Five-layer systems analysis of Leishmania stage differentiation reveals an essential role for protein degradation in parasite development
- Institut Pasteur, Université Paris Cité, INSERM 1347, Unité de Parasitologie moléculaire et Signalisation, France
- Institut Pasteur, Université Paris Cité, CNRS UAR 2024, Proteomic Platform, Mass Spectrometry for Biology Unit, France
- Institut Pasteur, Université Paris Cité, Biostatistics and Bioinformatics Hub, France
- Glasgow Centre for Parasitology, University of Glasgow, United Kingdom
- Weizmann Institute of Science, Department of Chemical and Structural Biology, Israel
- Bar-Ilan University, Mina and Everard Goodman Faculty of Life Sciences and Advanced and Nanotechnology Institutee, Israel
- Université Paris-Saclay, CEA, Centre National de Recherche en Génomique Humaine, France
Figures
Figure 1 with 1 supplement
Comparative genomic analysis of L. donovani lesion-isolated amastigotes (ama) and derived promastigotes (pro) (N = 3 biological replicates).
(A) Schematic overview of the parasite samples used for the various systems-level analyses presented in this study. A more detailed overview is shown in Figure 1—source data 1. (B–D) Genomic analyses. (B) Heatmap showing the somy score of three individual differentiation experiments using hamster-isolated amastigotes (ama1, ama2, and ama3) and their corresponding, culture-derived promastigotes analyzed at passage 2 (pro1, pro2, and pro3). Samples and chromosomes are indicated on the x- and y-axis, respectively. The somy scores were calculated as described in Materials and methods and correlate to the gray level according to the shown legend. (C) Ratio plot showing the gene coverage ratio ama vs pro (y-axis) for all genes across the 36 chromosomes (x-axis) as calculated based on median read depth normalized by the somy score. Each color represents one individual differentiation experiment using ama isolates from individual hamsters. The red dotted lines indicate the ratio values corresponding to 1.5 (upper line) and 0.5 (lower line), indicating, respectively, gain or loss of one gene copy in the ama samples. Fluctuations between 0.5 and 1.5 were not considered significant. (D) Correlation plots representing the individual (histograms on the diagonal) and pairwise (off-diagonal scatterplots) distributions of SNP frequencies for the indicated samples. Of note, only SNPs with a frequency above 10% were plotted. Due to bottleneck events, some low-frequency SNPs appear to be unique in one or the other stage as they are either ‘lost’ (filtered out) or ‘gained’ (passing the 10% cutoff) between the ama and pro samples. The absence of SNPs at 100% is explained by the heterozygous nature of the Ld1S genome.
-
Figure 1—source data 1
Table presenting the details of the samples used in this study.
Each L. donovani infected hamster is identified by the cage number, as are the hamster-derived amastigotes (ama) and corresponding promastigotes (pro).
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig1-data1-v1.xlsx
-
Figure 1—source data 2
DNA – somy score DNA – gene copy number variation (gCNV).
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig1-data2-v1.xlsx
-
Figure 1—source data 3
DNA – gene copy number variation (gCNV).
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig1-data3-v1.xlsx
-
Figure 1—source data 4
DNA – frequency of SNV.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig1-data4-v1.xlsx
Figure 1—figure supplement 1
Somy score and SNP analyses.
(A) Box plots representing the median somy score for all the chromosomes. Each box corresponds to one of three biological replicates for ama and pro as indicated in the graph. Sample identifiers are detailed in the legend of Figure 1A and Figure 1—source data 1. (B) Frequency distribution plots representing, for each biological replicate and each chromosome, a kernel density estimate of the distribution of SNP frequencies in the amastigote and promastigote sample.
Figure 2 with 1 supplement
Stage-specific transcript profiling (N = 4 biological replicates).
