Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity

A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.

Introduction would like to acknowledge FCT for supporting JF (SFRH/BD/79712/2011) and IL (SFRH/BD/64528/ 2009). IL was also supported by the European Community's Seventh Framework Programme (KINDRED-PR300102-BD). JT is an Investigator FCT funded by National funds through FCT and co-funded through European Social Fund within the Human Potential Operating Programme. NS is supported by a fellowship from the European Community's Seventh Framework Programme under grant agreements No. 602773 (Project KINDRED). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
suggest that Asn homeostasis could be differently regulated among trypanosomatids. These parasites present different amino acid requirements for either energetic or osmotic functions in different stages of their life cycles and as a reflex of the different environmental stimuli they receive in the vector or mammalian host [34]. Across trypanosomatids' species, the amino acid transporters (AAT) repertoire has a high interspecific variation, regarding number, affinity, specificity and capacity [34]. For instance, in the case of cysteine, a crucial amino acid for thiol biosynthesis, Leishmania major contrarily to T. brucei, fails to uptake it at a rate that ensures the intracellular pool is enough for optimal growth. Therefore, these parasites rely mainly on pathways that enable cysteine synthesis [35].
In this work, we have biochemically characterized L. infantum AS-A (LiAS-A), and to gain further insights on AS-A essentiality across different Leishmania species, we have performed gene replacement studies in L. infantum.

Ethics statement
All experiments were carried out in accordance with the IBMC.INEB Animal Ethics Committee and the Portuguese National Authority for Animal Health (DGAV) guidelines, according to the statements on the directive 2010/63/EU of the European Parliament and of the Council. DGAV approved the animal experimentation presented in this manuscript under the license DGAV number 25268/2013-10-02.

Expression and purification of poly-His-tagged recombinant LiAS-A
The LiASA gene was excised from the pGEM-T Easy vector (using NdeI/EcoRI), and subcloned into pET28a(+) expression vector (Novagen). The resulting construct presented a poly-His tag (6x Histine residues) at the N-terminal and was transformed into E. coli BL21DE3. The recombinant protein was expressed by induction of log-phase cultures with 0.5 mM of IPTG at 18°C O/N. Bacteria were harvested and resuspended in buffer A (0.5 M NaCl, 20 mM Tris.HCl, pH 7.6). The sample was sonicated, according to the following conditions: output 4, duty cycle 50%, 10 cycles with 15 s each (Branson sonifier 250), followed by centrifugation to obtain the bacterial crude extract. For enzymatic activity experiments and rabbit polyclonal antibody production, the recombinant enzyme was purified in one step using Ni 2+ resin (Qiagen) pre-equilibrated in buffer A. The column was washed sequentially with buffer A, bacterial crude extract, and buffer A with increasing concentrations of imidazole. LiAS-A was eluted in the fractions of buffer A containing 100 to 500 mM of imidazole. Dialysis was performed against PBS.
For additional activity tests, oligomeric form and Stokes' radius assessment, a deeper purification was performed. Firstly, the enzyme was purified by affinity chromatography, using a Histrap HP column (GE Healthcare), charged with nickel sulphate and equilibrated in buffer A, and posteriorly mounted in an AKTAPrimer Plus (GE Healthcare) system, at 4°C. Secondly, it was purified by size exclusion chromatography, in a Hiprep 26/60 Sephacryl S-200 column (GE Healthcare), previously equilibrated with running buffer (150 mM NaCl, 20 mM Tris, pH 7.6). The last purification step was a preparative ion exchange chromatography, using an UNO Q-1 (Bio-Rad, Cat. No 720-0001) column, mounted in a BioLogic DuoFlow (Bio-Rad) device, at 4°C. The fractions were finally analysed by analytic size exclusion chromatography and analytic ion exchange chromatography, using AktaPurifier10 system (GE Healthcare), using Superose 12 10/300GL (GE Healthcare) column and a UNO Q-1 (Bio-Rad, Cat. No 720-0001) column, respectively. The final fractions were concentrated using Millipore centrifugal filter 30K (Amicon Ultra).
Concentration was determined measuring the absorbance at 280 nm using the theoretical molar extinction coefficient of 46910 M -1 .cm -1 for LiAS-A, making use of NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). The purified recombinant protein was resolved in SDS/PAGE and stained with Coomassie Brilliant Blue G-250 (Biorad).
For estimation of the LiAS-A oligomeric state the purified recombinant protein was analysed by analytic size exclusion chromatography, using the above described conditions. Blue dextran (2,000 kDa), catalase (MW 232 kDa, Stokes radius (SR) 5.22 nm), aldolase (MW 158 kDa, SR 4.81 nm), albumin (MW 67 kDa, SR 3.55 nm), ovalbumin (MW 43 kDa, SR 3.05 nm), chymotrypsinogen A (MW 25 kDa, SR 2.09 nm) and ribonuclease (MW 13.7 kDa, SR 1.64 nm) were used as standards. A calibration curve relating Log (MW) or Log(SR) with K av was performed (K av is (V e -V 0 )/(V t -V 0 ), in which V e is elution volume, V 0 is the exclusion volume given by blue dextran and V t is the total volume of the column).

