Comparative Proteomic Analysis of Lysine Acetylation in Trypanosomes

Protein acetylation is a post-translational modification regulating diverse cellular processes. By using proteomic approaches, we identified N-terminal and ε-lysine acetylated proteins in <i>Trypanosoma cruzi</i> and <i>Trypanosoma brucei</i>, which are protozoan parasites that cause significant human and animal diseases. We detected 288 lysine acetylation sites in 210 proteins of procyclic form, an insect stage of <i>T. brucei</i>, and 380 acetylation sites in 285 proteins in the form of the parasite that replicates in mammalian bloodstream. In <i>T. cruzi</i> insect proliferative form we found 389 ε-lysine-acetylated sites in 235 proteins. Notably, we found distinct acetylation profiles according to the developmental stage and species, with only 44 common proteins between <i>T. brucei</i> stages and 18 in common between the two species. While K-ac proteins from <i>T. cruzi</i> are enriched in enzymes involved in oxidation/reduction balance, required for the parasite survival in the host, in <i>T. brucei</i>, most K-ac proteins are enriched in metabolic processes, essential for its adaptation in its hosts. We also identified in both parasites a quite variable N-terminal acetylation sites. Our results suggest that protein acetylation is involved in differential regulation of multiple cellular processes in Trypanosomes, contributing to our understanding of the essential mechanisms for parasite infection and survival

Volunteer gene expression trends visualized as heatmaps.

<p>Gene expression trends are shown for two differentially expressed BTMs and one gene set linked to parasitemia. Gene-wise expression levels of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199392#pone.0199392.g004" target="_blank">Fig 4</a> DE modules are visualized as heatmap. Column color bars indicate grouping of subjects into early (red), average (green) and late (blue) group, based on time point of parasitemia detection. Each row corresponds to one gene.</p

DE genes determined by limma pairwise visit comparison.

<p>DE was pronounced at a BH-adjusted p-value < 0.05 and >1.5 fold expression change. (a) DE genes (red: up-regulated genes, blue: down-regulated genes) identified for each tested contrast are visualized as bars. The number of DE genes per contrast is indicated and additionally emphasized by the length of the bars. The bar width / x-axis indicates the log2 fold expression change of each DE gene. The Venn diagrams display the overlaps between (b) all DE genes of contrasts 5/0 and 9/5, (c) the 5/0 up-regulated and 9/5 down-regulated DE genes and (d) the 5/0 down-regulated and 9/5 up-regulated DE genes.</p

Image_2_Longitudinal immune profiling after radiation-attenuated sporozoite vaccination reveals coordinated immune processes correlated with malaria protection.pdf

BackgroundIdentifying immune processes required for liver-stage sterilizing immunity to malaria remains an open problem. The IMRAS trial comprised 5x immunizations with radiation-attenuated sporozoites resulting in 55% protection from subsequent challenge.MethodsTo identify correlates of vaccination and protection, we performed detailed systems immunology longitudinal profiling of the entire trial time course including whole blood transcriptomics, detailed PBMC cell phenotyping and serum antigen array profiling of 11 IMRAS radiation-attenuated sporozoite (RAS) vaccinees at up to 21 timepoints each.ResultsRAS vaccination induced serum antibody responses to CSP, TRAP, and AMA1 in all vaccinees. We observed large numbers of differentially expressed genes associated with vaccination response and protection, with distinctly differing transcriptome responses elicited after each immunization. These included inflammatory and proliferative responses, as well as increased abundance of monocyte and DC subsets after each immunization. Increases in Vδ2 γδ; T cells and MAIT cells were observed in response to immunization over the course of study, and CD1c+ CD40+ DC abundance was significantly associated with protection. Interferon responses strongly differed between protected and non-protected individuals with high interferon responses after the 1st immunization, but not the 2nd-5th. Blood transcriptional interferon responses were correlated with abundances of different circulating classical and non-classical monocyte populations.ConclusionsThis study has revealed multiple coordinated immunological processes induced by vaccination and associated with protection. Our work represents the most detailed immunological profiling of a RAS vaccine trial performed to date and will guide the design and interpretation of future malaria vaccine trials.</p

Volunteer gene expression trends visualized as boxplots.

<p>Gene expression trends are shown for two differentially expressed BTMs and one gene set linked to parasitemia. Boxplots with gene-wise baseline-subtracted expression values are shown separately for subjects with early (red), average (green) and late (blue) detection of blood stage parasitemia.</p

GSEA using camera (limma), visualized as heatmap.

<p>Statistical significance is pronounced at a p-value and FDR < 0.05. Red: up-regulated, blue: down-regulated.</p

Image_1_Longitudinal immune profiling after radiation-attenuated sporozoite vaccination reveals coordinated immune processes correlated with malaria protection.pdf

BackgroundIdentifying immune processes required for liver-stage sterilizing immunity to malaria remains an open problem. The IMRAS trial comprised 5x immunizations with radiation-attenuated sporozoites resulting in 55% protection from subsequent challenge.MethodsTo identify correlates of vaccination and protection, we performed detailed systems immunology longitudinal profiling of the entire trial time course including whole blood transcriptomics, detailed PBMC cell phenotyping and serum antigen array profiling of 11 IMRAS radiation-attenuated sporozoite (RAS) vaccinees at up to 21 timepoints each.ResultsRAS vaccination induced serum antibody responses to CSP, TRAP, and AMA1 in all vaccinees. We observed large numbers of differentially expressed genes associated with vaccination response and protection, with distinctly differing transcriptome responses elicited after each immunization. These included inflammatory and proliferative responses, as well as increased abundance of monocyte and DC subsets after each immunization. Increases in Vδ2 γδ; T cells and MAIT cells were observed in response to immunization over the course of study, and CD1c+ CD40+ DC abundance was significantly associated with protection. Interferon responses strongly differed between protected and non-protected individuals with high interferon responses after the 1st immunization, but not the 2nd-5th. Blood transcriptional interferon responses were correlated with abundances of different circulating classical and non-classical monocyte populations.ConclusionsThis study has revealed multiple coordinated immunological processes induced by vaccination and associated with protection. Our work represents the most detailed immunological profiling of a RAS vaccine trial performed to date and will guide the design and interpretation of future malaria vaccine trials.</p

