed by six to eight washes in PBS. The images were then visualized by the confocal laser scanning microscope (Olympus FluoView TM FV1000 with objective lenses PLAPON ϫ60 O, NA-1.42) at an excitation wavelength of 495 nm. DAPI was used to stain the nuclei of both the parasite and host macrophages. Immunoprecipitation and Western Blotting Analysis-Immunoprecipitation of the LdTyrRS was performed using the culture supernatants as previously described (58). Briefly, the culture supernatants from uninfected macrophages, infected macrophages, and promastigote cultures were collected by centrifugation. The culture supernatants were neutralized with 5 N NaOH and concentrated using an Amicon Ultra-15 centrifugal filter unit (Millipore). The concentrated culture supernatants were incubated overnight at 4°C with anti-LdTyrRS antibodies. Protein A-Sepharose beads (Sigma) were then added to each sample for 30 min, following which the beads were washed with Leishmania lysis buffer (50 mM Tris, pH 8, 137 mM NaCl, complete protease inhibitor mixture (Roche)). The immunoprecipitated proteins were eluted from the beads using 0.2 M glycine buffer. SDS-PAGE was used to separate the eluted immunoprecipitated proteins from the culture supernatant, which were then detected by Western blotting analysis.
Protein samples for Western blotting analysis were fractionated on a 10% SDS-PAGE gel and blotted onto a nitrocellulose membrane using electrophoretic transfer cell (Bio-Rad). After blocking with 5% skimmed milk, the membrane was incubated for 2 h at room temperature with the anti-LdTyrRS antibody (1:1000). The membrane was then washed with PBS containing 0.05% Tween 20 (PBS-T) and incubated with the horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Cell Signaling Technology, catalogue number 7076S; 1:5000). The blot was developed by the enhanced chemiluminescence (ECL) kit (Amersham Biosciences) according to the manufacturer's protocol.
The quantification of LdTyrRS in the immunoblot was carried out as described earlier (59,60). Briefly, different concentrations of rLdTyrRS were analyzed using anti-LdTyrRS antibody by Western blotting analysis. Band intensities for rLdTyrRS were measured densitometrically. A standard curve of protein load versus band intensity was then produced (data not shown). The band intensity of LdTyrRS from the parasite lysate was calibrated against the standard curve generated for the recombinant protein. 30 ng of LdTyrRS was estimated to be present in ϳ10 6 parasites. In our present set of experiments with mammalian cells, the minimum quantity of LdTyrRS used was 6 ng, which is equivalent to the amount of protein present in ϳ2 ϫ 10 5 Leishmania parasites. Because the experimental infections in mice are done using 10 5 -10 7 Leishmania parasites (49,61), it would appear that the amount of LdTyrRS (6 ng) used is within the physiological range.
Chemotaxis Assays-For the neutrophil chemotactic assay, mice neutrophils were isolated with a Ficoll-Paque (Sigma) centrifugation method, as previously described (62). The final preparation consisted of Ͼ98% neutrophils, as determined by Wright-Giemsa staining. The cells were resuspended in che-motaxis media (RPMI 1640 containing 10% FBS) at a concentration of 5 ϫ 10 6 cells/ml. 200 l of this cell suspension (1 ϫ 10 6 cells) was plated in the upper well of a 24-well transwell Boyden chemotaxis plate (Costar). Recombinant LdTyrRS (0.1 nM or 6 ng), fMLP (10 Ϫ8 M), and mutant LdTyrRS protein (ELR/ AAA) (0.1 nM or 6 ng) were diluted in 600 l of the chemotaxis medium and placed in the lower well. The filled chemotaxis transwell plates were then incubated at 37°C in a humidified CO 2 incubator for 4 h. After the incubation, non-migratory cells on the upper surface of the filter were removed by wiping with a cotton swab. The number of neutrophils that migrated into the lower chamber was determined. For quantification, the myeloperoxidase activity of neutrophils was measured as previously described (63). Briefly, neutrophils sedimented by light centrifugation were solubilized in 0.1% Triton X-100. To this lysate, 300 l of the o-dianisidine solution containing 0.003% sodium perborate was added. The color of the reaction was developed for 30 min and terminated by the addition of 50 l of 0.5 N HCl. The absorbance of the reaction was measured at 405 nm. The number of neutrophils that migrated to the lower chamber was calculated from a standard curve obtained with the lysates of a known number of neutrophils. Migration was plotted either as the mean number of neutrophils migrated in the lower chamber or as migration index (MI ϭ number of migrating cells in each condition/number of migrating cells in basal medium).
