LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity

Cells.Leishmania mexicana M379 cells were cultured at 25°C 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 macrophages were grown at 37°C in Dulbecco’s modified Eagle’s medium (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.

Monitoring morphological changes in cells exposed to conditions known to induce axenic differentiation and following their reversal to conditions typical of promastigote growth.Promastigotes from all L. mexicana cell lines were seeded at a concentration of 5 × 10⁵/ml and grown for 3 days to reach their late log phase of growth (the concentrations reached were ∼3.6 × 10⁷ cells/ml for the wild type, ∼3.5 × 10⁷ cells/ml for Cas9/T7, ∼1.8 × 10⁷ cells/ml for the LeishIF4E1^–/– mutant, and ∼3.5 × 10⁷ cells/ml for add-back cells). The cells were washed twice with phosphate-buffered saline (PBS) and resuspended in M199 adjusted to pH 5.5 (by the addition of 0.5 M succinic acid) and supplemented with 25% FCS, 5 μg/ml hemin, 0.1 mM adenine, 40 mM HEPES, pH 5.5, 4 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were allowed to grow and differentiate at 33°C for 4 days under gentle-shaking conditions.

To allow the transformation of L. mexicana axenic amastigotes back to promastigotes, the cells were washed twice with PBS, resuspended in the medium used for promastigote growth (M199, pH 7.4), and transferred to 25°C.

CRISPR-Cas9-mediated deletion of LeishIF4E1.Plasmids developed for the CRISPR system in Leishmania were obtained from Eva Gluenz (University of Oxford, UK) (16). The pTB007 plasmid contained the genes encoding the Streptococcus pyogenes CRISPR-associated protein 9 endonuclease and the T7 RNA polymerase gene (Cas9/T7), along with the hygromycin resistance gene. pTB007 was transfected into L. mexicana promastigotes, and transgenic cells stably expressing Cas9 and the T7 RNA polymerase were selected for hygromycin resistance (200 μg/ml).

Generation of the LeishIF4E1 deletion mutant by CRISPR-Cas9.To generate LeishIF4E1 null mutants, we used three PCR-amplified products: the two 5′ and 3′ sgRNAs designed to create double-strand breaks upstream and downstream of the LeishIF4E1 coding region and the LeishIF4E1 repair cassette fragment containing the G418 resistance marker. The three PCR products 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 (44). Thus, the LeishIF4E1^–/– line may be polyclonal.

The sgRNA sequences that were used to delete the LeishIF4E1 gene were obtained from http://leishgedit.net/ (45). The sgRNAs contained the highest-scoring 20-nucleotide sequence within 105 bp upstream or downstream of the target gene. The sequences of the sgRNAs were subjected to a BLAST search against the L. mexicana genome in TriTrypDB to verify the specificities of the sgRNAs for LeishIF4E1 (E values = 0.001 and 8e−5). We also ran a BLAST analysis with the drug resistance repair cassette that contained the sequence with homology to the UTR of LeishIF4E1 to specifically target the insertion of the selection marker. The repair cassette showed an E value of 5e−9, suggesting a very high specificity of the system. The sgRNA target sequences and the homology arms on the repair cassette fully matched the target sequence of LeishIF4E1.