Table_1_Longitudinal immune profiling after radiation-attenuated sporozoite vaccination reveals coordinated immune processes correlated with malaria protection.xlsx

BackgroundIdentifying immune processes required for liver-stage sterilizing immunity to malaria remains an open problem. The IMRAS trial comprised 5x immunizations with radiation-attenuated sporozoites resulting in 55% protection from subsequent challenge.MethodsTo identify correlates of vaccination and protection, we performed detailed systems immunology longitudinal profiling of the entire trial time course including whole blood transcriptomics, detailed PBMC cell phenotyping and serum antigen array profiling of 11 IMRAS radiation-attenuated sporozoite (RAS) vaccinees at up to 21 timepoints each.ResultsRAS vaccination induced serum antibody responses to CSP, TRAP, and AMA1 in all vaccinees. We observed large numbers of differentially expressed genes associated with vaccination response and protection, with distinctly differing transcriptome responses elicited after each immunization. These included inflammatory and proliferative responses, as well as increased abundance of monocyte and DC subsets after each immunization. Increases in Vδ2 γδ; T cells and MAIT cells were observed in response to immunization over the course of study, and CD1c+ CD40+ DC abundance was significantly associated with protection. Interferon responses strongly differed between protected and non-protected individuals with high interferon responses after the 1st immunization, but not the 2nd-5th. Blood transcriptional interferon responses were correlated with abundances of different circulating classical and non-classical monocyte populations.ConclusionsThis study has revealed multiple coordinated immunological processes induced by vaccination and associated with protection. Our work represents the most detailed immunological profiling of a RAS vaccine trial performed to date and will guide the design and interpretation of future malaria vaccine trials.</p

DataSheet_1_Longitudinal immune profiling after radiation-attenuated sporozoite vaccination reveals coordinated immune processes correlated with malaria protection.zip

BackgroundIdentifying immune processes required for liver-stage sterilizing immunity to malaria remains an open problem. The IMRAS trial comprised 5x immunizations with radiation-attenuated sporozoites resulting in 55% protection from subsequent challenge.MethodsTo identify correlates of vaccination and protection, we performed detailed systems immunology longitudinal profiling of the entire trial time course including whole blood transcriptomics, detailed PBMC cell phenotyping and serum antigen array profiling of 11 IMRAS radiation-attenuated sporozoite (RAS) vaccinees at up to 21 timepoints each.ResultsRAS vaccination induced serum antibody responses to CSP, TRAP, and AMA1 in all vaccinees. We observed large numbers of differentially expressed genes associated with vaccination response and protection, with distinctly differing transcriptome responses elicited after each immunization. These included inflammatory and proliferative responses, as well as increased abundance of monocyte and DC subsets after each immunization. Increases in Vδ2 γδ; T cells and MAIT cells were observed in response to immunization over the course of study, and CD1c+ CD40+ DC abundance was significantly associated with protection. Interferon responses strongly differed between protected and non-protected individuals with high interferon responses after the 1st immunization, but not the 2nd-5th. Blood transcriptional interferon responses were correlated with abundances of different circulating classical and non-classical monocyte populations.ConclusionsThis study has revealed multiple coordinated immunological processes induced by vaccination and associated with protection. Our work represents the most detailed immunological profiling of a RAS vaccine trial performed to date and will guide the design and interpretation of future malaria vaccine trials.</p

Evaluation of the genetic differentiation between isolates of <i>T. evansi</i> and <i>T. equiperdum</i> and subspecies of <i>T. brucei</i> using principal component analysis (PCA) of microsatellite data.

<p>PCA was performed in R using the package adegenet. Within subspecies of <i>T. brucei</i>, the differentiation between temporally and geographically cohesive subgroups was estimated using DEST and calculated with the program smogd. Points representing individual genotypes are connected by a line to the centroid of an ellipse, which circumscribes a region encompassing 95% of the variance observed within six trypanosome subgroups identified: major <i>T. evansi</i> and <i>T. equiperdum</i> group (grey and pink), <i>T. b. rhodesiense</i> (red), <i>T. b. brucei</i> Kiboko (dark blue), <i>T. b. brucei</i> non-Kiboko (light blue), <i>T. b. gambiense</i> group 1 (dark green), <i>T. b. gambiense</i> group 2 (light green). <i>T. evansi</i> and <i>T. equiperdum</i> isolates that fell outside the major group are shown as black data points. The wide distribution of <i>T. evansi</i> and <i>T. equiperdum</i> isolates among distinct subgroups strongly supports multiple independent origins for these dyskinetoplastic strains. The first two principal components (PC1 and PC2) explain 29.2% and 8.4% of the total variance in the data, respectively. Data for isolates other than <i>T. evansi</i> and <i>T. equiperdum</i> had been published previously <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003404#pntd.0003404-Balmer1" target="_blank">[40]</a>.</p