Cells

Leishmania mexicana M379 cells were routinely cultured at 25 ^oC in Medium 199 (M199), pH 7.4, supplemented with 10% fetal calf serum (FCS, Biological Industries), 5 μg/mL hemin, 0.1 mM adenine, 40 mM Hepes, 4 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin.

RAW 264.7 macrophage cells were grown at 37 ^oC in DMEM supplemented with 10% FCS, 4 mM L-glutamine, 0.1 mM adenine, 40 mM Hepes pH 7.4, 100 U/mL penicillin and 100 μg/mL streptomycin, in an atmosphere of 5% CO2.

Affinity purification of recombinant LeishIF4E2

The open reading frame (ORF) of LeishIF4E2 was amplified with primers LeishIF4E2-His forward and LeishIF4E2-His reverse (S1 Table) and cloned in pET28 vector and expressed in the Rossetta strain of E. coli. The bacterial cells were grown to OD₆₀₀ of 0.5–0.7 and expression was induced by the addition of 0.3 mM IPTG at 25 ^oC for 8 hrs. The cells were harvested and re-suspended in lysis buffer [20 mM HEPES-KOH pH 7.5, 100 mM KCl, 2 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), 0.1 mM EDTA, 0.01% Triton X-100, a cocktail of protease inhibitors (Sigma) and 5 μg/mL DNaseI. Ni-NTA beads (Cube Biotech, 5 mL) were pre-washed with two column volumes (CVs) of Binding Buffer [20 mM HEPES-KOH pH 7.5, 150 mM KCl, 2 mM TCEP]. The cells were disrupted using a French Press at 1500 psi, followed by centrifugation at 45,000 rpm (Beckman 70 Ti rotor) for 45 min. The supernatant was further incubated with Ni-NTA beads that were washed four times with two CVs of Wash Buffers (WB) containing 20 mM Hepes-KOH pH 7.5, 2 mM TCEP, 10 mM imidazole, in 2 CVs and gradually decreasing KCL concentrations (750 mM, 500 mM, 250 mM and 100 mM). Finally, the beads were eluted with a single CV of PBS containing 300 mM imidazole in 20 mM Hepes-KOH pH 7.5, 100 mM KCl, 2 mM TCEP. The recombinant protein was dialyzed overnight at 4 ^oC against the Binding Buffer.

CRISPR-Cas9 mediated deletion of a single allele of LeishIF4E2

Plasmids developed for the CRISPR system in Leishmania were obtained from Dr. Eva Gluenz (University of Oxford, UK, [24]). The pTB007 plasmid used contained the genes encoding the Streptococcus pyogenes CRISPR-associated protein 9 endonuclease and the T7 RNA-polymerase (Cas9/T7), along with the hygromycin resistance gene. pTB007 was transfected into L. mexicana promastigotes and transgenic cells stably expressing the Cas9 and T7 RNA-polymerase were selected for 200 μg/mL hygromycin resistance.

Generation of a LeishIF4E2(+/-) mutant by CRISPR-Cas9

To generate LeishIF4E2(+/-) mutants we used the two 5’ and 3’ sgRNAs designed to create double-strand breaks (DSBs) upstream and downstream of the LeishIF4E2 coding region and the LeishIF4E2 repair cassette fragment containing the G418 resistance marker. The three PCR products (4 μg of each) were transfected into mid-log phase transgenic cells expressing Cas9 and T7 RNA-polymerase and cells were further selected for resistance to 200 μg/mL G418 [25].

The sgRNA sequences used to delete the LeishIF4E2 gene were obtained from LeishGEdit.net [26]. The sgRNAs contained the highest-scoring 20 nt sequence within 105 bp upstream or downstream of the target gene. The sequences of the sgRNAs were blasted against the L. mexicana genome in TriTrypDB to verify that the sgRNAs were specific for LeishIF4E2 exclusively (E value = 0.001 and 8e^-5). We also ran a BLAST analysis with the drug resistance repair cassette that contained the homology sequence to the UTR of LeishIF4E2 to specifically target the insertion of the selection marker. The repair cassette showed an E value of 5e^-9 suggesting very high specificity of the system. The sgRNA target sequences and the homology arms on the repair cassette fully matched the target sequence of LeishIF4E2.

PCR amplification of sgRNA templates

DNA fragments encoding LeishIF4E2 specific 5’ and 3’ guide RNAs for cleavage upstream and downstream to the LeishIF4E2 target gene were generated. All primers are listed in S1 Table. The template for this PCR reaction consisted of two fragments: one contained the common sgRNA scaffold fragment; the other contained a primer that included the T7 RNA polymerase promoter (small letters) fused to the gRNA (5’ or 3’), targeting LeishIF4E2 (capital letters) and a short sequence overlapping with the scaffold fragment (small letters, 5’gRNA (LmxM.19.1480) or 3’gRNA (LmxM.19.1480, S1 Table). Each of these two fragments (1 μM each) was annealed to the partially overlapping scaffold fragment and further amplified with two small primers (2 μM each) derived from the T7 promoter (G00F) and the common scaffold fragment (G00R, S1 Table). The reaction mixture consisted of dNTPs (0.2 mM), HiFi Polymerase (1 unit, Phusion, NEB) in GC buffer with MgCl₂ (NEB), in a total volume of 50 μL. The PCR conditions included an initial denaturation at 98°C for 2 min, followed by 35 cycles of 98°C for 10 s, annealing at 60°C for 30 s and extension at 72°C for 15 s. All PCR products were gel-purified and heated at 94°C for 5 min before transfection.

PCR amplification of the LeishIF4E2 replacement fragment

A DNA fragment designed to repair the DSB surrounding the LeishIF4E2 target gene was amplified by PCR. The LeishIF4E2 specific primers were derived from the 5’ and 3’ endogenous untranslated regions (UTR) sequences upstream and downstream to the LeishIF4E2 gene based on the LeishGEdit database (http://www.leishgedit.net/Home.html) and the sequences of the antibiotic repair cassette, The primers were Upstream Forward (LmxM.19.1480) and Downstream reverse (LmxM.19.1480, S1 Table). Capital letters represent the UTR sequences of the LeishIF4E2 gene and small letters represent the region on the pT plasmid that flanks the UTR adjacent to the antibiotic resistance gene. The PCR for generating the fragment used for repair of the DSBs on both sides of the gene targeted for deletion was performed using the pTNeo plasmid as a template. The resulting fragments promote the integration of the drug resistance marker by homologous recombination at the target site. The reaction mixture consisted of 2 μM of each primer, dNTPs (0.2 mM), the template pTNeo (30 ng), 3% (v/v) DMSO, HiFi Polymerase (1 U of Phusion, NEB) in GC buffer (with MgCl₂ to a final concentration of 1.5 mM) in a total volume of 50 μL. PCR conditions included initial denaturation at 98°C for 4 min followed by 40 cycles of 98°C for 30 Seconds (s), annealing at 65°C for 30 s and extension at 72°C for 2 min 15 s. The final extension was performed for 7 min at 72°C. All PCR products were gel purified and heated at 94°C for 5 min before transfection.