(A) Volcano plots (left panels) showing differential transcript abundance between ama and pro samples as assessed by RNA-seq analysis. The dotted lines indicate fold change (FC)=2 (vertical line) and p-value=0.01 (horizontal line). Transcripts with FC<2 or adjusted p-value>0.01 are represented by black dots. Transcripts with significantly increased abundance FC≥2 and adjusted p-value<0.01 in ama and pro are indicated, respectively, in dark cyan and dark gray for all transcripts (left panel) or transcripts of only coding genes (middle panel). Transcripts for non-coding genes with significantly increased abundance FC≥2 and adjusted p-value<0.01 in ama and pro are indicated in the right volcano plot (orange dots). The pie chart (right panel) depicts the percentage of transcripts for each of the categories. (B) Gene Ontology (GO) term enrichment analysis of these transcripts for the category ‘biological process’ was performed, and ‘cluster efficiency’ (blue) and ‘enrichment score’ (orange) were plotted for pro (left panel) and ama datasets (all transcripts, middle panel; transcripts for coding genes, right panel). The graphs show some of the most significantly enriched GO terms associated with a Benjamini and Hochberg (BH) p-value<0.05 that were identified using the BiNGO plug-in in Cytoscape. (C) Volcano plot corresponding to the differential expression of snoRNA between pro and ama stages. Cyan dots (ama) and gray dots (pro) represent signals with FC≥2 and adj. p-value<0.05. Black dots represent nonsignificant changes. (D) Localization of differentially modified rRNA sites on the Leishmania ribosome. The RNA modifications are indicated as space filling in the Leishmania cryo-EM structure (PDB: 8RXH). The identity of the RNA modification is defined by the label (Ψ, pseudouridine; Gm and Cm refer to guanine and cytosine methylation, respectively; numbers indicate residues). Upregulated and downregulated RNA modifications in amastigotes according to Rajan et al., 2024, are indicated in red and blue, respectively.
-
Figure 2—source data 1
RNA – transcriptomic analysis ama vs pro.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig2-data1-v1.xlsx
-
Figure 2—source data 2
RNA – snoRNAs.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
Analysis of RNA-seq data.
(A) Hierarchical clustering of the samples according to RNA-seq expression profiles. The dendrogram is built using the Ward method. The normed data was log-transformed first and at max 5000 most variable features were selected. (B) Principal component analysis. The two main components are represented. Ama and pro samples correspond to blue and orange dots, respectively.
Figure 3 with 1 supplement
Systems analysis of the stage-specific transcriptome and proteome (N = 4 biological replicates).
(A) Volcano plot (left panel) showing differential protein abundance between four ama and pro samples as assessed by label-free, quantitative proteomics analysis. The dotted lines indicate fold change (FC)=2 (vertical line) and false discovery rate (FDR)=0.01 (horizontal line). Proteins with FC<2 or FDR>0.01 are represented by light gray dots. Proteins that were reproducibly detected in only one of the two stages (considered unique) are represented by the lateral histograms plotting their relative abundance according to the iBAQ value. These proteins, together with proteins showing differential abundance of FC≥2 and adjusted p-value<0.01, are indicated for ama in dark cyan and for pro in dark gray (see Figure 3—source data 1). Gene Ontology (GO) term enrichment analysis (right panels) for the category ‘biological process’ was performed, and the resulting values for ‘cluster efficiency’ (blue) and ‘enrichment score’ (orange) were plotted for ama (middle panel) and pro (right panel). The graphs show the main GO terms identified using the BiNGO plug-in in Cytoscape and associated with a BH p-value<0.05. (B) Double ratio plot showing the log2 FC between ama and pro in transcript (x-axis) and protein (y-axis) abundances. Red dots correspond to changes in both transcript and protein abundance defined either by significant differential abundance at both levels (p-value<0.01) or by significant RNA changes (p-value<0.01) corresponding to proteins detected in only one of the two stages. The dotted lines indicate FC=2. Word cloud enrichment performed with the L. donovani LdBPK orthologs is presented for each of the four quadrants, Q1 to Q4, and a detailed GO enrichment analysis is given in Figure 3—source data 2. The font size is proportional to the number of genes per GO term, and the gray scale refers to the p-value calculated for each GO term. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) map for ribosome biogenesis. Each gene associated with expression changes indicated in Q1 to Q4 (see Figure 2C) and annotated with the GO term ‘ribosome biogenesis’ has been projected on the ribosome biogenesis KEGG map. Each gene is represented by a square with the left segment showing FC between ama and pro at transcript and the right segment showing FC at the protein level, with the differential abundance indicated by the color and its intensity (red, increase; blue, decrease). Only genes quantified in both transcriptome and proteome analyses and associated with an adjusted p-value<0.01 were considered.
