Table 1.
Oligonucleotide sequences.
Fig 1.
Quantitative proteomic analysis of cell death-induced L. major.
A- Volcano plot of the quantified proteome of L. major treated with 25µM miltefosine for 3 h, B- 25µM staurosporine for 6 h and C- statPh promastigotes, always compared to untreated, logPh promastigotes. A-C- Volcano plot visualized with VolcaNoseR [46], proteins with commonly increased abundance are labelled (Table 2). Cutoffs were set at log2FC < -1.5, > 1.5 and p-value < 0.01. Visible gaps in the fold change separate protein hits that revealed increased abundance in one condition but had not been detected in the other condition. In order to plot these proteins, we set an arbitrary value for each undetected protein. D- Venn diagram displaying the number of proteins with significantly increased abundance in each labeled condition versus the untreated logPh control. Intersections represent proteins detected as more abundant in several conditions. Template created using BioVenn [47]. Analysis based on 7 biological replicates and 3 technical replicates each.
Table 2.
Common proteins with increased abundance in L. major undergoing cell death; annotations extracted from UniProt [49]. All peptides detected for p1/s1 could be assigned to the conserved C-terminal region of proteins in the cluster (Fig 2A).
Fig 2.
Ecto-nucleotidase and -endonuclease activities of L. major increase in the stationary-phase.
A- Depiction of the genomic p1/s1 cluster of six genes (LmjF.30.1460 – 1510) sharing a conserved domain but encoding either a signal sequence or a transmembrane domain (TMD). Indicated 5’sgRNA and 3’sgRNA were used for CRISPR Cas9-mediated inducible knockout of the cluster. Created in BioRender. Schmelzle, S. (2026) https://BioRender.com/mlghi1j. B- Western blot to determine p1/s1-His6 localization in 12x106 L. major promastigotes overexpressing a C-terminally His6-tagged LmjF.30.1500 construct (clones H9 and F3) and a control parental strain (par.). Secreted, tagged proteins were enriched with magnetic Ni-NTA beads from the corresponding culture supernatant. a-Tubulin was used as loading control for pelleted parasites. Representative blot of n = 3. C- Superimposition of the AlphaFold-predicted structure of Q4Q7F3 (orange, encoded by LmjF.30.1510) onto the experimentally determined structure of a homologous enzyme in Arabidopsis thaliana (green; PDB ID: 3W52). The active site residues and the bound adenine base (cyan) in 3W52 are shown as sticks. Coordinated zinc ions are represented as gray spheres, and the sulfate ion trapped among the zinc ions is shown in ball-and-stick representation. D- Multiple sequence alignment of Q4Q7F8-F3, as encoded in the LmjF.30.1460-1510 cluster. The five regions corresponding to homologous active sites in homologous enzymes are highlighted in yellow boxes. E- Ecto-3’-nucleotidase activity of 2x106 intact logPh or statPh L. major and secreted enzymes in the corresponding supernatant (SN) was measured in a colorimetric assay as µM Pi released from 3’-AMP in one hour. n = 4. F- Ecto-5’-nucleotidase activity of 2x106 intact logPh or statPh L. major and secreted enzymes in the corresponding supernatant (SN) was measured in a colorimetric assay as µM Pi released from 5’-AMP in one hour. n = 3. G- Relative endonuclease activity measured by degradation of double-stranded M13 phage genome on 2x106 intact logPh or statPh L. major promastigotes. Pellet and supernatant were separated and circular, double-stranded M13 DNA (dsM13) added for 60 min. Remaining intact substrate was assessed on a 0.8% TAE-agarose gel, semi-quantified using ImageJ and normalized to intact substrate not incubated with parasites. n = 3. H- Representative gel of G, box denotes area of semi-quantification. dsM13: undigested product; Lm: control with parasites but no substrate; DNase: Substrate incubated with DNase, first and last lane: DNA ladder.
Fig 3.
Presence of adenosine or 3‘-AMP during L. majorDsRed infection of hMDM decreases TNFα secretion and PBL proliferation.