Diagnostic PCR to confirm the deletion of LeishIF4E2

Genomic DNA from the drug-resistant cells was isolated 14 days post transfection using DNeasy Blood & Tissue Kit (Qiagen) and analyzed for the presence of the LeishIF4E2 gene, using specific primers derived from the UTRs of LeishIF4E2. The primers used were LeishIF4E2 (5’UTR) forward (P1) and LeishIF4E2 (3’UTR) Reverse (P2). A parallel reaction was performed to detect the presence of the G418 resistance gene with primers derived from its ORF: G418 Forward (P3) and G418 Reverse G418 Reverse (P4). An additional reaction confirming the insertion of the G418 cassette at the target site was performed with primers G418 Forward P3, and LeishIF4E2 (3’UTR) Reverse P2. All primers are listed in S1 Table. Genomic DNA from Cas9/T7 L. mexicana cells was used to detect the presence of the LeishIF4E2 gene. The reaction mixture consisted of 2 μM of each primer, gDNA (100 ng), dNTPs (0.2 mM), HiFi Polymerase (1 U Phusion, NEB) in GC buffer with MgCl₂ (NEB) in a total volume of 50 μl. PCR conditions included initial denaturation at 98°C for 4 min followed by 35 cycles of 98°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 2 min 15 s. Final extension was done for 7 min at 72°C. PCR products were separated on 1% agarose gels.

Generation of LeishIF4E2 add-back parasites and SBP-tagged LeishIF4E2 expressers

The hemizygous mutant LeishIF4E2(+/-) cells were transfected with an episomal vector that restored full-length LeishIF4E2_1-281 expression. The plasmid was derived from pTPuro [24] which confers resistance to puromycin. The LeishIF4E2 add-back gene from L. mexicana was tagged with the Streptavidin binding peptide (SBP, ~4 kDa), enabling its further identification in the transgenic parasites by antibodies against the SBP tag [13]. The tagged LeishIF4E2 was amplified with primers LeishIF4E2_1-281 Forward and LeishIF4E2_1-281 Reverse, with BamHI and XbaI sites introduced at the 5’ ends of these primers (S1 Table). The BamHI/XbaI cleaved PCR product of LeishIF4E2 was cloned into the BamHI and XbaI sites of pTPuro, replacing the pre-existing LeishIF4E1-SBP that was already cloned in that plasmid between two intergenic regions derived from the HSP83 genomic locus. The resulting pTPuro-LeishIF4E2-SBP expression vector was transfected into the LeishIF4E2(+/-) mutant, and cells were selected for their resistance to puromycin (100 μg/mL).

To express the full-length LeishIF4E2-SBP_1-281 and the C-terminal truncated protein LeishIF4E2-SBP_1-217 in transgenic parasites, the two ORFs were cloned into the BamHI/XbaI sites of the pX-based vector, designed to tag the proteins at their N-terminus between two intergenic regions derived from the HSP83 genomic locus (pX-H-N-SBP-H) [13]. The two resulting vectors, pX-H-N-SBP-LeishIF4E2_1-281-H and pX-H-N-SBP LeishIF4E2_1-217-H, were transfected into WT cells that were selected for resistance to G418 (200 μg/mL G418).

Growth analysis

L. mexicana M379 WT, Cas9/T7 expressing cells, the LeishIF4E2(+/-) mutant, the LeishIF4E2 add-back, LeishIF4E2-SBP_1-281 and LeishIF4E2-SBP_1-217 cells were cultured as promastigotes at 25 ^oC in M199 medium with supplements (above). Cells were seeded at a concentration of 5 X 10⁵ cells/mL, and counted daily during 5 consecutive days. The curves were obtained from three independent repeats.

Western analysis

Cells in the mid-log phase of growth (10 mL) were harvested and washed twice with phosphate buffered saline (PBS). The cell pellet was resuspended in 300 μl of PBS supplemented with a 2 X cocktail of protease inhibitors (Sigma) and 4 mM iodoacetamide (Sigma) with (+) phosphatase inhibitors: 25 mM sodium fluoride, 55 mM β-glycerophosphate and 5 mM sodium orthovanadate. Cells were lysed by the addition of 65 μl of 5 X Laemmli sample buffer and heated at 95°C for 5 min. Cell extracts (40 μL) were resolved over 10% SDS-PAGE, blotted and probed using specific primary and secondary antibodies.

Antibodies against LeishIF4E2 (rabbit polyclonal, 1:2,000, Adar Biotech) and against the SBP tag (mouse monoclonal, 1:10,000, Millipore), were used to detect the endogenous and tagged LeishIF4E2 proteins, respectively. The blots were developed by incubation with specific peroxidase-labelled secondary antibodies against rabbit (KPL, 1:10,000 for LeishIF4E2) and mouse (KPL, 1:10,000 for SBP).

Translation assay

Global translation levels were monitored using the non-radioactive SUnSET (Surface SEnsing of Translation) assay. This assay is based on the incorporation of puromycin, an amino-acyl tRNA analog, into the A site of translating ribosomes [27]. Cells were incubated with puromycin (1 μg/mL, Sigma) for 1 hr and then washed twice with PBS. Cell pellets were resuspended in 300 μl of PBS, denatured in Laemmli sample buffer and boiled for 5 min. Cells treated with cycloheximide (100 μg/mL) prior to the addition of puromycin served as a negative control. Samples were resolved over 10% SDS-Polyacrylamide gel electrophoresis (SDS-PAGE). The gels were blotted and subjected to western analysis using monoclonal mouse anti-puromycin antibodies (DSHB, 1:1,000) and secondary peroxidase labeled anti-mouse antibodies (KPL, 1:10,000).