Differential scanning fluorimetry
In a 96-well, thin-walled white PCR plate, 5 μl of LiAS-A (2.4 μM) were mixed with 5 μl of 10x SYPRO Orange (λ exc 485 nm; λ em 625 nm) and 40 μl of water or the ligands and ligands combinations to be tested. Plates were then sealed and placed into a BioRad iCycler5 PCR instrument. Measurements were taken every minute in 0.5°C increments from 25°to 95°C. Subsequent analysis of the fluorescent data using Biorad iCycler iQ Optical System Software Version 3.1 yielded the protein melting temperature (T m ) for LiAS-A.

Enzymatic assay
A 150 μl enzymatic mixture containing 85 mM Tris.HCl, 8.4 mM magnesium and varying concentrations of aspartate, ammonia and ATP was assayed. The assay was performed as previously described [28], and ultimately absorbance at 340 nm was measured [45]. To determine the optimal conditions for kinetic parameters determination, reaction linearity was checked by varying enzyme concentration and time. The final reaction conditions used 7.5 μg of enzyme per assay and 15 min incubation at 37°C. A pH range of 7.0 to 9.0 was assessed, and pH 7.6 was selected as the optimal one to perform the following enzyme assays. To determine the K m of each substrate a certain range of concentration was used and the remaining substrates were maintained in excess. For aspartate, ammonia, ATP and glutamine, the following concentrations were used: 1.25 to 20, 0.78 to 50, 0.625 to 10 and 1.56 to 25 mM, respectively. neomycin phosphotransferase (NEO) or hygromycin phosphotransferase (HYG) genes using a fusion PCR approach. The 5' and 3' UTR were amplified using primers 1 + 2 and 3 + 4 (S2 Table), respectively. NEO and HYG were amplified from pSP72αNEOα and pGL345HYG templates, using primers 5 + 6 and 7 + 8 (S2 Table) respectively, which possess around 30 nucleotides of the 5' UTR in the sense primer and the first 30 nucleotides of the 3' UTR in the antisense primer. 5' UTR_NEO_3' UTR and 5' UTR_HYG_3' UTR constructs were obtained using primers 1 + 4 (S2 Table). Mid-log promastigotes were transfected with approximately 10 μg of linear construct, obtained by fusion PCR, using an AMAXA Nucleofector II device with Human T-cell nucleofector kit (Lonza). The day after transfection drug selection was carried out at 20 μg/mL of G418 (Invitrogen) and 50 μg/mL of hygromycin B (InVivoGen). Parasite cloning was performed by diluting the parasite suspension to a concentration of 0.5 cells/ well, using SDM culture medium. The drug concentrations for clone maintenance correspond to half of the selection concentrations.

Generation of LiASA overexpressor (OE) and null mutants' complementation
LiASA gene was excised from the pGEM-T Easy vector (using XbaI/NdeI) and subcloned into pSP72αBLASTα vector. Mid-log promastigotes, WT and dKO mutants, were transfected with approximately 10 μg of plasmid DNA as above in order to generate an overexpressing line (OE) or complemented null mutants, respectively. Drug selection was carried out at 30 μg/ml of blasticidin (InVivoGen).

In vitro growth of LiASA mutants
Cultures were launched and monitored microscopically every 24h for 8 days or were maintained in log phase by subculturing every 2 days and cumulative growth was assessed for 5 consecutive passages. The growth experiments were performed in complete RPMI (cRPMI) or Asn depleted cRPMI (cRPMI + L-asparaginase) obtained by cRPMI O/N incubation with 1250 U/L of L-asparaginase at 37°C. Growth curves were also undertaken in a serum-free RPMI (sfRPMI [46]) incubated with L-asparaginase, that was removed afterwards by flowing the medium through a 3 kDa Millipore centrifugal filter (Amicon Ultra), generating an Asn free medium (sfRPMI + L-asparaginase). Asn was directly added to the Asn free sfRPMI (cf = 380 μM) to generate complemented sfRPMI (sfRPMI + L-Asparaginase + Asn). Finally, growth curves were performed in complete M199 (cM199) [29] or cM199 supplemented with Asn (cf = 380 μM). Growth curves of LiASA mutants and WT were seeded at 1 x 10 6 parasites/ml at 26°C, except in the case of sfRPMI, whose initial parasite density was 2 x 10 6 parasites/ml, grown with agitation. Before launching growth curves, the parasites were maintained in log phase for 2-3 passages in the absence of selection drugs.