-
Figure 3—source data 1
Protein – proteomic analysis ama vs pro.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig3-data1-v1.xlsx
-
Figure 3—source data 2
RNAxProtein – post-transcriptional regulation of gene expression.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig3-data2-v1.xlsx
Figure 3—figure supplement 1
Metabolomics analysis.
(A) Cluster analysis of metabolites showing differential abundance in ama and pro samples. (B) Quantification of amino acids or metabolites involved in glycolysis, TCA cycle, and mannogen cycle. The y-axis represents the intensity for each metabolite as detected by MS. (C–D) Correlation between metabolomic and proteomic analyses for TCA cycle (C) and glycolysis/gluconeogenesis (D). Fold changes in protein abundance (squares) and metabolite abundance (circles) with adjusted p-values of, respectively, <0.01 and <0.05 were projected on the respective Kyoto Encyclopedia of Genes and Genomes (KEGG) maps. The color intensity reflects the fold change value as indicated by the legends in the graphs. (E) Representation of the mannogen pathway. Metabolites are represented by a square, with red-lined squares corresponding to the metabolites that accumulate at the ama stage. Proteins are represented by a circle, with fold changes in protein abundance between ama and pro datasets indicated by color and intensity (red, increase; blue, decrease).
Figure 4 with 4 supplements
Analysis of stage-specific proteasomal protein turnover (N = 4 biological replicates).
(A) Overview of experimental workflow. To assess early and late steps of differentiation (left part of the graph in black), comparative, quantitative proteomics analyses were performed on tissue-isolated amastigotes (ama), amastigotes at 18 hr into the transition to promastigotes in culture (ama-18h) and fully differentiated promastigotes after one passage in vitro (pro). To gain insight into stage-specific and constitutive protein degradation (right part of the graph in red), amastigotes and promastigotes were treated with the irreversible inhibitor lactacystin (ama-lacta and pro-lacta). (B) Microscopic images of amastigotes at 18 hr in the absence of lactacystin treatment (amastigotes, left), after 48 hr in the absence ((-) lacta) or the presence of the inhibitor ((+) lacta). (C) Western blot analysis. Protein extracts were obtained from 2×106 parasites after 3, 6, 18, and 48 hr of differentiation from amastigotes to promastigotes in the presence (+) or the absence (-) of lactacystin. Extracts were separated by electrophoresis, transferred onto PVDF membrane and the presence of PFR2 was revealed using an anti-PFR2 antibody (upper panel). A Coomassie blue stain of the same gel is shown as loading control (lower panel). (D) Double ratio plot comparing the log2 fold changes (FC) in protein abundance between ama-18hr/ama-lacta (x-axis) and ama/ama-lacta (y-axis). Dashed lines indicate FC = 2. (E) Projection of the proteins stabilized in the presence of the inhibitor in ama (ama-lacta, red) and pro (pro-lacta, green) onto the double ratio plot shown in Figure 3B. Note that ama-specific proteins are rescued from degradation in pro-lacta, while pro-specific proteins are rescued from degradation in ama-lacta. (F) List of the protein kinases stabilized after lactacystin treatment in pro or ama (columns lacta rescued) and either not detected in the total proteome (column ama vs pro, beige cell) or specifically quantified at the ama (red cell) or the pro (blue cell) stage. (G) Venn diagram showing the number of proteins specifically stabilized in ama or pro or stabilized in both stages after lactacystin treatment.
-
Figure 4—source data 1
Protein – global proteomic analysis of lactacystin-treated ama and pro vs untreated parasites.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig4-data1-v1.xlsx
-
Figure 4—source data 2
Protein – proteins modulated during the first 18 hr of ama to pro differentiation.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig4-data2-v1.xlsx
-
Figure 4—source data 3
Protein – protein stabilization in ama and pro in the presence of lactacystin.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig4-data3-v1.xlsx
-
Figure 4—source data 4
List of protein kinases stabilized in the presence of lactacystin.
Their corresponding orthologs in L. donovani strain LdBPK and L. mexicana are indicated, as well as the knockout phenotype as published by Baker et al., 2021.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig4-data4-v1.xlsx
-
Figure 4—source data 5
PDF file containing original western blot and SDS-PAGE for Figure 4C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig4-data5-v1.zip
-
Figure 4—source data 6
Source images of the western blot and SDS-PAGE presented in Figure 4C.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig4-data6-v1.zip
Figure 4—figure supplement 1
Label-free quantitative proteomic analyses in the presence or absence of lactacystin.