Phase contrast microscopy of Leishmania promastigotes

Late log-phase cells from different lines were harvested, washed in cold PBS, fixed in 2% paraformaldehyde in PBS and mounted on glass slides. Phase contrast microscope images were captured at X100 magnification with a Zeiss Axiovert 200M microscope equipped with an AxioCam HRm CCD camera.

Flow cytometry analysis of Leishmania

Cell viability was verified by incubation of the cells with 20 μg/mL propidium iodide (PI) for 30 min. The stained cells were analyzed using the ImageStream X Mark II Imaging flow cytometer (Millipore) with an X60/0.9 objective. Data from channels representing bright field, and fluorescence (PI) emission at 488 nm (to evaluate cell viability) were recorded for 20,000 cells for each analyzed sample. IDEAS software [28] generated the quantitative measurements of the focused, single live cells for all four examined cell strains. Cell shape was quantified using circularity and length features applied to the bright field image processed by an Adaptive Erode mask. Representative scatter plots are shown for single cells and for circularity (cell shape). Recorded emission of the PI in the gated population evaluated cell viability.

Measurements of flagellar length

Changes in the flagellar length for each cell were recorded using IDEAS software. A customized mask based on subtraction of the Adaptive Erode mask from the skeleton mask was created for measuring only the flagella without the cell soma. Based on this mask we created a length feature on the Brightfield Image and used it to compare flagella length between different samples (S2 Table).

Data analysis

IDEAS software [28] was used to generate the quantitative measurements of images recorded for the examined cell population. The focus quality of each cell was first determined by measuring the gradient root mean square (RMS) value. The cells with high RMS value in the histogram were gated to select cells in focus. Next, single cell populations were gated from the scatter plot of aspect ratio/area to exclude cell aggregates; the intensity of PI staining was used to exclude dead cells. The remaining living single cells in focus were subjected to image analysis to determine cell morphology. To obtain cell shape a customized Adaptive Erode mask was used on the bright field channel, with a coefficient of 78. We further customized this mask to exclude the flagellum from the cell shape analysis, to measure, circularity and elongation features. A predetermined threshold value of 4 was set to define circularity. Elongation values represent the ratio between cell length and width. Representative scatter plots are presented for focused single cells and for circularity. All data shown are from a minimum of three biological replicates.

In vitro macrophage infection assay

L. mexicana LeishIF4E2(+/-) mutants and add-back cells, WT and transgenic parasites expressing Cas9/T7, were seeded at a concentration of 5 X 10⁵ cells/ mL and allowed to grow for 5 days to reach stationary growth phase. WT, Cas9/T7 expressers, LeishIF4E2(+/-) and add-back cells were washed with DMEM (Dulbecco’s Modified Eagle Medium) and labeled by incubation with 10 μM carboxyfluorescein succinimidyl ester (CFSE) in DMEM at 25°C for 10 min. The cells were washed with DMEM, counted and used to infect RAW 264.7 macrophages. The macrophages (5 X 10⁵) were pre-seeded a day in advance in chambered slides (Ibidi) and incubated with the parasites at a ratio of 10:1 for 1 h in 300 μl medium. The cells were washed three times with PBS and once in DMEM to remove extracellular parasites. The infected macrophages were either fixed immediately for further analysis by confocal microscopy, or incubated for a further 24 h at 37°C in an atmosphere containing 5% CO₂. The infected macrophages were then processed for confocal microscopy. A single representative section of Z-projections (maximum intensity) produced by Image J software is presented in all the figures. The infectivity values were determined using the cell counting plugin in ImageJ. We first counted the number of infected cells in a total of 100 macrophages, and then counted the number of internalized parasites within the infected cells, 1 or 24 hr following infection. Statistical analysis was performed using GraphPad Prism 5. We used the non-parametric Kruskal-Wallis test to determine significant differences in the infectivity and in the average number of parasites per infected macrophage.

Confocal microscopy of Leishmania promastigotes

Infected macrophages, following 1 or 24 hr of infection were washed with PBS, fixed in 2% paraformaldehyde for 30 min, washed once with PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Nucleic acids were stained with 4′,6-diamidino-2-phenylindole (1 μg/mL DAPI, Sigma) and the cells were washed three times with PBS. The slides were observed using an inverted Zeiss LSM 880 Axio-observer Z1 confocal laser scanning microscope with Airyscan detector. Cells were visualized using Zeiss Plan-Apochromat oil objective lens of X 63 and numerical aperture of 1.4. Z-stacked images were acquired with a digital zoom of X 5.58 (X 1.8 for broad fields), using the Zen lite software (Carl Zeiss microscopy). Images were processed using Image J software package. A single representative section of the compiled Z-projections produced by Image J software is presented in all the figures.

Mass spectrometry analysis

To characterize the proteomic differences between the LeishIF4E2(+/-) mutant and the Cas9/T7 control cells we performed mass spectrometry (MS) analysis of total cell lysates. Total cell lysates from mid log stage promastigotes of Cas9/T7 and LeishIF4E2(+/-) were resuspended in a buffer containing 100 mM Tris HCl pH 7.4, 10 mM DTT, 5% SDS, 2 mM iodoacetamide and a cocktail of protease inhibitors (Sigma). Cell lysates were precipitated using 10% trichloro-acetic acid (TCA) and the pellets were washed with 80% acetone. The mass spectrometric analysis was performed by the Smoler Proteomics Center at the Technion, Israel.

Mass spectrometry (MS)

Proteins were reduced using 3 mM DTT (60°C for 30 min), followed by modification with 10 mM iodoacetamide in 100 mM ammonium bicarbonate for 30 min at room temperature. This was followed by overnight digestion in 10 mM ammonium bicarbonate in trypsin (Promega) at 37°C. Trypsin-digested peptides were desalted, dried, resuspended in 0.1% formic acid and resolved by reverse phase chromatography over a 30 min linear gradient with 5% to 35% acetonitrile and 0.1% formic acid in water, a 15 min gradient with 35% to 95% acetonitrile and 0.1% formic acid in water and a 15 min gradient at 95% acetonitrile and 0.1% formic acid in water at a flow rate of 0.15 μl/min. MS was performed using a Q-Exactive Plus Mass Spectrometer (Thermo) in positive mode set to conduct a repetitively full MS scan followed by high energy collision dissociation of the 10 dominant ions selected from the first MS scan. A mass tolerance of 10 ppm for precursor masses and 20 ppm for fragment ions was set.

