Fig 1.
Quantitative label-free mass spectrometry was used to quantify the Leishmania spp. and M. musculus proteomes at seven postinfection timepoints.
a, Three Leishmania spp. were used to infect M. musculus BMDMs in quadruplicate. Whole-cell lysates were prepared at seven different timepoints post infection and quantified via mass spectrometry. The bar plot represents the total number of M. musculus (gray) or Leishmania spp. (green, pink, and blue for L. infantum, L. major, and L. mexicana, respectively) quantified proteins in each experiment. b, Venn diagrams showing the overlap between the different Leishmania spp. OrthoMCL IDs and between the different M. musculus protein IDs obtained in the L. infantum (green), L. major (pink), and L. mexicana (blue) experiments. c, Scatter plot showing the first two components of the principal component analysis (PCA), which together explain 36.74%, 34.11%, and 34.67% of the variance in the data for the L. infantum (green), L. major (pink), and L. mexicana (blue) experiments, respectively. The amount of variance explained by each PC is indicated on each axis. The samples are represented as dots and color-coded according to a grayscale that increases in darkness on the basis of their postinfection timepoints. A 95% confidence level multivariate t distribution of each timepoint is represented as an ellipse, with colors corresponding to the grayscale used for sample representation.
Fig 2.
Proteome dynamics reveal different protein expression profiles for the three Leishmania spp. experiments.
a, Heatmap of quantified proteins for the L. infantum (green), L. major (pink), and L. mexicana (blue) experiments. Each row represents the abundance (z score [LFQ]; yellow-to-blue scale) of each quantified protein across the postinfection timepoints (grayscale). Proteins are annotated as either M. musculus (gray) or belonging to Leishmania spp. (green, pink, and blue for L. infantum, L. major, and L. mexicana, respectively). b, Scatter plot of quantified proteins for the L. infantum (green), L. major (pink), and L. mexicana (blue) experiments. Each dot represents the abundance (mean [log2]; x-axis) and dynamicity (Gini score; y-axis) of each quantified protein. The colors of the dots reflect the density distribution (bluescale). Highlighted dots (larger, blue) represent known dynamic and stable M. musculus (gray) or Leishmania spp. (green, pink, and blue for L. infantum, L. major, and L. mexicana, respectively) proteins. c, Line plot of highlighted proteins for the L. infantum (green), L. major (pink), and L. mexicana (blue) experiments. Each dotted line represents the protein abundance (mean [log2]; y-axis) across the hours postinfection (x-axis). Dotted lines are color coded using grayscale for M. musculus proteins and greenscale, pinkscale, or bluescale for L. infantum, L. major, and L. mexicana proteins, respectively.
Fig 3.
Clustering analysis revealed differences and similarities between the expression profiles of the M. musculus proteins.
a, Bar plot displaying the number of M. musculus proteins within each self-organizing map (SOM) cluster. The fractions of protein per bar from the L. infantum (green), L. major (pink), and L. mexicana (blue) experiments are indicated. b, Scatter plot showing the first two components of the principal component analysis (PCA), which together explain 81.87% of the data variance. The variance explained by each principal component is labeled on its respective axis. Protein IDs are depicted as dots and colored on the basis of their SOM cluster assignment. c, SOM cluster panel. The line plot depicts the protein abundance (z score [LFQ]; y-axis) across the hours post infection (x-axis) for each protein ID (gray) assigned to the cluster. The mean abundance of a cluster is represented by its designated color. An adjacent Venn diagram depicts the overlap between the M. musculus protein IDs from the L. infantum (green), L. major (pink), and L. mexicana (blue) experiments assigned to the cluster.
Fig 4.