(A–E) Volcano plots representing changes in protein abundance for the different comparisons (left panels) and Gene Ontology (GO) term enrichment analyses for the indicated sample (right panels). Proteins identified by at least two peptides in at least three out of four biological replicates were considered. For volcano plots, colored dots indicate values with false discovery rate (FDR)<0.01 and fold change (FC)≥2 (see Figure 4—source data 1). The light gray dots indicate nonsignificant expression changes. The colored bars indicate unique protein identifications in one (green) or the other (orange) condition, with relative abundance indicated by the iBAQ value (left panels). Proteins quantified in only one condition or showing a significant increase in abundance (FC≥2 and adjusted p-value<0.01) were used to perform the GO analysis for the category ‘biological process’. The histogram plots show ‘cluster efficiency’ in blue and ‘enrichment score’ in orange (right panels) for a selection of GO terms identified using the BiNGO plug-in in Cytoscape and associated with a BH p-value<0.05. (A) ama vs ama-18h; (B) ama-18h vs pro; (C) ama vs ama-lacta; (D) ama-18h vs ama lacta; (E) pro vs pro-lacta.
Figure 4—figure supplement 2
Systems analyses of expression changes.
(A) Functional network enrichment analysis for proteins showing increased abundance in ama-18h compared to ama. The network was generated with the L. infantum orthologs using the STRING plug-in of the Cytoscape software package and considering the full STRING network with a confidence score cutoff of 0.4. The Gene Ontology (GO) terms associated with each protein are represented by the colored segments according to the legend. Only GO terms associated with a p-value<0.05 were considered. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) map for ribosome biogenesis showing changes in abundance protein between ama and ama-18h. Only proteins with an adjusted p-value<0.01 were considered. Differential abundance is indicated by the color and its intensity (red, increase; blue, decrease).
Figure 4—figure supplement 3
Analyses of lacatcystin-treated parasites.
(A) Viability assessment of the indicated amastigote (upper panel) and promastigote (lower panel) samples after lactacystin treatment by FACS analysis using propidium iodide or YoPro staining. (B) Microscopic images of promastigotes at 18 hr in the absence (upper image) or the presence of 10 µM of lactacystin (lower image). (C) Western blot analysis of protein extracts obtained from ama (upper image) and pro (lower image) in the presence or the absence of lactacystin treatment. 10 µg of proteins were labeled with Cy5, separated on NuPAGE 4–12%, and transferred onto PVDF membrane. Membranes were subsequently incubated with a monoclonal antibody against ubiquitin and revealed with HRP-conjugated secondary antibodies. Images of Cy5-labeled proteins (left images), ubiquitinated proteins (middle images), and an overlay of both images (right images, Cy5-labeled proteins in green, ubiquitinated proteins in red) are presented. The table shows the relative quantification after normalization using the Cy5-labeled proteins of untreated samples as a loading control. (D) Western blot analysis of protein extracts obtained at the indicated time points after in vitro differentiation from hamster-derived amastigotes to promastigotes. 10 µg of protein were separated on NuPAGE 4–12%, transferred onto PVDF membrane, subsequently incubated with antibodies against paraflagellar rod proteins 1 and 2 and alpha-tubulin, and revealed with HRP-conjugated secondary antibodies (left image). Micrographs showing hamster-derived amastigotes during in vitro differentiation to promastigotes (right image). Parasites were collected at the time points indicated, fixed in 4% PFA, and seeded on poly-L-lysine-treated coverslips. Parasites were observed with a 63× oil immersion objective, and images were processed with AxioVision Rel.4.8 software. Differential interference contrast (DIC) microscopy was performed with Axioplan 2 imaging microscope using a 63× oil immersion objective, Axiovision software, and AxioCam MRm camera (Carl Zeiss). The scale bars correspond to 1 µm.