hMDM (GM-CSF+IFNγ) were infected with statPh L. majorDsRed promastigotes.3 hours post infection (pi), extracellular parasites were washed off and autologous peripheral blood lymphocytes (PBLs) stained with CellTrace Far Red were added in a 1 macrophage: 5 PBLs ratio. During infection and coculturing, adenosine (Ado) or 3’-AMP were maintained in the indicated concentrations. A- Soluble TNFα secreted from hMDM (GM-CSF + IFNγ) was determined in supernatant collected 3 hours post infection with MOI = 10 using ELISA and normalized to the infected sample without additives. n = 5-7 donors in 4 independent experiments. Absolute values available in Fig BC in S1 Appendix. B- Relative parasite burden measured in flow cytometry as DsRed mean fluorescence intensity of infected hMDM (GM-CSF + IFNγ) in a hMDM:PBL coculture and normalized to the infected control without additives, determined 4 days pi with a MOI = 20 of L. majorDsRed. Absolute values available in Fig BD in S1 Appendix. C- Percentage of proliferated PBLs in a hMDM:PBL coculture was determined as viable CellTracelow lymphocytes 4 days pi with a MOI = 20 of L. majorDsRed and normalized to the infected control without additives. Absolute values available in Fig BE in S1 Appendix. D- Pearson correlation analysis of relative PBL proliferation against relative TNFα secretion and E- relative PBL proliferation and relative parasite burden. r: Pearson correlation coefficient. B-E: n = 9-10 donors in 4 independent experiments.
Fig 4.
DiCre based inducible knockout approach yields L. major p1/s1 null mutants.
A- Schematic visualization of the inducible, dimerizable Cre (diCre) system used to generate L. majorΔp1/s1. Full floxed parasites with flox cassettes integrated up- and downstream of the p1/s1 locus (top) expressing a diCre construct were induced with rapamycin to initiate Cre-mediated excision of the p1/s1 cluster alongside the eGFP gene. Successful recombination (bottom) can be traced by the loss of eGFP signal. Created in BioRender. Schmelzle, S. (2026) https://BioRender.com/62mz7co. B, C- eGFP and mCherry signal (circles) upon diCre induction with rapamycin (+rapa) measured in flow cytometry until 9 days post induction in two p1/s1 full floxed clones E10 and F4. As controls, respective strains not induced with rapamycin (-rapa, squares) were included. D- Diagnostic PCR to detect eGFP and p1/s1 loci in genomic DNA of single cell clones derived from floxed clones E10 and E- F4 post rapamycin induction. Confirmed L. majorΔp1/s1 clones used for further experiments are marked in gray and further named as E10 and F4 induced knockouts (iKO). L. majorCas9/T7/diCre (DiCre) and induced, floxed parasites before limiting dilution (pre.dil.) and were used as controls. F- Cell numbers of viable and dead L. majorCas9/T7/diCre/3‘flox, L. majorΔp1/s1 clones E10 & F4 and L. majorp1/s1 addback were counted on day 3 (logPh) and 7 (statPh) post passaging for viable and dead parasites. All strains were grown under selection pressure of the same antibiotics and were seeded as 0.5x106 viable parasites on day 0. n = 3 of consecutive passages.
Fig 5.
L. major p1/s1 null mutants show reduced 3’-nucleotidase/nuclease activity.
A- 3‘-nucleotidase activity was measured as µM released Pi from 3‘-AMP on 2x106 total, intact L. major statPh promastigotes (L. majorCas9/T7/diCre/3‘flox L. majorΔp1/s1 clones E10 & F4 and L. majorp1/s1 addback) and the corresponding supernatant (SN). Parasite pellet and supernatant were separated and 3‘-AMP as substrate added for 1 h, Pi content was determined with a malachite green based assay. n = 4. B- Endonuclease activity measured by degradation of double-stranded M13 phage genome on 2x106 total L. major statPh promastigotes (L. majorCas9/T7/diCre/3‘flox L. majorΔp1/s1 clones E10 & F4 and L. majorpSaddback). Pellet and supernatant (SN) were separated and circular, double-stranded M13 DNA added for 90 min. Remaining intact substrate was assessed on a 0.8% TAE-agarose gel, semi-quantified using ImageJ and normalized to intact substrate not incubated with parasites (dsM13). Representative gel shown. Also included are controls containing only parasites (Lm) and dsM13 incubated with DNase. n = 3. Uncropped gel available in Fig C in S1 Appendix. C, D- Endonuclease activity on L. major pellet and supernatant (SN) fraction was determined as in B, but on single-stranded M13 phage genome (ssM13) for the stated reaction time. n = 3. E- Representative gels of C & D.
Fig 6.
L. major p1/s1 null mutants bypass loss of p1/s1 via redundant purine salvage.
A- Ecto-5’-nucleotidase activity of 2x106 intact statPh L. major promastigotes (L. majorCas9/T7/diCre, L. majorΔp1/s1 clones E10 & F4) and secreted enzymes in the corresponding supernatant (SN) was measured in a colorimetric assay as µM Pi released from 5’-AMP in one hour. n = 3. B- Principal component analysis of the quantitative proteome data generated on L. majorΔp1/s1 clones E10 & F4 and a parental strain (L. majorCas9/T7/diCre/3‘flox) by label-free mass spectrometry. C- Venn diagram showing the number of proteins with increased abundance in L. majorΔp1/s1 clones compared to their parental strain. Template created using [43]. D, E- Volcano plot of quantified protein abundance of L. majorΔp1/s1 clones E10 & F4, respectively, in comparison to their parental strain. Cutoffs were set at log2FC < -0.5, > 0.5 and p-value < 0.01. Denoted are highly abundant proteins of the purine salvage pathway listed in Table 3 Volcano plots visualized with VolcaNoseR [42]. B-E Data analysis based on n = 7 biological replicates in 3 technical repeats each.