In vivo infectivity of LiASA mutants
Five to six weeks old female BALB/c mice were obtained from Charles River. For each mouse injection, 1 x 10 8 promastigotes recovered from 4 days old stationary culture were washed, resuspended in PBS, and injected intraperitoneally. Mice of each group (n = 4) were sacrificed at 2 weeks post-infection. The parasite burden in the spleen and liver was determined by limiting dilution as previously described [47].

Digitonin fractionation
For each sample condition, 1 x 10 8 promastigotes were washed once with cold trypanosome homogenisation buffer (THB), composed by 25 mM Tris, 1 mM EDTA and 10% sucrose, pH 7.8. Just before cell lyses, peptidase inhibitor (Roche) and different digitonin (Calbiochem) quantities (final concentrations of 12.5, 25, 50, 100, 200, 500 and 1000 μg/ml) were added to 250 μl of cold THB, for cell pellet resuspension. Untreated cells and those completely permeabilised (total release, the result of incubation in 1% Triton X-100) were used as controls. Each sample was incubated 60 min on ice, and then centrifuged at 13,000 rpm, 4°C, for 10 min. Supernatants were taken off into new pre-cold tubes and 250 μl of cold THB was added to each pellet and then mixed. All fractions were analysed by WB.

Immunofluorescence
L. infantum mid-log promastigotes were fixed, permeabilised and stained as previously described [48]. Cells were incubated with primary antibody O/N at 4°C. The following primary antibodies were used: rabbit anti-LiAS-A (1:1000) and sheep anti-LiTDR1 (thiol-dependent reductase 1, 1:2000). Subsequently, slides were incubated for 1h at RT in a dark humidified atmosphere with a secondary antibody (1:500). The following secondary antibodies were used: goat anti-rabbit Alexa Fluor 488 or 568 and donkey anti-sheep Alexa Fluor 488 (Molecular probes, Life Technologies). In the case of Mitotracker Orange (Invitrogen), we stained the parasites by adding 1 μM to culture medium (without FBS) for 1h at 26°C, prior to the above described procedure. Slides were stained and mounted with Vectashield-DAPI (Vector Laboratories, Inc.). Images were captured using fluorescence microscope AxioImager Z1 (Carl Zeiss), equipped with a Axiocam MR v. 3.0 camera (Carl Zeiss), using either 63x (Plan-Apochromat 63x/1.40 Oil DIC) or 100x (Plan-Apochromat 100x/1.40 Oil DIC) objective. Images analysis and deconvolution was performed using ImageJ software (v. 1.47) and image deconvolution lab plugin (2010 Biomedical Imaging Group, EPFL, Switzerland) with Richardson-Lucy algorithm.

LiAS-A and LmAS-A sequence alignment and homology models
The open reading frames (ORFs) encoding putative AS-A and AS-B enzymes were identified in the genomes of L. infantum JPCM5 (LinJ.26.0790; LinJ.29.1590) and L. major Friedlin (LmjF.26.0830; LmjF.29.1490) [42][43][44]. The ASA amplified sequence from L. infantum strain matched 100% the annotated sequence from JPCM5 genome. To obtain structural and functional insights on AS-A enzymes, we have performed in silico analysis using the L. infantum (LiAS-A), L. major (LmAS-A), T. brucei (TbAS-A), T. cruzi (TcAS-A) and E. coli (EcAS-A) sequences that generate polypeptides containing 353, 353, 351, 348 and 330 residues, respectively ( Fig 1A). Overall, the sequence alignment shows a high conservation of the main structural features, including the active site residues (Fig 1A). Indeed, the amino acids involved in Asn binding are strictly conserved across species, whereas in the case of AMP binding pocket, the majority of residues are conserved with a few exceptions. For instance, in the case of LiAS-A and LmAS-A, there is a sole residue replacement, namely EcAS-A L109, corresponding to I111 in both cases. This residue is not involved in polar interactions with AMP molecule, but instead integrates the outer wall of the nucleotide binding pocket [15].
Analysing the homology models of LiAS-A and LmAS-A ( Fig 1B) obtained by superimposition with EcAS-A crystal structure (PDB 12AS [15]), there is a divergent region highlighted with a dashed rectangle ( Fig 1B) in L. infantum and L. major enzymes, which is strictly conserved in these two species. This region also exists in trypanosomes, although little conservation is found when comparing to Leishmania sp. (Fig 1A, [28,29]). EcAS-A (NCBI-GeneID:948258/pdb:12AS). A preestablished colour pattern was used, according to ALSCRIPT Calcons (Aline version 011208): red, identical residues; orange to blue, scale of conservation of amino acid properties in each column alignment; white, dissimilar residues). Secondary structure components of EcAS-A crystal structure (black) are represented above the alignment. In all sequences, binding residues for several ligands were represented: AMP (circles), asparagine (squares), ATP (triangle) and aspartate (inverted triangle). B) Superposition of EcAS-A structure (green) (PDB accession code 12AS), with LiAS-A (blue) and LmAS-A (purple) homology models (obtained from the SWISS-MODEL server, using PDB 12AS as a template). The dashed box points a structurally divergent region.