Statistical analysis for enriched proteins

Raw MS data were analyzed by the MaxQuant software, version 1.5.2.8 [29]. The data were searched against the annotated L. mexicana proteins from the TriTrypDB [30]. Protein identification was set at less than a 1% false discovery rate. The MaxQuant settings selected were a minimum of 1 razor/unique peptide for identification with a minimum peptide length of six amino acids and a maximum of two mis-cleavages. For protein quantification, summed peptide intensities were used. Missing intensities from the analyses were substituted with values close to baseline only if the values were present in the corresponding analyzed sample. The log₂ of LFQ intensities [31] were compared between the three biological repeats of each group on the Perseus software platform [32], using a t-test. The enrichment threshold was set to a log₂ fold change > 0.8 and p < 0.05. The annotated proteins were categorized manually and by GO annotation (below).

Categorization of enriched proteins by the gene ontology (GO) annotation via TriTrypDB

Enriched proteins were classified by the GO Annotation tool in TriTrypDB, based on Biological Functions. The threshold for the calculated enrichment of proteins based on their GO terms was set at 3 fold, with a p <0.05. This threshold eliminated most of the general groups that represented parental GO terms. GO terms for which only a single protein was annotated were filtered out as well. In some cases, GO terms that were included in other functional terms are not shown leaving only the representative terms.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 5. Each experiment was performed independently at least three times and the individual values were presented as dots. For experiments with a higher number of repeats results are expressed as Mean ± SD. Statistical significance was determined using Wilcoxon paired t-test for matched pairs test or Kruskal-Wallis with Dunn’s multiple comparison test for comparing three or more groups. Significant P values were marked with stars (*, **, ***) representing P <0.05, P <0.01 and P <0.001, respectively.

LeishIF4E2 is a cytoplasmic protein with an extended C-terminal region

Given the multiple eIF4E paralogs in Leishmania, and the assumption that they vary in their functional assignments, we examined the sequences of the different LeishIF4E paralogs in search for non-conserved gaps and extensions. We evaluated the homology between LeishIF4E2 and the mammalian eIF4E, as well as its homology with the other LeishIF4E paralogs. Fig 1 and S1A show that LeishIF4E2 has a non-conserved C-terminal extension whereas LeishIF4E3 and LeishIF4E4 contain extended N-terminal regions. These N-terminal extensions have been widely examined for phosphorylation sites [18,33,34] and structural features [35]. The different LeishIF4Es differ from their mammalian counterpart, showing only 29.4–40.4% similarity. LeishIF4E2 from different Leishmania and Trypanosoma species are also diverged from their mammalian ortholog (Human eIF4E) (S1B and S1C Fig) and show a sequence variability among themselves as well when compared to LeishIF4E1, with 26.4–30.9% similarity (S1D Fig).

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Fig 1. Multiple sequence alignment of the LeishIF4E2.

(A)The ORFs of LeishIF4E2 from different Leishmania and Trypanosoma specis along with mammalian eIF4E1 were aligned using Jalview (2.10.5). The sequences were retrieved from L. mexicana (L. mex, LmxM.19.1480), L. major (L. maj, LmjF.19.1500); L. donovani, (L. don, LdBPK_191520.1), T. brucei (T. bruc, Tb927.10.16070) and T. congolense (T. cong, TcIL3000_0_03820). The alignment file was saved in FASTA format. The final alignment showing the predicted secondary structure of LeishIF4E2 was developed using the downloaded PDB file (5EHC, DOI: 10.2210/pdb5EHC/pdb) of Homo sapiens eIF4E1 (https://www.rcsb.org/), along with the FASTA alignment file, using the online ESPript 3 tool [60]. Secondary structure elements of the aligned sequences are (α: alpha helices, ƞ: 3₁₀-helix, β: beta-strands, TT: strict β-turns). White letters over a red background indicate identical residues and red letters over a white background indicate sequence similarity.

https://doi.org/10.1371/journal.pntd.0008352.g001

In Fig 1, the ORFs of LeishIF4E2 from different Leishmania and Trypanosoma species were aligned using Jalview (2.10.5). The final alignment also demonstrates the secondary structure which was developed using the 3D structure of Homo sapiens eIF4E1 from PDB (https://www.rcsb.org/), along with the FASTA alignment file generated using the online ESPript 3 tool. Despite the partial sequence similarities, the structural core of the eIF4E orthologs (1–217,) is conserved (Fig 1 and [36]). The predicted secondary structure of LeishIF4E2 is composed of alpha helices, beta strands and beta turns. LeishIF4E2 from all Leishmania species have an extended C-terminal region which is absent from the trypanosome orthologs (T. brucei and T. congolensi in Fig 1). The Leishmania C-terminus appears to be highly disordered as predicted by the DISOPRED2 server through the XtalPred site [37]. There are four adjacent proline residues after amino acid 217 which are known to break secondary structures. These appear within the extended C-terminus (S1A Fig).

We examined the possibility that the disordered region at the C-terminus is subject to proteolytic cleavage both in vivo and in vitro. For monitoring cleavage in vivo, we generated Leishmania lines expressing the full length LeishIF4E2_1-281 and the shorter version devoid of the C-terminal extension (LeishIF4E2_1-217), both tagged with SBP at their N-terminus, so that the protein could be detected with anti-SBP antibodies irrespective of C-terminal cleavage (S2A and S2B Fig). The transgenic cell lines expressing full length LeishIF4E2 (1–281) and the shorter version LeishIF4E2_1-217 were validated by western analysis using antibodies specific for the SBP tag (S2A Fig). Western blots of the total proteins versus the supernatant of disrupted cells (S2B Fig), show a reduced reaction with anti-SBP antibodies in both lines. However, the full-length protein (LeishIF4E2_1-281) exhibits partial degradation (S2B Fig) a feature not observed for the shorter version of LIF4E2_1-217. This reduction is compatible with protein cleavage, especially given the appearance of a smaller band in the western blots of the full-length protein. Tagging the protein at its N-terminus enabled us to visualize the bands that could represent LeishIF4E2 partially degraded from its C-terminus. LeishIF4E2_1-217 does not show any degradation like the full-length protein (LeishIF4E2_1-281), supporting our interpretation that degradation occurs at the C-terminus.

