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

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

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

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

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

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

Diversity of a-type and b-type VSG between <i>T. b. brucei</i> TREU 927/4 and <i>T. evansi</i> STIB805 compared.

<p>Histograms showing a-type VSG (A.) or b-type VSG (B.) distributions of strain-specific clade size in <i>T. b. brucei</i> (black bars) and <i>T. evansi</i> (red bars) as defined by the phylogeny (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003404#pntd.0003404.s010" target="_blank">S10 Fig</a>.). Frequency distributions of a-type VSG (C.) or b-type VSG (D.) synonymous (<i>Ks</i>) and non-synonymous (<i>Ka</i>) substitution rates per site, and the ω (<i>Ka/Ks</i>) for orthologous pairs of <i>VSG</i> (a-type n = 151; b-type n = 112), as defined by the phylogeny, in relation to values for unambiguous non-VSG orthologous pairs (n = 6331).</p

Bayesian phylogeny of <i>Trypanozoon</i> isolates based on the dihydrolipoamide dehydrogenase gene (LipDH; Tb927.11.16730).

<p>Panel A shows a mid-point rooted tree based on an alignment of 32 unique LipDH haplotypes, assembled from sequences derived from 13 <i>T. b. brucei</i> (Tbb), 3 <i>T. b. gambiense</i> type 1 (Tbg1), 4 <i>T. b. rhodesiense</i> (Tbr), 15 <i>T. evansi</i> (Tev) and 5 <i>T. equiperdum</i> (Teq) samples. Scale units for the phylogeny are substitutions per site. The chart illustrates the distribution of each haplotype among samples from each <i>Trypanozoon</i> taxon. Phylogenetic analysis grouped all but three of the haplotype sequences into one of five major clusters with strong support (posterior probabilities ≥0.9), which are referred to as clades V, W, X, Y and Z. Eight unique Tev/Teq genotypes were found, as summarized in panel B. Discounting minor sequence differences (1–2 SNPs) these were reduced to four major genotypes based on the position of haplotypes in the tree, which mirrored the type of mutation (A281Δ, M282L, A273P, WT) in the C-termini of the ATP synthase subunit γ in these the samples.</p

Pulse-field gel electrophoresis comparing chromosomes of <i>T. evansi</i> STIB805 (Tev) and <i>T. b. brucei</i> TREU 927/4 (927).

<p>While the sizes of megabase chromosomes are largely similar, differences in the intermediate and minichromosomes (825 kbp and smaller) are evident between <i>T. evansi</i> and <i>T. brucei</i>. Although <i>T. brucei</i> chromosomes I-XI were not unambiguously identified, labels to the left of the gel indicate bands consistent with the expected <i>T. brucei</i> chromosome sizes <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003404#pntd.0003404-Melville2" target="_blank">[46]</a>, as well as intermediate and minichromosomes. The signal at the top of the gel is from the well, as indicated.</p

Schematic of reads from <i>T. b. brucei</i> TREU 927/4 and <i>T. evansi</i> STIB805 mapped on to the procyclin loci of <i>T. brucei</i>.

<p>CDS are denoted by yellow arrows, with Tb927 GeneIDs above and gene names in parentheses. Reads that could be uniquely mapped to the reference are colored blue (intact paired reads), green (single forward reads) or red (single reverse reads). Reads that could be mapped to more than one position in the Tb927 reference were placed randomly and are colored yellow. Red asterisks denote mutated or absent CDS in <i>T. evansi</i>. Both PAG5 and PAG2* are missing in <i>T. evansi</i>, and mutations are present in all copies of EP2, PAG3, and GRESAG2.</p