PCR amplification of sgRNA templates.DNA fragments encoding LeishIF4E1-specific 5′ and 3′ guide RNAs for cleavage upstream and downstream of the LeishIF4E1 target gene were generated. The template for this PCR consisted of two fragments; one contained the common sgRNA scaffold fragment (5′-AAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3′), and the other contained the T7 RNA polymerase promoter (lowercase letters at the beginning of the sequence below) fused to the gRNA (5′ or 3′) targeting LeishIF4E1 (capital letters below) and a short sequence overlapping the scaffold fragment (lowercase letters at the end of the sequence below). The two individual template fragments for targeting a double-strand break at the 5′ end of LeishIF4E1 was (5′-gaaattaatacgactcactataggCTCTTCTTCGTTTCGCGCCAgttttagagctagaaatagc-3′), and the template fragment targeting a double-strand break at the 3′ end was (5′-gaaattaatacgactcactataggGTGTGCATATCATCTTGCTGgttttagagctagaaatagc-3′). 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, 5′-TTAATACGACTCACTATAGG-3′) and the common scaffold fragment (G00R, 5′-GCACCGACTCGGTGCCACTT-3′). The reaction mixture consisted of deoxynucleoside triphosphates (dNTPs) (0.2 mM) and HiFi polymerase (1 unit of Phusion; NEB) in buffer suitable for templates rich in GC (guanosine and cytosine) with MgCl₂ (NEB) in a total volume of 50 μl. PCR conditions included initial denaturation at 98°C for 2 min, which was followed by 35 cycles of 98°C for 10 s, annealing at 60°C for 30 s, and an 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 LeishIF4E1 replacement fragment.A DNA fragment designed to repair the double-strand breaks surrounding the LeishIF4E1 target gene was amplified by PCR. The LeishIF4E1-specific primers were derived from the 5′ and 3′ endogenous UTR sequences upstream and downstream of the LeishIF4E1 gene and the sequences from the antibiotic repair cassette, based on sequences in the LeishGEdit database (http://www.leishgedit.net/Home.html). The primers were (5′-CTAGATCATCGCCTTACGCACCCCCCTCCCgtataatgcagacctgctgc-3′ [forward]) and 5′-CACAACACGTAGACAAGCAAACATCAACCAccaatttgagagacctgtgc-3′ [reverse]). Capital letters represent the UTR sequences of LeishIF4E1, and lowercase 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 double-strand breaks on both sides of the gene targeted for deletion was performed using the pTNeo plasmid as a template. The resulting fragments enable the integration of the drug resistance marker by homologous recombination at the target site. The reaction mixture consists of 2 μM each primer, dNTPs (0.2 mM), the template (pTNeo at 30 ng), 3% (vol/vol) dimethyl sulfoxide (DMSO), and HiFi polymerase (1 unit of Phusion; NEB) in GC buffer (containing 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, which was followed by 40 cycles of 98°C for 30 s, annealing at 65°C for 30 s, and an 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 LeishIF4E1.Genomic DNA from the drug-resistant cells was isolated 14 days posttransfection using a DNeasy blood and tissue kit (Qiagen) and analyzed for the presence of the LeishIF4E1 gene, using specific primers derived from the open reading frame (ORF) of LeishIF4E1. The primers used were LeishIF4E1 forward (5′-GGATCCATGTCATCTCCATCTTCAG-3′) and LeishIF4E1 reverse (5′-TCTAGAAGACGCCTCGCCGTGCTT-3′). A parallel reaction was performed to look for the presence of the G418 resistance gene, with primers derived from its ORF: G418 F (5′-GCCCGGTTCTTTTTGTCAAGAC-3′) and G418 R (5′-GTCACGACGAGATCATCATCGCCG-3′). Genomic DNA from Cas9/T7 L. mexicana cells was used as a positive control for the presence of the LeishIF4E1 gene. The reaction mixture consisted of 2 μM each primer, genomic DNA (gDNA) (100 ng), dNTPs (0.2 mM), and HiFi polymerase (1 unit of 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, which was followed by 35 cycles of 98°C for 30 s, annealing at 60°C for 30 s, and an extension at 72°C for 2 min 15 s. A final extension was done for 7 min at 72°C. PCR products were separated over 1% agarose gels.

Generation of LeishIF4E1 add-back parasites.The transgenic LeishIF4E1^–/– deletion mutant cells were transfected with an episomal transfection vector that promoted the recovery of LeishIF4E1 expression. The plasmid was derived from pTPuro, which confers resistance to puromycin. The added-back LeishIF4E1 gene from L. mexicana was tagged with the streptavidin-binding peptide (SBP; ∼4 kDa), which enabled its further identification in the transgenic parasites by antibodies against the SBP tag (14). Tagged LeishIF4E1 was cloned between two intergenic regions derived from the HSP83 (H) genomic cluster. Stably transfected cells were selected for resistance to puromycin. The pTPuro-LeishIF4E1 plasmid was generated as follows. The ORF of LeishIF4E1 from L. mexicana was amplified using the forward 5′-ggatccATGTCATCTCCATCTTCAG-3′ and reverse 5′-tctagaAGACGCCTCGCCGTGCT-3′ primers, with BamHI and XbaI sites introduced at the 5′ ends of these primers (lowercase letters). The BamHI/XbaI PCR product was cloned into the BamHI and XbaI sites of the pX-H-SBP-H expression cassette between two intergenic regions derived from the HSP83 genomic locus (46, 47). The fragment containing the SBP-tagged LeishIF4E1 ORF and the two flanking HSP83 intergenic regions was cleaved by HindIII, blunted, and cloned into the blunted SfoI site of pT-Puro (16). The resulting pTPuro-LeishIF4E1-SBP expression vector was transfected into the LeishIF4E1^–/– deletion mutant, and cells were selected for their resistance to puromycin (200 μg/ml).

