Gene Fusion Analysis in the Battle against the African Endemic Sleeping Sickness

<div><p>The protozoan <i>Trypanosoma brucei</i> causes African Trypanosomiasis or sleeping sickness in humans, which can be lethal if untreated. Most available pharmacological treatments for the disease have severe side-effects. The purpose of this analysis was to detect novel protein-protein interactions (PPIs), vital for the parasite, which could lead to the development of drugs against this disease to block the specific interactions. In this work, the Domain Fusion Analysis (Rosetta Stone method) was used to identify novel PPIs, by comparing <i>T. brucei</i> to 19 organisms covering all major lineages of the tree of life. Overall, 49 possible protein-protein interactions were detected, and classified based on (a) statistical significance (BLAST e-value, domain length etc.), (b) their involvement in crucial metabolic pathways, and (c) their evolutionary history, particularly focusing on whether a protein pair is split in <i>T. brucei</i> and fused in the human host. We also evaluated fusion events including hypothetical proteins, and suggest a possible molecular function or involvement in a certain biological process. This work has produced valuable results which could be further studied through structural biology or other experimental approaches so as to validate the protein-protein interactions proposed here. The evolutionary analysis of the proteins involved showed that, gene fusion or gene fission events can happen in all organisms, while some protein domains are more prone to fusion and fission events and present complex evolutionary patterns.</p></div

Phylogenetic trees showing examples of the four categories of evolutionary events.

<p>These trees show examples of all the gene evolutionary events observed in this study: A) Gene fusion event detected in <i>Cryptococcus neoformans</i>; this represents a unique fusion event which most likely happened before the diversification of unikonts. B) Gene fusion event detected in <i>Methanobrevibacter smithii</i>; this represents a unique fission event that probably occurred in the <i>Eukaryotes</i> superkingdom. C) Gene fusion detected in <i>Caenorhabditis elegans</i>; this represents a multiple gene event, including gene fusions and gene fissions. D) Gene fusion detected in <i>Oryza Sativa</i>; this was classified as a non-conclusive gene event, as there was not enough sequencing data to support any hypothesis regarding specific gene fusion or fission events. The colored dots along the tree branches represent the state of the protein in each lineage, based on BLAST analysis. <i>Red</i>: the protein pair is separate (two different proteins), <i>Green</i>: the protein pair is fused, <i>Blue</i>: only one part of the fused protein is conserved, either the first or the second member of the protein pair, <i>Grey</i>: Absence of either proteins, or not enough data to conclude the presence of the protein pair. The highlighted oval shape indicates the species in which the fusion protein was identified. For a full phylogenetic profile of every result in this study, please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068854#pone.0068854.s006" target="_blank">Table S6</a>.</p

Details of the organisms used in this study.

<p>For each species, the database used as the source of the data is shown, the strain that the data corresponds to, as well as the number of genes estimated for each genome, and the number of protein sequences annotated for each proteome at the specific database source. Pathogenic organisms are indicated by (p) and the disease they cause is shown. Finally, the number of fusion events detected by the SAFE software and verified in this study is given.</p

Evolutionary relationships of the organisms used in this study.

<p>The selected organisms represent all major eukaryotic and prokaryotic lineages along the tree of life. Colored boxes in the different tree nodes correspond to the colors used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068854#pone-0068854-t001" target="_blank">Table 1</a>. Species names shown in grey were examined in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068854#pone.0068854-Dimitriadis1" target="_blank">[5]</a> based on which we have excluded the Rhizaria from the analysis as there is not enough sequence data and the Amoebozoa as the two completed available genomes have already been analyzed for fusion events by this method. The tree is based on the model proposed by Dacks and Field <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068854#pone.0068854-Dacks1" target="_blank">[49]</a>; the tree is schematic i.e. the order of the branching events delineating organismal relationships is retained, but distances are not drawn to scale for clarity of presentation.</p

Selected gene fusions identified in this study.

<p>Schematic alignment of the <i>T. brucei</i> protein pair with the fused protein in another organism, showing the amino acid positions that delineate the beginning and end of the alignment, relative to the full protein length, as well as the % identity and the E-value given by BLAST for each alignment. Panel A: G6PD-6PGL Bifuctional enzyme fusion, detected in <i>P. falciparum</i>. Panel B: NAD oxidoreductase fusion detected in <i>O. sativa</i>. Panel C: Centromere binding protein -nucleolar protein fusion, detected in <i>S. hellenicus</i>. Panel D: Peptide Methionine Sulfoxide Reductase (PMSR) fusion, detected in <i>B. anthracis</i>. Panel E: Protein kinase ck2 regulatory subunit - hypothetical protein fusion, detected in <i>O. sativa</i>. Panel F: CHORD-SGT1 domains fusion, detected in <i>A. fumigatus</i>. Further details are discussed in the text.</p

Immunofluorescence vessel imaging.