Enzymatic characterization of LiAS-A
Recombinant LiAS-A, comprising a 6 histidine N-terminal tag, was expressed in E. coli and purified by affinity chromatography in native conditions in order to evaluate and characterize its enzymatic activity. The protein presented the expected MW for the monomer,~42 kDa, as presented on Fig 2A and 2B, with either Coomassie staining or Western-blot analysis with an anti-HisTag antibody, respectively. Subsequently, LiAS-A was further purified sequentially by size exclusion and ion exchange chromatographies, and the final fractions were analysed by analytic size exclusion chromatography (Fig 2C). Using the latter chromatography and calibration standards, Stokes' radius (~3.52 nm, Fig 2D) and MW (~78.8 kDa, Fig 2E) were extrapolated in the protein native state. LiAS-A corresponds to a homodimer, as predicted.
For the characterization of the enzymatic activity of recombinant LiAS-A, a specific colorimetric assay that quantifies Asn formation was used [28,45]. The optimal pH for the enzymatic activity was 7.6. The kinetic characterization of the enzyme was undertaken in steadystate conditions, using a fixed concentration of 8.4 mM of Mg 2+ (Table 1). LiAS-A displayed ammonia and glutamine dependent activity When comparing K m values for ammonia and glutamine, no statistical significant difference is found (p = 0.03), but there is significance in the differences found in k cat (p = 1.80 x 10 −4 ). In order to discard the possibility that utilization of glutamine as a substrate was an artefact resulting from contamination with EcAS-B (EcAS-B~120 kDa), highly purified fractions of LiAS-A (LiAS-A~84 kDa) were tested and glutamine utilization were clear in all protein samples tested.
Differential scanning fluorimetry was also used in order to further understand the relevance of the different substrates for thermal stabilization of the enzyme. AS-A forms a crucial β-Aspar-tylAMP-Mg 2+ intermediate, which then undergoes a nucleophilic attack of ammonia, forming Asn and releasing AMP e pyrophosphate [15]. According to our data, ammonia can be free or glutamine-derived, although the glutaminase domain of LiAS-A remains to be identified. Looking at the differential scanning fluorimetry data, AMP leads to a 10 degrees shift in LiAS-A T m , thermally stabilising this protein (Fig 2F) in a concentration dependent fashion (Fig 2G).
LiASA null mutants generation by targeted gene replacement A targeted gene replacement strategy was used for inactivation of the ASA gene of L. infantum. Two constructs, obtained by fusion PCR, linking NEO or HYG to the 5' and 3' UTRs of the LiASA gene were used to remove the first and second LiASA allele, respectively. Two sKO mutants (clones A and B) were transfected with the HYG construct. We successfully obtained 5 dKO mutants, 3 from clone A (A1, A2 and A3) and 2 from clone B (B1 and B2). The integration of the resistance markers in the expected locus was confirmed by PCR using primers upstream of the 5' UTR or downstream of the 3'UTR coupled with primers in their ORFs (the strategy is illustrated on Fig 3A). NEO 5' and 3' integration was positive in both sKO and dKO mutants, as for HYG, only in dKO parasites, as expected (Fig 3B). Also by PCR analysis, we could not amplify LiASA ORF in null mutants (a non-related gene-LiRPIB-was amplified as control- Fig 3B).
Southern-blot analysis confirmed the genotypes: the expected fragments upon digestion with SacI and NdeI are represented on Fig 3A. A first hybridisation was performed using 5' UTR as a probe: in WT a single band of~1696 bp corresponding to LiASA was generated, with twice the intensity observed in the sKO mutants that possess a single copy, and absent in the dKO mutants, confirming the successful gene removal (Fig 3C). In both sKO and dKO clones, a band of~2973 bp was generated corresponding to NEO, and then only in dKO clones, a band of~2052 bp corresponding to HYG was observed (Fig 3C). The blot was then stripped and reprobed three additional times to confirm each one of the bands (faint bands of incomplete stripping can be observed), sequentially using LiASA, NEO and HYG. All the mutants were also analysed by Western-blot, showing a protein reduction in sKO mutants and a complete absence in the dKO clones ( Fig 3D). LiASA gene was cloned into a pSP72αBLASTα vector in order to obtain an overexpressor mutant (OE) as well (Fig 3D).