Growth analysis.L. mexicana M379 wild-type and Cas9/T7-expressing cells, along with the LeishIF4E-1^–/– deletion mutant and the LeishIF4E-1 add-back cells, were cultured as promastigotes at 25°C in M199 containing all supplements (see above). Cells were seeded at a concentration of 5 × 10⁵ cells/ml, and the cells were counted daily for five consecutive days. The curves were obtained from three independent repeats.

Western blot analysis.Cells at their mid-log phase of growth (10 ml) were harvested and washed twice with PBS and once with post-ribosomal supernatant (PRS) buffer (35 mM HEPES, pH 7.5, 100 mM KCl, 10 mM MgCl₂, 1 mM dithiothreitol [DTT]). The cell pellet was resuspended in PRS+ (300 μl), which was supplemented with a 2× cocktail of protease inhibitors (Sigma) and 4 mM iodoacetamide (Sigma), along with the following 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× Laemmli sample buffer and heated at 95°C for 5 min. Cell extracts (40 μl) were resolved by 10% SDS-PAGE, blotted, and further probed using specific primary and secondary antibodies.

Antibodies against LeishIF4E1 (rabbit polyclonal, 1:2,000) and against the SBP tag (Millipore; monoclonal, 1:10,000) were used to detect the endogenous and tagged LeishIF4E1 proteins, respectively. These were further detected by specific peroxidase-labeled secondary antibodies against rabbit (KPL; 1:10,000 for LeishIF4E1) and mouse (KPL; 1:10,000 for SBP).

Translation assay.Global translation was monitored using the SUnSET (surface sensing of translation) assay. This assay is based on the incorporation of puromycin, a tRNA analog, into the A site of translating ribosomes (48). Puromycin (1 μg/ml; Sigma) was added to cells for 30 min, which were then washed twice with PBS and once with PRS+. Cell pellets were resuspended in 300 μl of PRS+ buffer, 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 by 10% SDS-PAGE. The gels were blotted and subjected to Western blot analysis using monoclonal mouse antipuromycin antibodies (DSHB; 1:1,000) and secondary peroxidase-labeled anti-mouse antibodies (KPL; 1:10,000).

XTT assay for measuring cell metabolism.The metabolic activities of the different cells were estimated using a cell proliferation assay kit based on 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2 H-tetrazolium-5-carboxyanilide inner salt (XTT; Biological Industries, Israel). XTT is reduced by mitochondrial dehydrogenases by metabolically active cells, resulting in an orange formazan compound. L. mexicana WT and LeishIF4E1^–/– deletion mutant cells were cultivated in 96-well plates in phenol red-free M199. Reaction solution containing an activation solution and XTT reagent was added to each well, and the plate was incubated at 26°C for 6 h. Absorbance at 450 nm, with a reference at 630 nm, was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

Phase-contrast microscopy of Leishmania promastigotes.Cells from different lines in their late-log phase of growth were harvested, washed, fixed in 2% paraformaldehyde in PBS, and mounted onto glass slides. Phase-contrast microscope images were captured at a ×100 magnification with a Zeiss Axiovert 200M microscope equipped with an AxioCam HRm charge-coupled device (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 a 60/0.9× objective. Data from channels representing bright-field as well as fluorescence (PI) emission at 488 nm (to evaluate cell viability) were recorded for 20,000 cells for each analyzed sample. IDEAS software generated the quantitative measurements of the focused single and live cells for all four examined cell strains. Cell shape was quantified using circularity and elongatedness features applied on the bright-field image processed by an adaptive-erosion mask. Representative scatterplots are shown for focused single cells and for circularity (cell shape). Recorded emission of the PI in the gated population evaluated cell viability.

Data analysis.IDEAS software 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 representing high RMS values in the histogram were gated to select cells in focus. In the second step, single-cell populations were gated from the scatterplot of the aspect ratio over the area to exclude cell aggregates. Further, 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-erosion 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. Further, circularity and elongatedness features were measured. A predetermined threshold value of 4 was set to define circularity. Elongatedness values represent the ratio between cell length and width. Representative scatterplots are presented for focused single cells and for circularity. Cell viability was measured by recording the emission of PI in the gated population. All data shown are from a minimum of three biological replicates. A similar approach was taken for generating a template adapted for measuring the structural features of axenic amastigotes. Furthermore, due to the tendency of axenic amastigotes to aggregate, different cell populations were gated to obtain only single rounded cells (resembling axenic amastigotes). The gated area excluded cell aggregates, debris, and elongated promastigotes in a specific area of the scatterplot. This template was further used to quantify the single, round, amastigote-like cells in all the cell lines that were analyzed.