-
Figure 4—figure supplement 3—source data 1
PDF file containing original western blots presented in Figure 4—figure supplement 3C and D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig4-figsupp3-data1-v1.zip
-
Figure 4—figure supplement 3—source data 2
Source images of the western blots presented in Figure 4—figure supplement 3C and D.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig4-figsupp3-data2-v1.zip
Figure 4—figure supplement 4
Functional network enrichment analysis for the proteins stabilized in the presence of lactacystin in promastigote (pro) and amastigote (ama) datasets.
The network was generated with the L. infantum orthologs using the STRING plug-in of the Cytoscape software package and considering the full STRING network with a confidence score cutoff of 0.4. The Gene Ontology (GO) terms associated with each protein are represented by the colored segments according to the legend. Only GO terms associated with a p-value<0.05 were considered.
Figure 5 with 2 supplements
Stage-specific phosphoproteomic profiling (N = 4 biological replicates).
(A) Volcano plots corresponding to the total proteome (right panel) and phosphoproteome (left panel) analyses. Proteins and phosphosites (P-sites) only identified in one stage are presented at each side of the volcano plots. Cyan dots (ama) and gray dots (pro) represent signals with fold change (FC)≥2 and false discovery rate (FDR)<1%. Light gray dots represent nonsignificant changes. (B) Relative phosphorylation change normalized to protein abundance. Ratio plot comparing the log2 FC in total protein abundance between ama and pro (x-axis) and phosphosite abundance in ama vs pro (y-axis). Dashed lines indicate an FC = 2. The color intensity of each dot reflects the p-value calculated for the relative phosphorylation change normalized to protein abundance as indicated in the graph. Confidence values were derived as described in Appendix 1. (C) Gene Ontology (GO) term enrichment analysis for the category ‘biological process’. Only stage-specific phosphosites were considered (i.e. sites that showed a significant increase in relative phosphorylation normalized to protein abundance, and sites that were only detected in one or the other stage, whether the protein was identified or not in the total proteome analysis). The histogram plots show ‘cluster efficiency’ in blue and ‘enrichment score’ in orange for the GO term enrichment analysis performed with the ama (middle panel) and pro (right panel) datasets. The main GO terms identified using the BiNGO plug-in in Cytoscape and associated with a BH p-value<0.05 are shown. (D) Phosphorylation pattern of MAPK and MAPKK proteins (left panel) or ubiquitin hydrolases and transferases (right panel) identified in the relative phosphorylation change normalized to protein abundance. The positions of the phosphorylated amino acid specific to ama (red) and pro (blue) are indicated. Only phosphopeptides identified in the relative phosphorylation change normalized to phosphorylation level (see Figure 5—source data 2) and detected either in one stage or the other or quantified with an FC≥2 were considered for the identification of the phosphosites.
-
Figure 5—source data 1
Phosphoprotein – phosphoproteomic analysis of ama vs pro.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig5-data1-v1.xlsx
-
Figure 5—source data 2
Phosphoprotein – stage-specific relative phosphorylation changes.
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig5-data2-v1.xlsx
-
Figure 5—source data 3
Phosphoprotein – stage-specific functional networks (STRING).
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig5-data3-v1.xlsx
Figure 5—figure supplement 1
Upset plot representing the number of phosphopeptides (y-axis, blue histograms, numbers are indicated) and the samples in which they were quantified (x-axis).
Pie chart of the phosphopeptide analysis showing the distribution of the phosphorylation sites according to the condition in which they were quantified as indicated in the legend of the graph.
Figure 5—figure supplement 2
Functional network analysis.
Networks for proteins that show unique or increased phosphorylation in ama (upper panel) and in pro (lower panel) datasets were generated with the STRING plug-in of the Cytoscape software package using L. infantum orthologs and considering the full STRING network with a confidence score cutoff of 0.4. The nodes represent phosphoproteins associated with one or more Gene Ontology (GO) terms (listed in the graph), with the respective gene identifier indicated. The GO terms associated with the proteins are represented by the colored segments around the nodes according to the legend shown in the graph. Only GO terms associated with a false discovery rate (FDR)<0.05 were considered.
Figure 6
Network analyses.