AS-A is localized in the cytosol of L. infantum promastigotes
Rabbit polyclonal antibodies produced against recombinant LiAS-A recognised a major band in total WT promastigotes extract with the expected molecular weight (~39.8 kDa [web.expasy.org/ protparam], S1A Fig), but not in a dKO mutant (dKO A2). Prior to immunolocalisation studies, LiAS-A antibody was also validated (S1B Fig) by performing an IFA and comparing the labelling intensity in WT promastigotes versus LiASA null mutants and OE. A positive correlation between protein level and fluorescence intensity was found on WT versus OE (S1C Fig). As expected, no specific labelling was detected for the LiAS-A null mutants (S1B and S1C Fig).
Using α−tubulin (~50 kDa) as loading control we compared the expression levels of LiAS-A in different developmental stages: promastigotes (logarithmic, early stationary and late stationary phase) and axenic amastigotes (Fig 4A). No significant differences were observed.
Immunofluorescence analysis showed that in promastigotes LiAS-A co-localises with LiTDR1 (thiol-dependent reductase 1), which is a cytosolic protein involved in thiol metabolism [49] (Fig 4B, upper panel). LiAS-A subcellular localisation in promastigotes was also assessed by digitonin fractionation. The fractioning profile was evaluated using antibodies for proteins present in different subcellular compartments, namely, anti-TbEnolase (LiEnolase versus TbEnolase 79% identity, LiEnolase 39.6 kDa) as cytosolic marker [50], and anti-LdHGPRT (hypoxanthine guanine phosphoribosyltransferase, 23.6 kDa) as glycosomal marker [51]. LiEnolase (Fig 4C) can be found in the supernatant for digitonin concentrations as low as 12.5 μg/ml, and retained in the pellets up to 25-50 μg/ml. LiHGPRT (Fig 4C), which localises to the glycosomes, is detected in the supernatant in appreciable amounts for higher digitonin concentrations and is retained longer in the pellet (up to 200-500 μg/ml, and residually at 1000 μg/ml of digitonin). As expected, LiAS-A presents a profile similar to LiEnolase, supporting a cytosolic location (Fig 4C). Intriguingly, LdAS-A was reported to have dual localisation between the cytoplasm and mitochondria in promastigote form [29]. Due to the high identity (~99%) between both enzymes, we investigated whether LiAS-A also localised to the mitochondria. Immunofluorescence analysis of LiAS-A subcellular distribution on promastigotes labelled with mitotracker showed no evidence of mitochondrial location (Fig 4B, lower panel). Moreover, by using tools for protein localisation prediction (TargetP, CELLO, MITOPROT and Predotar), mitochondria localisation seems unlikely and actually, CELLO predicts cytoplasmic localisation. In conclusion, our data shows LiAS-A localises to the cytosol.