In addition to in vivo expressed LeishIF4E2, we monitored the potential cleavage of the disordered C-terminus in vitro. The full length recombinant LeishIF4E2, with an N-terminal histidine tag, was expressed in E. coli and affinity purified over a nickel column. The eluted fractions were examined by western analysis using an antibody against histidine. The affinity purified protein migrated as a major band of >25 kDa, and a minor higher band of <35 kDa. (S2C Fig, Size differences between the in vivo and in vitro assays could be due to the different tags fused to LeishIF4E2 or to post translational modifications in the different hosts). The appearance of the 25 kDa band supports our in vivo observation of the inherent instability of the C-terminal disordered region.

We further investigated the localization of LeishIF4E2 within Leishmania cells by examining transgenic parasite cells expressing the SBP-tagged LeishIF4E2 by immunohistochemical confocal microscopy. Mid-log cells (1.2 x 10⁷ cells/mL) were harvested, washed and fixed with paraformaldehyde. The fixed cells were incubated with the primary anti-SBP antibody followed by a secondary anti-mouse antibody labeled with a fluorophore (Alexa Fluor 488 green). Nuclear and kinetoplast DNA were stained with DAPI. S3 Fig shows that LeishIF4E2 is non-uniformly distributed occurring mostly in the cytoplasm.

Deletion of a single copy of LeishIF4E2 by CRISPR-Cas9

LeishIF4E2 is the least studied cap-binding protein paralog and we therefore attempted to delete its two alleles and examine how this deletion affects the parasite phenotype. Specific sgRNAs that targeted LeishIF4E2 at its 5’ and 3’ UTRs were transfected into the L. mexicana Cas9/T7 expressing line [19, 24]. The sgRNAs were designed to cleave around the target gene promoting its replacement with the G418 or Blasticidin repair fragments. The LeishIF4E2 deletion cell line was selected in the presence of G418 (200 μg/mL) and a diagnostic PCR analysis indicated that a single allele was eliminated. Attempts to delete the other copy of LIF4E2 with the Blasticidin replacement cassette did not result in viable cells.

Diagnostic PCR was performed with genomic DNA of the mutant using several primer pairs. Primers derived from the 5’ and 3’ UTRs of the LeishIF4E2 transcript were used to monitor the presence of the LeishIF4E2 gene (primers P1/P2, Fig 2A, left panel). The reaction generated two products: a 1.2 Kb product representing the endogenous LeishIF4E2 gene, and a second product of ~2.1 Kb corresponding to the integrated G418 resistance gene flanked by the 5’ and 3’ LeishIF4E2 UTRs that replaced the LeishIF4E2 gene. A similar PCR control reaction using the Cas9/T7 control gDNA as a template yielded only a single product (~1.2 Kb), representing the endogenous LeishIF4E2 gene (Fig 2A left panel). The presence of the G418 selection marker in the genome was verified by primers P3/P4 that were derived from the G418 gene and gave a 450 bp product only in the mutant of LeishIF4E2 and not in the Cas9/T7 control (Fig 2A middle panel). To validate that the G418 selection marker replaced the LeishIF4E2 gene, the integration site was amplified using primers P2/P3, P3 was derived from the G418 ORF and P2 was derived from the 3’ UTR of LeishIF4E2. This PCR product (1.4 kb) was observed only in the mutant and not in the Cas9/T7 control DNA (Fig 2A right panel). Fig 2B shows the positions of different primers used for the diagnosis PCR. The different PCR reactions confirmed the deletion of a single copy of the LeishIF4E2 gene from the genome of Leishmania cells expressing Cas9/T7.

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Fig 2. CRISPR-Cas9 mediated hemizygous deletion of LeishIF4E2.

(A) Diagnostic PCR was performed to confirm the deletion of single allele of LeishIF4E2. Genomic DNA was extracted from L. mexicana Cas9/T7 cells and from the LeishIF4E2(+/-) mutant. PCR was performed using different combinations of primers derived from the LeishIF4E2 5’ UTR (Forward) and 3’ UTR (Reverse) (P1/P2 –left panel); G418 ORF (Forward) and Reverse (P3/P4, middle panel); and a primer set derived from the G418 resistance gene, forward and the 3’ UTR (Reverse) (P3/P2). (B) Schematic representation of LeishIF4E2 locus and primers (represented by arrows). The PCR was applied to test the presence or absence of the LeishIF4E2 gene and the G418 resistance marker. Primers derived from the LeishIF4E2 UTRs are shown in blue and primers derived from the ORF of G418 are shown in red. (C) Western analysis monitoring the protein level of LeishIF4E2 in the LeishIF4E2(+/-) mutant and in Cas9/T7 control was performed using LeishIF4E2 specific antibodies. LeishIF4A-1 served as a loading control. (D) Dot plot representing the densitometry analysis of the LeishIF4E2 protein levels in the LeishIF4E2(+/-) mutant and in the Cas9/T7 controls.

https://doi.org/10.1371/journal.pntd.0008352.g002

The effect of the LeishIF4E2 hemizygous deletion on target protein expression was examined by western analysis of cell extracts obtained from the LeishIF4E2(+/-) mutant, as compared to control cells expressing Cas9/T7. The blots were reacted with LeishIF4E2 specific antibodies (Fig 2C upper panel). Antibodies against LeishIF4A served as a loading control (Fig 2C lower panel). Fig 2D shows a densitometry analysis of the LeishIF4E2 expression in the deletion mutant as compared to the Cas9/T7 control. The LeishIF4E2(+/-) mutant shows a 40% reduction in the protein levels as compared to control Cas9/T7 cells. We observed two bands in the western analysis using antibodies specific for LeishIF4E2, one at ~31 kDa and another above 25 kDa. A pattern that could fit protein cleavage was also observed with the recombinant LeishIF4E2 tagged at its N-terminus (S2C Fig) most probably due to cleavage at the C-terminus.

Expression of LeishIF4E2 was recovered in an add-back line. The LeishIF4E2(+/-) cells were transfected with a pTPuro-derived plasmid that contained the SBP-LeishIF4E2 gene flanked by HSP83 intergenic regions. The cells were selected for their resistance to puromycin, and expression of the newly introduced gene was verified by western analysis using antibodies against the SBP-tag (S4 Fig).