(A) Model of Leishmania gene expression regulation during stage differentiation. (B–C) Network analysis. Networks, restricted to the protein kinases (blue nodes), proteins implicated in ribosome biogenesis and ribosomal proteins (violet nodes), and proteins linked to ubiquitination/deubiquitination (orange nodes), identified based on normalized phosphorylation levels conducted in ama (B) or pro (C), were generated with the STRING plug-in of the Cytoscape software package using L. infantum orthologs and full STRING network with a confidence score cutoff of 0.4. Each node represents a phosphoprotein with the respective gene identifier indicated. Gene Ontology (GO) terms associated with the proteins are represented by the colored segments around the nodes according to the legend shown in the graph. Only GO terms associated with a p-value<0.05 were considered.
-
Figure 6—source data 1
Phosphoprotein – stage-specific interaction networks for selected phosphoproteins (STRING).
- https://cdn.elifesciences.org/articles/111115/elife-111115-fig6-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Leishmania donovani 1S2D) | L. donovani | Originally obtained from Henry Murray, Weill Cornell Medical College, New York, USA | Strain 1S2D MHOM/SD/62/1S-CL2D | Maintained in hamsters (Mesocricetus auratus) |
| Strain, strain background (L. donovani amastigotes) | Amastigotes | This paper | Prepared from hamster infected spleens | |
| Strain, strain background (L. donovani promastigotes) | Promastigotes | This paper | Derived from splenic amastigotes | |
| Antibody | Mouse monoclonal antibody anti-PFR 1+2 (clone L13D6) | Philippe Bastin, Institut Pasteur, Paris | WB (1:50) | |
| Antibody | Mouse monoclonal antibody anti-tubulin alpha (clone B-5-1-2) | Sigma | T6074 | WB (1:25,000) |
| Antibody | Mouse monoclonal antibody anti-ubiquitin (clone p4d1) | Abcam | AB303664 | WB (1:1000) |
| Antibody | Goat secondary antibody anti-mouse IgG-HRP conjugated | Invitrogen | 32230 | WB (1:10,000) |
| Antibody | Goat secondary antibody anti-rabbit IgG-HRP conjugated | Invitrogen | 31462 | WB (1:10,000) |
| Commercial assay or kit | DNeasy Blood and Tissue Kit | QIAGEN | 69504 | |
| Commercial assay or kit | Nucleospin RNA plus | Macherey Nagel | 740984 | |
| Commercial assay or kit | RCDC Protein assay Kit II | Bio-Rad | 5000122 | |
| Commercial assay or kit | Quick Stain Kit | Cytiva | RPN4000 | |
| Commercial assay or kit | rDNase set | Macherey Nagel | 740963 | |
| Commercial assay or kit | SuperSignal West Pico PLUS Kit | Thermo Scientific | 34577 | |
| Commercial assay or kit | ECL prime western blotting detection reagent | Cytiva | RPN2232 | |
| Commercial assay or kit | Quant-IT kits | Invitrogen | Q33267 (DNA) Q10213 (RNA) | |
| Commercial assay or kit | TruSeq DNA PCR-Free Library Preparation Kit | Illumina | 20015963 | |
| Commercial assay or kit | Illumina Stranded mRNA Prep | Illumina | 20040534 | |
| Chemical compound, drug | YO-PRO | Invitrogen | Y3306 | |
| Chemical compound, drug | Propidium iodide | Sigma-Aldrich | P4864 | |
| Chemical compound, drug | Lactacystin | Sigma-Aldrich | L6785 | |
| Chemical compound, drug | Protease inhibitor cocktail cOmplete | Roche | 11836170001 | |
| Chemical compound, drug | Phosphatase inhibitor cocktail PhosStop | Roche | 04906845001 | |
| Software, algorithm | Sequana | Cokelaer et al., 2017 | ||
| Software, algorithm | Sequana RNA-seq | doi: https://doi.org/10.5281/zenodo.19456198 | ||
| Software, algorithm | GIP | Späth and Bussotti, 2022 | ||
| Software, algorithm | Cytoscape | Shannon et al., 2003 | ||
| Software, algorithm | UCSF Chimera-X | Pettersen et al., 2021 |
Additional files
-
Supplementary file 1
Table for Ld1S, LdBPK, and L. infantum gene/protein IDs equivalence.
- https://cdn.elifesciences.org/articles/111115/elife-111115-supp1-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/111115/elife-111115-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Five-layer systems analysis of Leishmania stage differentiation reveals an essential role for protein degradation in parasite development
eLife 15:RP111115.
https://doi.org/10.7554/eLife.111115.3