In vitro macrophage infection assay.L. mexicana LeishIF4E1^–/– deletion mutants and add-back cells and wild-type and transgenic parasites expressing Cas9/T7 polymerase were seeded at the concentration of 5 × 10⁵ cells/ml and allowed to grow for 5 days to reach their stationary growth phase. Parasites (WT, ∼5 × 10⁷/ml; Cas9/T7, ∼4.9 × 10⁷/ml; LeishIF4E1^–/– mutant, ∼4.4 × 10⁷/ml; add-back cells, ∼5.1 × 10⁷/ml) were washed with DMEM and labeled by incubation with 10 μM carboxyfluorescein succinimidyl ester (CFSE) in DMEM at 25°C for 10 min. The cells were then washed with DMEM, counted, and used to infect RAW 264.7 macrophages at a ratio of 10:1. The macrophages (5 × 10⁵) were preseeded a day in advance in chambered slides (Ibidi). The macrophages were incubated with the parasites for 1 h in 300 μl and then 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 further incubated for 24 h at 37°C in an atmosphere containing 5% CO₂. The infected macrophages were then processed for confocal microscopy as described below. A single representative section of Z-projections (maximum intensity) produced by Image J software is presented in all the figures. With the cell-counting plug-in in Image J, the infectivity values were determined. We first counted the number of infected cells in a total of 200 macrophages and then counted the number of internalized parasites within the infected cells 1 or 24 h following infection. Statistics were generated using GraphPad Prism 5. We used the nonparametric Kruskal-Wallis test to determine significant differences in the infectivities and in the average numbers of parasites per infected macrophage.

Confocal microscopy of Leishmania promastigotes.Following 1 or 24 h of infection, the macrophages 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. We stained nucleic acids with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml; Sigma), and finally, 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 an Airyscan detector. Cells were visualized using a Zeiss Plan-Apochromat oil lens objective of 63× and a numerical aperture of 1.4. Z-stacked images were acquired with a digital zoom of 8× (1.8× for broad fields), using the Zen lite software (Carl Zeiss microscopy). Images were processed using the 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 LeishIF4E1^–/– deletion mutant and wild-type cells, we carried out mass spectrometry analysis of total cell lysates of LeishIF4E1^–/– mutant cells. Total cell lysates from mid-log-stage promastigotes (WT, ∼3.4 × 10⁷/ml; Cas9/T7 cells, ∼3 × 10⁷/ml; LeishIF4E1^–/– cells, ∼1 × 10⁷/ml; and add-back cells, ∼3.2 × 10⁷/ml) 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. Cell lysates were precipitated using 10% trichloroacetic acid (TCA), and the pellets were washed with acetone. The mass spectrometric analysis was performed by the Smoler Proteomics Center at the Technion in Israel.

(i) Mass spectrometry.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. Mass tolerances of 10 ppm for precursor masses and 20 ppm for fragment ions were set.

(ii) Statistical analysis for enriched proteins.Raw mass spectrometric data were analyzed by the MaxQuant software, version 1.5.2.8 (49). The data were searched against the annotated L. mexicana proteins from TriTrypDB (50). Protein identification was set at less than a 1% false-discovery rate. The MaxQuant settings selected were a minimum of 1 razor per unique peptide for identification, a minimum peptide length of 6 amino acids, and a maximum of two miscleavages. For protein quantification, summed peptide intensities were used. Missing intensities from the analyses were replaced with values close to baseline only if the values were present in the corresponding analyzed sample. The log₂ values of label-free quantification (LFQ) intensities (51) were compared between the three biological repeats of each group on the Perseus software platform (17), using a t test. The enrichment threshold was set to a log₂ fold change of >1.6 and a P of <0.05. The annotated proteins were first categorized manually.

(iii) Categorization of enriched proteins by the GO annotation via TriTrypDB.Enriched proteins were classified by the GO annotation tool in TriTrypDB, based on molecular functions. The threshold for the calculated enrichment of proteins based on their GO terms was set at 2.5-fold, with a P of <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 GO term.

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