Clustering analysis revealed differences and similarities between the expression profiles of the Leishmania spp. proteins.
a, Bar plot displaying the number of Leishmania spp. proteins within each self-organizing map (SOM) cluster. The fractions of L. infantum (green), L. major (pink), and L. mexicana (blue) proteins per bar are indicated. b, Scatter plot showing the first two components of the principal component analysis (PCA), which together explain 63.73% of the data variance. The variance explained by each principal component is labeled on its respective axis. Protein IDs are depicted as dots and colored on the basis of their SOM cluster assignment. c, SOM cluster panel. The line plot depicts the protein abundance (z score [LFQ]; y-axis) across the hours post infection (x-axis) for each protein ID (gray) assigned to the cluster. The mean abundance of a cluster is represented by its designated color. An adjacent Venn diagram depicts the overlap between the OrthoMCL IDs assigned to L. infantum (green), L. major (pink), and L. mexicana (blue) Leishmania spp. protein IDs in the cluster.
Fig 5.
Pairwise differential expression analysis revealed Leishmania spp. and M. musculus protein IDs with significantly modulated expression profiles.
a, Bar plot showing the number of significantly up- or downregulated Leishmania spp. (right panel) and M. musculus (left panel) proteins (p value < 0.05 and abs[log2(FoldChange)] > 1) for the L. infantum (green), L. major (pink), and L. mexicana (blue) experiments. b, Venn diagram depicting the overlap between OrthoMCL IDs assigned to the significant Leishmania spp. protein IDs (upper panel) and the significant M. musculus protein IDs (lower panel) from the L. infantum (green), L. major (pink), and L. mexicana (blue) experiments. c, Heatmap displaying the OrthoMCL IDs assigned to the significant Leishmania spp. protein IDs (upper panel) and the significant M. musculus protein IDs (lower panel) from the L. infantum (green), L. major (pink), and L. mexicana (blue) experiments. Each row represents the abundance (z score [LFQ]; yellow-to-blue scale) of each quantified protein across pairwise hours after infection (grayscale).
Fig 6.
Knockout of two upregulated L. mexicana proteins results in reduced infectivity and a distinct differentiation phenotype.
a, Fluorescent signal of endogenously tagged LmxM.28.2260 overlapping with the glycosomal marker (GAPDH) signal in both promastigotes and amastigotes (scale bar: 5 µm). b, Label-free quantification values for LmxM.28.2260 and orthologs in L. major and L. infantum show species-dependent relative protein levels throughout the infection. c, Axenic differentiation to amastigotes is affected by knockout of LmxM.28.2260 (blue square), with a cell density not exceeding 6*106 c/ml. Rescue of the knocked out gene (blue triangle) re-established the growth phenotype to WT conditions (blue circle). d, Infection experiments with ∆LmxM.28.2260 in BMDMs revealed significantly reduced infectivity compared with that of the positive control (Cas9) starting at 24 h (unpaired t test, p < 0.05). The rescue cell line partially restored the infection phenotype of the positive control after 48 h (unpaired t test, p < 0.05). A significant difference in the infection rate between the rescue and knockout cell lines was not detected after 72 h. e, The fluorescent signal of endogenously tagged LmxM.10.0130 (magenta) does not overlap with the GAPDH signal (green) in promastigotes or amastigotes (scale bar: 5 µm). The LmxM.10.0130 signal is irregularly dispersed throughout the cell body, with a relatively high intensity near the kinetoplast. f, Relative protein levels of LmxM.10.0130 were low throughout infection and increased only at 72 h. In comparison, the protein levels of PFR-2 were high until 24 h and then decreased. g, LmxM.10.0130 knockout cells are unable to differentiate under axenic conditions (blue square) when the cell density does not exceed 106 c/ml. Rescuing the knocked out gene (gray triangle) enables the cells to reestablish their differentiation phenotype to WT conditions (blue circle). h, Infection experiments with ∆LmxM.10.0130 revealed a significant difference in infection rates compared with the positive control (Cas9) at 72 h (unpaired t test, p < 0.005). The rescue cell line partially restored the infection phenotype of the positive control, which was significantly different from that of the knockout cell line.