The LeishIF4E2(+/-) mutant shows altered promastigote morphology

Mid log phase (Day 2) promastigotes of LeishIF4E2(+/-), Cas9/T7 expressers, WT cells, and LeishIF4E2 add-back parasites were grown at 25 ^o, washed with PBS and fixed with 2% paraformaldehyde. The slides were visualized by phase contrast microscopy at 100 X magnification. The LeishIF4E2(+/-) mutant showed a defective morphology; the cells had become round, flagellum length was reduced and deviated from the typical promastigote form. Control WT and Cas9/T7 expressing cells exhibited normal promastigote features with a typical elongated shape and a long protruding flagellum. Normal promastigote morphology of elongated cells and flagellum growth were restored when expression of LeishIF4E2(+/-) was recovered in the add-back cells by episomal transfection of SBP tagged-LeishIF4E2 with pT-Puro-H-LeishIF4E2_1-281-SBP-H. Restoration of LeishIF4E2 expression led to recovery of promastigote-like cell morphology and flagellum growth nearly similar to control WT and Cas9/T7 cells (Fig 3A right panel and S5 Fig for the broad field, S6 and S7 Figs).

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Fig 3. The LeishIF4E2(+/-) mutant shows altered promastigote morphology.

(A) Mid-log phase (Day 2) promastigotes of WT, Cas9/T7 expressers, LeishIF4E2(+/-) cells, and LeishIF4E2 add-back cells were fixed with 2% paraformaldehyde and visualized by phase contrast microscopy at 100x magnification. WT, Cas9/T7 expressing cells showed normal promastigote morphology while LeishIF4E2(+/-) were round with reduced flagellar length. (B) All the cell lines were stained with 20 μg/mL propidium iodide (PI) for 30 min and cell viability was analyzed using the ImageStream X Mark II Imaging flow cytometer (Millipore). 20,000 cells were analyzed for each sample and percent viable cells were determined. (C). The circularity of single, viable and focused cells from each of the cell lines was quantified using flow cytometry, and is shown as percentage of the total number of cells measured. Data from three independent experiments are shown.

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We further used flow cytometry imaging to analyze the cell shape and viability of the cell populations as previously described [28, 38] (S6 Fig). The circularity parameter is calculated for each cell as the average radius divided by the radial variance. Typical round cells exhibit low radial variance, whereas ruffled or elongated cells have high variance of radii that results in lower circularity parameter. Cells were gated on the basis of being viable, single (non-aggregated) and focused (S6A and S6B Fig), with 20,000 cells from each group analyzed. We measured cell viability by incubating with Propidium Iodide (PI) for 30 min. The viability of the LeishIF4E2(+/-) mutant was similar to that of control cells and cell viability was comparable between all the groups tested (Fig 3B and S6A). In parallel we analyzed and quantified the changes in cell shape. In the LeishIF4E2(+/-) mutant, ~83% cells were deviated from their normal promastigote morphology being round and with a reduced and shortened flagellum. In control WT and Cas9/T7 expressing cells, only 5.14% and 11.8% were round, respectively. As expression of LeishIF4E2 was restored in the add-back cells, we noticed a recovery from their mutant phenotype, with only 35% round cells (Fig 3C and S6C Fig).

Changes in the flagellar length for each cell were also recorded using the acquisition software that was then analyzed by the IDEAS software (S7 Fig). We created the custom mask based on subtraction of the Adaptive Erode mask from the skeleton mask, thus measuring only the flagella without the cell soma. Based on this custom mask we created a length feature on the Brightfield Image and used it to compare flagella length between different samples. In WT and CAS9/T7 expressers we could identify a flagellum in 91.8–93.1% of focused cells compared with only 40.6% of the LeishIF4E2(+/-) mutants. S2 Table shows that 81.2% of the add-back cells had restored flagella. For cells in which we could identify a flagellum there was a substantial difference in the flagellum length between the LeishIF4E2(+/-) (5.50 micrometer) and the WT or Cas9/T7 control cells (12.72 and 10.19 micrometers, respectively). The flagellar length of the add-back cells recovered and attained 9.93 micrometer S2 Table.

Global translation and growth are not affected by the deletion of a single LeishIF4E2 allele

To investigate how the reduced level of LeishIF4E2 expression in the hemizygous mutant affected overall translation we used the SUnSET assay which is based on the incorporation of puromycin into the growing polypeptide chains. Puromycin is a structural analogue of amino acyl tRNA that occupies the ribosomal A site. Its integration into the polypeptide chains blocks their elongation and results in translation termination. Mid-log LeishIF4E2(+/-) mutant, control WT and Cas9/T7 expressing cells, LeishIF4E2-SBP_1-281 and LeishIF4E2-SBP_1-217 transgenic cells were incubated with puromycin (1μg/mL) for 1 hr. The cells were then harvested, extracted and resolved on SDS-PAGE. Puromycin incorporation was monitored by western analysis using anti-puromycin antibodies. A cycloheximide treated sample served as a negative control with no active puromycin incorporation.

Global translation in the LeishIF4E2(+/-) mutants was hardly affected by the hemizygous deletion as compared to control Cas9/T7 expressing cells (Fig 4A). Densitometry analysis was normalized to the total protein load (Fig 4B) and showed that translation levels in Cas9/T7 and LeishIF4E2(+/-) mutants were 48% and 44.7%, respectively, as compared to WT (100%, Fig 4C). Translation of the transgenic parasites expressing the full length and truncated proteins (1–281 and 1–217, respectively) did not alter their translation efficiency of 53.6 and 55.7%, respectively. Reduced translation in the control Cas9/T7 as compared to WT has been previously reported [15] and could be due to the competition over the use of the translation machinery between the overexpressed and endogenous proteins. S8 Fig demonstrates that the global translation in cells expressing the non-related Chloramphenicol acetyltransferase (CAT) reporter was reduced (68.8%).

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Fig 4. Global translation and growth are not altered in the LeishIF4E2(+/-) mutant cells.