LiAS-A is required for promastigotes growth only in asparagine limiting conditions
All mutants displayed similar growth patterns comparing to WT promastigotes in cRPMI ( Fig  5A). However, in Asn depleted medium, achieved upon L-asparaginase treatment (cRPMI + Lasparaginase), the behaviour was quite different for some of the mutants. Parasites overexpressing LiAS-A displayed a significant higher growth during log phase when comparing to the WT, whereas the dKO mutants (clones A2 and B1) displayed a major growth defect (Fig 5B). The complementation of these null mutants with an episome (pSP72αBLASTα) carrying LiASA gene rescued the growth (Fig 5B). Moreover, an upregulation in LiAS-A levels could be observed in these mutants in Asn limiting conditions (Fig 5C). Western-blot analysis also showed that in the same conditions, an upregulation in LiAS-A could also be observed over time in the sKO parasites (clones A and B), enabling the growth recovery in these mutants ( Fig  5B-5D and S3 Table). This recovery was faster in sKO clone B that had higher basal levels of LiAS-A than clone A (Fig 5B and 5C). We also evaluated the cumulative growth under constant multiplicative conditions, in which high amino acids levels are required. For that, parasites were maintained in log phase in Asn replete or Asn depleting conditions, and the same patterns were observed (S2 Fig). To ensure the defective growth phenotype of sKO and dKO parasites in L-asparaginase treated medium was due to Asn depletion, we supplemented this medium with Asn. Surprisingly the addition of this amino acid to L-asparaginase treated RPMI medium fails to reverse the observed growth delay/arrest phenotype. The fact that L-asparaginase was not inactivated or neutralized, and consequently may have remained active, may explain this result. Consequently, we used another strategy by undertaking growth curves in a serum free medium (sfRPMI [46]) incubated with L-asparaginase that was removed afterwards using a 3 kDa Amicon column. In sfRPMI devoid of Asn (sfRPMI + L-asparaginase), the same growth defect of the sKO and dKO mutants was observed (Fig 5E). And then again, in the sKO clones the upregulation of LiAS-A allowed the growth rescue (Fig 5G and S3 Table). When adding back Asn (sfRPMI + L-Asparaginase + Asn), all mutants grew in a similar fashion (Fig 5F). In the absence of drug pressure and in normal conditions, parasites provided of an episome carrying LiASA (OE) hardly overexpress AS-A, however, under Asn depleting conditions, they upregulate its expression (comparing to the levels in the WT, there is an increase from~130% tõ 300% and from~110 to~130%, in panels C and G, respectively, and S3 Table). Moreover, besides the experiments using L-asparaginase treatment, we have also performed growth curves in a medium formally lacking Asn-complete M199 (cM199)-in order to further confirm Asn auxotrophy upon ASA ablation. In this medium, null mutants presented a growth defect comparable to the one observed in cRPMI + L-asparaginase, which again was reversed when these mutants were complemented with an ectopic copy of ASA gene (Fig 5B  versus 5H). The addition of Asn to the final concentration of 380 μM (like in RPMI) rescues the growth defect displayed by the null mutants in cM199 (Fig 5I). Interestingly, the experiments to assess LiAS-A essentiality in L. donovani were performed in cM199 [29], therefore the inability to generate LdASA null mutants may be due to the performance of those attempts in Asn limiting conditions.
In conclusion, ASA deletion renders parasites auxotrophic to Asn, but is dispensable for parasite growth in normal conditions. In vitro growth of LiASA mutants in normal or Asn depleted medium and respective LiAS-A expression levels. A and B) L. infantum promastigote growth curves in cRPMI or cRPMI + L-asparaginase, respectively, including dKO mutants (clones A2 and B1) complemented with pSPαBLASTα carrying LiASA gene. In B, a significant growth difference in comparison to WT (p < 0.05, Graphpad Prism 5.0 version) was found for sKO A, sKO B, dKO A2 and B1, dKO A2 + LiAS-A, dKO B1 + LiAS-A and OE mutants for days 1 to 8, days 1 to 6, days 1 to 8, days 2 to 6, days 2 to 7 and day 3, respectively. C) Western-blot analysis of LiAS-A expression in 7 days old LiASA mutants (versus WT) cultured in cRPMI and cRPMI + L-asparaginase. D) Western-blot analysis of LiAS-A expression levels over time in WT, and clones A and B cultured in cRPMI (lane L-asparaginase -) or cRPMI + L-asparaginase (lane L-asparaginase +). E and F) L. infantum promastigote growth curves in sfRPMI + L-asparaginase or sfRPMI + L-asparaginase + Asn, respectively. sfRPMI was Asn depleted through incubation with L-asparaginase, which was then removed by flowing the medium through an Amicon Column of 3 kDa pore (sfRPMI + L-asparaginase). Asn was then added directly to the medium (sfRPMI + L-asparaginase + Asn). In E, a significant growth difference in comparison to WT (p < 0.05, Graphpad Prism 5.0 version) was found for sKO A, sKO B and dKO A2 and B1 mutants for days 2 to 7, days 2 and 5 to 7 and days 2 to 7, respectively. G) Western-blot analysis of LiAS-A expression in 7 days old LiASA mutants (versus WT) cultured in sfRPMI + L-asparaginase (lane Asn -) and sfRPMI + Lasparaginase + Asn (lane Asn +). H and I) L. infantum promastigote growth curves in cM199 and cM199 + Asn, respectively, including dKO mutants complemented with pSPαBLASTα carrying LiASA gene. In H, a significant growth difference in comparison to WT (p < 0.05, Graphpad Prism 5.0 version) was found for dKO A2 and B1, dKO A2 + LiAS-A and dKO B1 + LiAS-A mutants for days 1 to 7, days 2 and 3, days 1 to 3, respectively. The results (A-I) are representative of 2 independent experiments. For the Western-blot analysis displayed in C, D and G, 1x10 7 parasites were used for total extract preparation and LiCS (cysteine synthase) was used as loading control. OE, overexpressor. In vivo infectivity of LiASA mutants Notwithstanding, we intended to evaluate the impact of LiASA ablation on, in vivo infectivity. Five to six old female BALB/c mice were infected and were sacrificed at 2 weeks post-infection. The parasite burden in the spleen ( Fig 6A) and liver (Fig 6B) was not statistically different in LiASA mutants when compared to the WT. The same scenario was observed for sKO A and dKO A2 mutants. No differences in LiAS-A expression levels were found when comparing parasites used in mice infection (Culture) to parasites recovered from spleen (S) or liver (L) ( Fig  6C). Thus, LiAS-A ablation does not compromise parasite infectivity in the context of an acute in vivo infection.