Cell Binding Assays-In vitro binding of the rLdTyrRS to mouse macrophages was performed by immunofluorescence analysis using standard protocols. The murine macrophage cell line J774 was cultured on coverslips in 6-well plates and maintained in RPMI 1640 medium containing 10% FBS. Recombinant LdTyrRS (0.1 nM or 6 ng) and mutant LdTyrRS protein (0.1 nM or 6 ng) (ELR/AAA) were incubated with the cells for 2 h at 4°C. The cells were then washed and incubated with 2% paraformaldehyde (Sigma) for 15 min. Following this, the primary (anti-LdTyrRS, 1:1000) and fluorescently conjugated secondary (Thermo Fisher Scientific catalogue number A-11071, 1:3000) antibodies were added. Fluorescence microscopy was performed with confocal laser scanning microscopy (Olympus FluoView TM FV1000 with objective lenses PLAPON ϫ60 O, NA-1.42).
In vitro binding of the rLdTyrRS to J774 cells was also analyzed by fluorescence-activated cell sorting (FACS) using standard protocols. Analytical flow cytometry was performed by indirect staining of the macrophages with the anti-LdTyrRS antibody (1:1000) and the fluorescently conjugated secondary antibodies (Thermo Fisher Scientific, catalogue number A-11071; 1:3000). The data obtained on BD FACS Calibur (BD Biosciences) were analyzed using CellQuest software.
Cytokine Secretion Assays-The concentrations of IL-6, TNF-␣, IL-12, IFN-␥, and IL-10 in culture supernatants were determined by ELISA. Briefly, J774 murine macrophages (5 ϫ 10 5 ) were cultured in RPMI 1640 medium with 10% FBS in 24-well plates. LPS, rLdTyrRS, and control proteins (rLdTyrRS mutant protein (ELR/AAA) and rLdLeuRS) were added to the cells at different concentrations. After 6, 12, and 24 h, the culture supernatants were collected, serially diluted, and the cytokine concentrations were quantified by ELISA. The assay was performed using a BD Pharmingen Opt EIA kit according to the manufacturer's instructions. To remove endotoxin contamination, recombinant proteins (rLdTyrRS, rLdTyrRS mutant protein (ELR/AAA), and rLdLeuRS) were passed through polymyxin beads (Sigma) before the assay. To confirm that the proteins were endotoxin-free, a LAL (limulus amebocyte lysate) assay (Thermo Fisher Scientific catalogue number 88282) was performed.
LdTyrRS Interaction with Murine Macrophages-J774 murine macrophages (5 ϫ 10 5 ) were cultured with rLdTyrRS in the absence or presence of different antibodies (anti-LdTyrRS or anti-CXCR2). Binding of rLdTyrRS was then tested by using a standard FACS protocol as described above. In related experiments, the cytokine profiling of these murine macrophages was also done. The cytokines released in the culture supernatants were detected by ELISA using commercially available kits according to the manufacturer's protocol (BD Pharmingen Opt EIA).
Statistical Analysis-Results for the aminoacylation activity, parasitemia, and the neutrophil migration index were entered as column data in GraphPad Prism and analyzed using Student's t test. Results for cytokine analysis were entered as grouped data in GraphPad Prism 5 and analyzed by two-way analysis of variance followed by Tukey's multiple comparison post test. The data are represented as mean Ϯ S.D. A p value of Ͻ 0.05 was accepted as an indication of statistical significance.
Author Contributions-S. A. conducted the experiments. R. M. B. designed the study, supervised the experiments, and edited the manuscript with contributions from S. A. Both the authors reviewed the manuscript.