(A) LeishIF4E2(+/-) cells, WT, Cas9/T7 expressing cells and transgenic parasites expressing the full length LeishIF4E2_1-281 and its truncated form devoid of the C-terminus, LeishIF4E2_1-217 were incubated with 1 μg/mL puromycin for 1 hr. Cycloheximide treated cells were used as a negative control for complete inhibition of translation. Puromycin treated cells were lysed and resolved over 12% SDS-PAGE and subjected to western analysis using antibodies against puromycin. (B) Ponceau staining was used to indicate comparable protein loads. (C) Densitometry analysis of puromycin incorporation in the different cells lines, was compared to WT cells (considered as 100%). Data from all three independent experiments are represented. (D) All cells were cultured at 25 ^oC in M199 containing essential nutrients. Cell counts were monitored daily during 4 consecutive days. The curves were obtained from three independent assays, error bars are also marked. Growth curves of WT cells are shown in yellow, Cas9/T7 are in brown, the LeishIF4E2(+/-) mutant cells are in green, cells expressing the full length LeishIF4E2 _1–281 are in purple, and the truncated LeishIF4E2_1-217 cells are in blue.

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We further monitored the growth rates of the LeishIF4E2(+/-) mutant, control WT and Cas9/T7 expressing cells, LeishIF4E2-SBP_1-281 and LeishIF4E2-SBP_1-217 transgenic cells. All lines were cultured at 25 ^oC in M199 containing essential nutrients, seeded at an initial concentration of 4 X 10⁵ cells/mL and counted on a daily basis during four consecutive days. The growth curves (Fig 4D) showed no difference between the proliferation of the LeishIF4E2(+/-) mutant cells and control lines (WT and Cas9/T7) or the transgenic lines expressing LeishIF4E2_1-281 or LeishIF4E2_1-217. This observation was in line with the results obtained in the SUnSET assay demonstrating that global translation in the LeishIF4E2(+/-) mutant cells was the same as in control cells. Overexpression of the two versions of LeishIF4E2 did not affect global translation indicating that the presumable C-terminal cleavage of LeishIF4E2 does not interfere with translation. Although additional analysis of translated transcripts is required, such a profile could be explained if LeishIF4E2 was involved in regulation of specific transcripts rather than global translation per sei.

The LeishIF4E2(+/-) mutant cells show reduced infectivity in cultured macrophages

Since the LeishIF4E2(+/-) mutant cells showed a defective morphology having short flagella and round body structure, we examined their ability to infect cultured murine macrophages using the RAW 267.4 line. Leishmania parasites from respective cell lines were pre-stained with carboxyfluorescein succinimidyl ester (CFSE), and used to infect the macrophages at a multiplicity of 10:1 parasites per macrophage. Infection lasted for 1 hr at 37°C. The macrophages were washed to remove unbound parasites and the cells were fixed with paraformaldehyde (2%) and processed for confocal analysis. DAPI staining was used to visualize the large macrophage nuclei. The infected macrophage cultures were examined by confocal microscopy either immediately (1 hr), or 24 hr post infection. These two time points allowed us to keep track of the initial entry of Leishmania and their subsequent multiplication inside the host macrophages. Infectivity of the LeishIF4E2(+/-) mutant cells was compared to that of WT and control Cas9/T7 expressing cells. We counted 100 macrophages from different fields to determine the infectivity index. The results, shown in Figs 5A and 5B, S9 Fig (for a broad field view), S10A and S10B Figs, indicate that infectivity of the LeishIF4E2(+/-) mutant was impaired, as observed at both 1 hr and 24 hrs post infection. Quantitation of the infection fields (S9 and S10 Figs and S3 Table) showed that the LeishIF4E2 mutant cells were able to infect only 48.5% of the macrophages after 1hr, with an average of 2.2 parasites per cell whereas WT and Cas9/T7 controls infected 87.8 and 91.5% of the cells, with 2.8 and 2.2 parasites per cell, respectively. The infectivity of the add-back cells was restored with 89.4% of the cells infected after 1 hr with an average of 3 parasites per macrophage (Figs 5A and 5B and S9 and S3 Table).

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Fig 5. The LeishIF4E2(+/-) mutant cells show reduced infectivity to macrophages.

Stationary phase (Day 5) L. mexicana WT, Cas9/T7 expressing cells, LeishIF4E2(+/-) mutant cells and the add-back LeishIF4E2 line were pre-stained with CFSE (green), counted, washed and used to infected RAW 264.7 macrophages, at a ratio of 10:1 for one hour. The cells were then washed to remove unattached parasites, and the macrophages were cultured for 1h (A) or 24h (B) at 37°C. Macrophage nuclei were stained with DAPI and the infected macrophage slides were processed for confocal microscopy, showing a Z-projection produced by Image J software. Fields containing 100 cells were further evaluated to quantify the infection. The scale bar represents 5 μm.

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A more pronounced difference was observed after 24 hr of infection. WT and Cas9/T7 controls infected 95–99% of the macrophages with 5.4 and 5 parasites per cell respectively, whereas the LeishIF4E2(+/-) mutant infected only 65% of the macrophages, with an average number of 1.8 parasites per cell, indicating that they did not multiply within the macrophages as in the control cells. The add-back parasites recovered their ability to infect the macrophages (97.8%) with an average of 4.8 parasites per cell showing that they recovered their ability to multiply within the macrophages. Thus, the original impaired infectivity of the mutant LIF4E2(+/-) was due to the reduced expression of the protein and once expression was restored infectivity recovered (Figs 5, S9 and S10 and S3 Table). Parasites per macrophage are represented after normalization to 1 hr uptake. This was achieved by dividing parasites per macrophages at the 24 hr time point with parasites per macrophages at 1 hr time point (S3 Table).

Proteomic analysis of the LeishIF4E2(+/-) mutant cells shows up- and down-regulation of proteins involved in specific cellular processes

To examine potential differences in the proteomic profile of LeishIF4E2(+/-) mutant we performed a MS analysis of the total cell extracts of the LeishIF4E2(+/-) mutant cells, as compared to Cas9/T7 control cells. Three independent cultures of mid-log cells were used for the proteome analysis and all samples were analyzed in the same run. The L. mexicana genome in TritrypDB was used to assign the peptides to their source proteins which were later quantified by the MaxQuant software. The proteomic comparison identified proteins that were increased relative to the Cas9/T7 control proteome (Fig 6) or decreased (S4 Table), using a threshold change of at least 1.7 fold, with p <0.05. Perseus software platform was used for the statistical analysis ([32] and S4 Table). The statistical analysis showed that 142 proteins were upregulated and 95 proteins were downregulated in LeishIF4E2(+/-) compared with Cas9/T7 control cells.