<p>(a) Representative photomicrographs of the matrigel implants containing eGFP-UCB ECFC derived cells and SS-AF-MSCs. (b-e) Representative photomicrographs of matrigel implant sections containing non eGFP tagged UCB ECFC derived cells and SS-AF-MSCs after staining with (b) DAPI (blue), (c) hCD31 (green), (d) following tomato lectin perfusion (red), and (e) mCD31 (white). (f) Co-localization of eGFP (green) with hCD31 (white) staining in matrigel implant sections containing eGFP-UCB ECFC derived cells and SS-AF-MSCs. (g) Representative photomicrographs of matrigel implant sections containing SS-AF-MSCs only stained for hCD31 (green) and mCD31 (white) antigens, but where hCD31 was not detected. (h) Representative photomicrographs of matrigel implant sections containing UCB ECFC derived cells only, stained for hCD31 (green) and mCD31 (white) antigens.</p

Role of IL-8, PDGF-AB/BB and MMP9 molecules from conditioned media in migration, proliferation and ability of tubule formation of UCB ECFC derived cells.

<p>(a) Histograms showing the migration of UCB ECFC derived cells towards SS-AF-MSC-conditioned medium (CM), EGM-2 medium, control medium (EBM-2, 0.5% (v/v) FCS), SS-AF-MSC-CM+IL8 neutralizing Ab, SS-AF-MSC-CM +PDGF-AB/BB neutralizing Ab or SS-AF-MSC-CM+MMP9 inhibitor (inh). Values are means ± S.D. for three independent experiments (*p<0.05 Student’s <i>t</i> test). (b) Examination of the proliferation rate in vitro of UCB ECFC derived cells under the same conditions. Control medium with recombinant (rec) IL-8 or PDGF-AB/BB was also included. Values are means ± S.D. for three independent experiments, (*p<0.05 Student’s <i>t</i> test). (c) In vitro angiogenesis matrigel assay for UCB ECFC derived cells under the respective conditions for estimation of the number of (i) tubules and (ii) junctions formed. Error bars indicate S.D. of the mean for 10 (5x) photographs from each group (*p<0.05 Student’s <i>t</i> test).</p

Analysis of angiogenic factors secreted by UCB ECFC derived cells in vitro.

<p>(a) Representative proteome profiler array for UCB ECFC derived cell-CM; (b) corresponding names of each molecule within the array summarized in tabular form; (c) Relative expression levels of angiogenic factors in UCB ECFC derived cell-CM. Values are normalized to positive controls. Values are means ± S.D. for three independent experiments, (*p<0.05 Student’s <i>t</i> test).</p

Analysis of angiogenic factors secreted by SS-AF-MSCs, BM-MSCs and hDFs in vitro using proteome arrays.

<p>(a-c) Representative proteome profiler arrays for (a) SS-AF-MSCs, (b) BMSCs and (c) hDFs respectively; (d) corresponding names of each molecule within the array summarized in tabular form.</p

Quantitating vessel formation in in vivo studies.

<p>(a) Histological evaluation of vessels containing SS-AF-MSCs and UCB ECFC derived cells, harvested 14 days post-implantation and stained (i) with hematoxylin/eosin and for (ii-iii) human CD31 antigen (brown stain). High-power view of a vessel containing red blood cells (arrowed) from (ii) is shown in (iii). (b) Microvessel density in matrigel implants containing combined SS-AF-MSCs, BMSCs or hDFs with UCB ECFC derived cells, SS-AF-MSCs only, BMSCs only, hDFs only or UCB ECFC derived cells only. Vessel number (vessels/mm²) was estimated using Image J 1.38× software. Statistical analysis was performed using Student’s <i>t</i> test. (c) Vessel diameter estimation in matrigel implants containing SS-AF-MSCs, BMSCs or hDFs and UCB ECFC derived cells, SS-AF-MSCs only, or UCB ECFC derived cells only using Image J 1.38× software, (*p<0.05 Student’s <i>t</i> test). A minimum of 15 fields of view (40x) were analyzed from each photograph. Error bars indicate S.D. of the mean for 10 photographs from each group.</p