Table 3.
Purine salvage proteins with increased abundance in both L. majorΔp1/s1 clones compared to a parental strain, ranked by p-value in E10 comparison.
Fig 7.
L. major p1/s1 null mutants do not show altered cell death hallmarks upon miltefosine treatment.
L. major logPh promastigotes (L. majorCas9/T7/diCre/3‘flox, L. majorΔp1/s1 clones E10 & F4 and L. majorp1/s1 addback) were treated with increasing concentrations of miltefosine (0.1-60µM) for 48 h and cell death hallmarks analyzed by different assays. A- Cellular metabolism of 5x106 parasites was quantified using a colorimetric MTT assay, absorbance at 540 nm was normalized to the sample containing 1.5% DMSO as carrier control; n = 3. B- Phosphatidylserine exposure on the cell surface of 2x106 parasites was stained using FITC labelled AnnexinV. Percentage of phosphatidylserine positive parasites (AnnV+) was quantified by flow cytometry; n = 3–5. C- 2x106 parasites were analyzed for fragmented DNA, as seen in the SubG1 phase, by stochiometric DNA staining using SYTOXGreen analyzed by flow cytometry; n = 3–5. D- Summarized EC50 values obtained from non-linear 4-parameter regression of all cell death assays for L. majorCas9/T7/diCre/3‘flox (par.), L. majorΔp1/s1 clones E10 & F4 and L. majorp1/s1 addback (addb.); ± 95% confidence interval.
Fig 8.
L. major p1/s1 null mutants cannot decrease PBL proliferation upon presence of 3’-AMP and show decreased degradation of neutrophil extracellular traps.
hMDM (GM-CSF + IFNγ) were infected with statPh L. majorCas9/T7/diCre/3‘flox, L. majorΔp1/s1 clones E10 or F4 promastigotes at an MOI=20. 3 hours post infection (pi), extracellular parasites were washed off and autologous peripheral blood lymphocytes (PBLs) stained with CellTrace Far Red were added in a 1 macrophage: 5 PBLs ratio for 4 days. During infection and coculturing, adenosine (Ado) or 3’-AMP were maintained at 100 µM, if applicable. Intracellular L. major were stained with α-Lm mouse serum and labelled with α-mouse-AlexaFluor647 antibody. A- L. major infection rate of macrophages in a hMDM:PBL coculture 4 days pi. The percentage of AlexaFluor647 + viable macrophages was determined in flow cytometry to give the share of infected macrophages. B- Parasite burden 4 days pi in a hMDM:PBL coculture. Parasite burden was measured as AlexaFluor647 mean fluorescence intensity of viable, infected macrophages and normalized to the control infected with parental strain. C- Percentage of proliferated PBLs in a hMDM:PBL coculture was determined as viable CellTracelow lymphocytes 4 days pi. D- Normalized share of proliferated PBLs in a hMDM:PBL coculture 4 days pi, relative to the respective strain’s infected control without adenosine or 3‘-AMP. Absolute percentage of PBL proliferation is provided in Fig FB in S1 Appendix. A-D: n = 6 donors in 3 independent experiments, Welsh’s t-test. Human primary neutrophils were used to study p1/s1 dependent degradation of neutrophil extracellular trap (NET) degradation. E- Nuclease activity measured by degradation of NET enriched supernatants on 2x106 intact L. major statPh promastigotes (P, L. majorCas9/T7/diCre/3‘flox L. majorΔp1/s1 clones E10 & F4) and the respective supernatant (SN). Pellet and supernatant were separated and 500µg NET DNA added for 2 hours. Remaining intact NET DNA scaffold was assessed on a 0.8% TAE-agarose gel, semi-quantified using ImageJ and normalized to substrate not incubated with parasites. n = 3 donors in three independent experiments. F- Representative gel of E, box indicates area used for semi-quantification with ImageJ. G- L. major statPh promastigotes (L. majorCas9/T7/diCre/3‘flox L. majorΔp1/s1 clones E10 & F4) were incubated with human neutrophils at MOI = 1, optionally in the presence of DNase, for 3 hours at 37 °C. CytochalasinD (CytD) inhibits phagocytosis of parasites into neutrophils. After further incubation at 27 °C for 2 days, motile, viable promastigotes were counted for each sample and normalized to the respective control incubated without neutrophils. n = 4–5 donors in 2–3 independent experiments.