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Fig 6. The categorized proteome of the upregulated proteins in LeishIF4E2(+/-) mutant cells as compared to Cas9/T7 control cells.

The proteomic content of LeishIF4E2(+/-) and Cas9/T7 cells was determined by LC-MS/MS analysis, in triplicates. Raw mass spectrometric data were analyzed and quantified using the MaxQuant software and the peptide data were searched against the annotated L. mexicana proteins listed in TriTrypDB. The summed intensities of the peptides that served to identify the individual proteins were used to quantify changes in the proteomic content of specific proteins. Statistical analysis was done using the Perseus software. Proteins that were upregulated in the LeishIF4E2(+/-) mutant by 1.7 fold as compared to Cas9/T7 cell extracts, with p<0.05 are shown. (A) Proteins in LeishIF4E2(+/-) that were upregulated (>1.7 fold) as compared to Cas9/T7 extracts were clustered manually into functional categories. The pie chart represents the summed intensities of upregulated proteins in each category, in the LeishIF4E2(+/-) mutant. Numbers in brackets indicate the number of proteins in each category, % represent their summed relative intensity in the analysis. (B) Enriched proteins were classified by the GO enrichment tool in TriTrypDB, based on Biological Function. The threshold for the calculated enrichment of proteins based on their GO terms was set for 2.5 fold, with p<0.05. This threshold eliminated most of the general groups that represented parental GO terms. GO terms for which only a single protein was annotated were filtered out as well.

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Fig 6A and S4 Table describe the manually categorized groups of the up- and down-regulated proteins compared to Cas9/T7 control cells. The upregulated proteins related to various cellular processes involved in cell metabolism, DNA repair and replication, signaling and microtubule-based movement. The amastin-like surface antigen was also upregulated; this protein family is known to increase in amastigotes. Signaling proteins that included various kinases and phosphatases were also upregulated.

The upregulated proteins were also evaluated by the Gene Ontology (GO) enrichment analysis through the TriTrypDB platform based on their biological functions. Enrichment threshold was set at 3 fold. Fig 6B and S4 Table highlight the GO enrichment of the upregulated protein groups, each containing at least 2 proteins. In line with the manually categorized proteins, the GO enrichment analysis also show that the upregulated proteins relate to general cellular and DNA metabolism and to microtubule-based movement. We note a strong increase in proteins involved in gluconeogenesis, which are typical of amastigotes.

The manually categorized proteins that were downregulated in the LeishIF4E2(+/) mutant as compared to the Cas9/T7 control cells (S4 Table) mostly related to the flagellar rod, the cytoskeleton dyneins and a few ribosomal proteins (L22, L18, S15 and S10). Other downregulated proteins in the LeishIF4E2(+/-) proteome included signaling and metabolic proteins. A number of proteins known to be involved in parasite virulence [39–42] such as surface proteins like GP46/PSA and hydrophilic acetylated surface proteins, cysteine and metallo-peptidases were also reduced. The GO enrichment analysis for the downregulated genes highlighted cellular amino acid biosynthetic processes and cyclic nucleotide biosynthetic processes, both know to decrease in amastigotes.

The LeishIF4E2(+/-) morphology resembles axenic amastigotes

The LeishIF4E2(+/-) mutant promastigotes were small and round and had a very short flagellum (Figs 7A and S7). This morphology resembles the changes observed during differentiation to axenic-like amastigotes under normal growth conditions. We therefore examined how the mutant cells responded to conditions known to generate axenic amastigotes. L. mexicana LeishIF4E2(+/-) promastigotes along with control WT and Cas9/T7 cells were grown to late log phase and transferred to conditions that induce differentiation to axenic amastigotes in vitro (pH 5.5, 33°C) for four days. The differentiated LeishIF4E2(+/-) cells maintained a similar morphology, except that they became smaller, resembling axenic amastigote structure (Fig 7B).

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Fig 7. LeishIF4E2(+/-) mutant cells easily transform to axenic amastigote-like cells.

(A) Promastigotes of the LeishIF4E2(+/-) mutant, control WT, and Cas9/T7 expressing cells grown under normal conditions are shown. (B) Morphology of cells transferred to conditions that induce differentiation to axenic-amastigotes (33°C/pH 5.5) during four days. Images were captured at 100x magnification with a Zeiss Axiovert 200M microscope equipped with AxioCam HRm CCD camera. The scale bar is 10 μm. (C) Shared upregulated proteins in the mutant LeishIF4E2(+/-) promastigotes (compared to Cas9/T7 cells) and in published amastigotes proteome. The total protein of the LeishIF4E2(+/-) mutant promastigotes was compared to the proteome of Cas9/T7 cells. The list of upregulated proteins was further compared with the proteins enriched in the amastigote proteome of the virulent L. amazonensis PH8 strain (de Rezende et al, PLOS NTD 2017) and of L. mexicana amastigotes (Paape et al, Mol Cell Prot 2008). L amazonensis gene IDs were converted to L. mexicana, for the sake of comparison.

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We compared the repertoire of proteins that were enriched in the LeishIF4E2(+/-) mutant promastigotes with the published proteome specific to amastigotes [43,44]. This comparison highlighted eight overlaps between the proteins that were upregulated in the LeishIF4E2(+/-) mutant and in amastigotes (Fig 7C and S5 Table), as reported previously [45,46]. The shared proteins included enzymes required for gluconeogenesis, such as the phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase. These two enzymes are key players in the gluconeogenesis pathways which are active in the amastigote stage of Leishmania and are required for the intracellular survival of the parasite within mammalian host cells [47]. We also noticed that the aldo-keto reductase was upregulated in LeishIF4E2(+/-). Aldo-keto reductase is suggested to have a role in the removal of the ketoaldehyde metabolites derived from lipids and trioses [46]. Other proteins upregulated in LeishIF4E2(+/-) included cystathionine beta-lyase (methionine biosynthesis) and signaling kinases like mitogen-activated protein kinase. Enzymes involved in lipid metabolism pathways were shown to increase in Leishmania amastigotes, as these change their energy source on entering mammalian macrophages [48]. Fewer overlaps were observed for down-regulated proteins. These included the peptidyl-prolyl cis-trans isomerase, putative, a protein implicated in protein folding [49]. Downregulation of a poly zinc finger protein 2 of unknown function was also shared.