Discussion
Despite being eukaryotes, trypanosomatids, present AS-A enzymes of bacterial origin. Moreover, these enzymes are aminoacyl-tRNA synthetase paralogues, displaying an AsnRS catalytic core with conserved class II motifs, yet lacking the tRNA binding domain [27]. In this work, we have demonstrated that LiAS-A is able to synthesize Asn using either ammonia or glutamine as nitrogen donors, as previously described for TbAS-A, TcAS-A and LdAS-A [28,29]. K m values for aspartate and ATP are close to the ones determined for TbAS-A and TcAS-A [27]. As for ammonia, the K m value found for LiAS-A is 5 fold lower in comparison to TbAS-A, TcAS-A and LdAS-A [27,28]. In the case of LdAS-A, the K m values for aspartate were around 10 fold lower [29] than the ones obtained for LiAS-A. Regarding the high conservation of the active sites among Leishmania AS-A enzymes, we cannot exclude that the observed kinetic differences may be due to the differences in the amount of protein that is properly folded, especially taking into account they are expressed in a heterologous system. Moreover, it is important to emphasize that the kinetic determinations for LdAS-A were performed using a different experimental set up. Importantly, TbAS-A and TcAS-A use preferably ammonia [28], whereas LiAS-A seems to use both roughly in the same extent (Table 1). AS-A activity in trypanosomatids more resembles AS-B enzymes, concerning both the optimal pH for enzymatic activity (7.6 instead of 8) and also the ability to use both nitrogen donors. AS-B enzymes use preferably glutamine, with exception of the human enzyme that presents approximately the same affinity for both nitrogen sources [18,19,25,[52][53][54][55][56][57]. This biochemical feature, so far only described for trypanosomatids AS-A enzymes [28,29], becomes particularly interesting in the context of the presence of an ORF encoding a hypothetical, yet non-classical, AS-B, in the genome of these organisms (L. infantum [LinJ. 29 [42][43][44]. These sequences contain a Pfam AS domain (pfam00733) and glutamine hydrolysing domains in the C and N-terminus, respectively. BLASTp analysis of L. infantum sequence, for instance, revealed several hits that corresponded to hypothetical proteins from a broad range of eukaryotes. However, we have no evidence AS-B is functional at all.
Much remains to be disclosed regarding the AS-A enzymes from trypanosomatids, for instance, we still lack information on their glutamine binding and hydrolysing sites. TbAS-A crystallisation only emphasised the high conservation of Asn and AMP binding pockets, as the only divergent region from EcAS-A (a 19 residues insertion, also present in LiAS-A and LmAS-A, Fig 1) was not visible in the experimental electron density maps and therefore likely disordered [29]. This insertion displays little conservation when comparing Leishmania and trypanosomes, and its role on a structural or functional level is still unclear.
AS-A is a key enzyme in Asn metabolism that was proposed as a potential drug target due to its absence in the human host. Moreover, AS-A was reported to be essential for L. donovani survival, contrasting with T. brucei bloodstream forms, as in the latter it was shown to be dispensable for both in vitro growth and infectivity. These findings pointed to a differently regulated Asn homeostasis across trypanosomatids. In L. infantum, our efforts to generate ASA null mutants were successful, indicating the gene is not essential for survival. Moreover, the null mutants did not present any growth or infectivity defect. Our in vitro growth data demonstrate that upon LiASA deletion, promastigotes become dependent on extracellular Asn for optimal growth (Fig 5). These results suggest that even if AS-B is functional, it does not compensate LiAS-A activity, as LiASA null mutants fail to grow in Asn limiting conditions. Additionally, WT parasites grew normally in Asn depleted medium without AS-A upregulation, suggesting Asn synthesis by basal AS-A suffices the cellular needs, although the mutants overexpressing this enzyme had a metabolic advantage in an Asn deprivation environment during log phase (S2B and S2E Fig). We can actually infer the parasite can both synthesise and take up this amino acid, and the latter fully compensates the former. Furthermore, our results indicate that LiAS-A levels are regulated according to Asn availability, and it was equally surprising to see how fast and efficiently sKO mutants were able to upregulate AS-A when cultured in Asn limiting conditions (Fig 5D). It is also noteworthy that the two sKO mutants displayed a substantial difference in AS-A levels, which has also been observed among other sKO mutants generated in this study. A possible explanation might be that the two allelic copies may differently affect ASA expression.
In trypanosomatids, much remains to be unravelled concerning amino acid transporters (AATs) and mostly the pathways involved in amino acid sensing and regulation of their synthesis and uptake [34]. Very few data is available in the literature concerning Asn transport in these parasites. In T. brucei, a protein presenting putative orthologues in Leishmania [42][43][44] was characterized as a transporter of several neutral amino acids, including asparagine (TbAATP1) [58]. In mammalian cells, AS-B is a transcriptional target of the well characterized GCN2/elF2α/ATF4 axis, in response to amino acid starvation [59,60]. The phosphorylation of elF2 leads to a repression of general protein synthesis, as well as an activation of gene-specific translation. In Saccharomyces cerevisiae, GCN2, which is activated by amino acid, glucose or purine deprivation, is the only elF2 kinase, contrasting with mammals that possess some additional three, HRI, PKR and PEK/PERK [61]. T. brucei and L. donovani PERK orthologues [62,63] have been implicated in the response to ER stress and their activation leads to a decrease in the overall translation [62]. At the moment, it is still not clear whether phosphorylation of elF2 in trypanosomatids would result in a downstream signalling cascade, as bZIP type transcription factors, that could act like GCN4 or ATF4, are absent in these organisms [64].
The close relation between L. infantum and L. donovani species and the 99% identity of AS-A between both makes the discrepant phenotype intriguing. In the literature, several cases in which knocking out a gene can have different impact on virulence depending on the species can be found. [65]. Nevertheless, to our knowledge, there is no documented example among cutaneous or among visceral species of a gene that is detrimental for survival in one species and dispensable in other closely related species. However, we did find a case of differences at a strain level for instance [66]. Nonetheless, firstly we must highlight that LdASA essentiality was claimed solely based on the consecutive failure in the removal of the second gene copy [29]. Secondly, our results suggest that the medium in which the experiments were performed, cM199, may explain this difference. The former lacks Asn and LiASA null mutants could not grow unless upon Asn supplementation (Fig 5H and 5I). These results reinforce the importance of the medium composition when attempting gene knock-out of metabolic enzymes, and supplementation may be detrimental when potentially generating auxotrophs [67,68].
LiASA dKO mutants displayed no compromised infectivity in mice, suggesting that in intracellular amastigote form, either AS-B is functional or, most likely, parasites are able to uptake Asn in such an extent that compensates the lack of intracellular synthesis. In vivo treatment with L-asparaginase, which induces a decrease in Asn bloodstream levels, has been successfully used for years in the treatment of acute lymphoblastic leukemia [69] and recently it was proposed as a promising strategy to treat bacteremia caused by group A Streptococcus and eventually other extracellular bacteria [13]. However, if for some extracellular pathogens, Lasparaginase treatment seems promising, in the case of an obligate intracellular microorganism, even when simultaneously inhibiting the microbial AS-A, several issues may arise, namely the potential contribution of the host cell for Asn de novo synthesis.
Taken all together, we conclude AS-A is not a suitable drug target candidate in L. infantum, and therefore, with regard to drug development, such a protein target becomes pointless against Leishmania.
Supporting Information S1 Table. Oligonucleotides sequences used to obtain LiAS-A recombinant protein (P1-P2) and pSPαBLASTαLiASA (P3-P4). (DOCX) S2 Table. Oligonucleotides sequences used to obtain gene replacement cassettes (P1-P8) and to confirm LiASA mutants' genotype (P9-P20). (DOCX) S3 Table. Semi-quantification of AS-A levels in LiASA mutants cultured in vitro in Asn replete or depleting conditions.  1000). B) Representative immunofluorescence images of different genotypes (WT, dKO clone A2 and OE) of mid-log L. infantum promastigotes, using rabbit polyclonal anti-LiAS-A antibody (1:1000). Upper and lower panels present brightfield and LiAS-A (green) + DAPI (blue) stained images, respectively. Images were acquired with a 100x objective, using a Zeiss AxioImager Z1. The scale bar corresponds to 5 μm. C) Fluorescence intensity quantification in WT, dKO clone A2 and OE parasites when stained with anti-LiAS-A antibody (1:1000). The values are expressed in CTCF (corrected total cell fluorescence), and background (BG) values are displayed as well. The quantification was performed on images acquired with 63x objective, using a Zeiss AxioImager Z1 and the same exposure time for all genotypes (LiAS-A 400 ms; DAPI 100 ms). Twenty different fields for each genotype were analysed in duplicate, and the fluorescence of an average of 50-100 parasites was quantified using ImageJ (v 1.47) software. Statistical analysis was performed using Graphpad Prism 5.0 version: statistical significance p < 0.05 ( Ã ), p < 0.01 ( ÃÃ ), p < 0.001 ( ÃÃÃ ), p < 0.0001 (  infantum promastigotes growth curves of LiASA mutants (versus WT), cultured in cRPMI and cRPMI + L-asparaginase, respectively. Parasites were maintained in logarithmic phase by subculturing every 2 days. The results correspond to mean values of duplicates ± standard deviation. Statistical analysis was performed using Graphpad Prism 5.0 version: statistical significance p < 0.05 ( Ã ), p < 0.01 ( ÃÃ ), p < 0.001 ( ÃÃÃ ), p < 0.0001 ( ÃÃÃÃ ). C and F) Western-blot analysis of LiAS-A expression levels in 8 days old promastigotes, cultured in cRPMI and cRPMI + L-asparaginase. In A-F panels, the results are representative of two independent experiments. For the Western-blot analysis displayed in C and F, 1x10 7 parasites were used for total extract preparation and LiCS (cysteine synthase) was used as loading control. (TIFF)