Taxonomic distribution and sequence properties of <i>ESAG</i> gene families in African trypanosomes.

<p>Note: <i>ESAG</i> family size is given for non-ES linked (i.e. core or subtelomeric) copies.</p>a<p>Plus and tilde symbols indicate the presence of orthologs and homologs respectively in <i>T. brucei</i> (Tb), <i>T. congolense</i> IL3000 (Tco), <i>T. vivax</i> Y486 (Tv) and <i>T. cruzi</i> CL Brener (Tc).</p>b<p>While the closest relatives of <i>ESAG2</i> are in <i>T. congolense</i>, orthologs cannot be precisely defined among the <i>T. congolense VSG</i> repertoire.</p>c<p>Reciprocal monophyly is confirmed where mutually exclusive clades of ES-linked and non-ES gene copies occur.</p>d<p>Average pair-wise sequence divergence estimated from Bayesian phylogenies using RAxML 7.0.4. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002121#pntd.0002121-Stamatakis1" target="_blank">[108]</a>.</p>e<p>Estimated using MEGA v5 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002121#pntd.0002121-Tamura1" target="_blank">[53]</a> where appropriate out-groups are present; the number of unique differences per lineage given is given in brackets.</p>f<p>Estimated using PhiPack <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002121#pntd.0002121-Bruen1" target="_blank">[54]</a> for separate alignments of <i>ESAGs</i> and related, non-ES gene copies (where non-ES copies are present).</p

A Cell-surface Phylome for African Trypanosomes

<div><p>The cell surface of <i>Trypanosoma brucei</i>, like many protistan blood parasites, is crucial for mediating host-parasite interactions and is instrumental to the initiation, maintenance and severity of infection. Previous comparisons with the related trypanosomatid parasites <i>T. cruzi</i> and <i>Leishmania major</i> suggest that the cell-surface proteome of <i>T. brucei</i> is largely taxon-specific. Here we compare genes predicted to encode cell surface proteins of <i>T. brucei</i> with those from two related African trypanosomes, <i>T. congolense</i> and <i>T. vivax</i>. We created a cell surface phylome (CSP) by estimating phylogenies for 79 gene families with putative surface functions to understand the more recent evolution of African trypanosome surface architecture. Our findings demonstrate that the transferrin receptor genes essential for bloodstream survival in <i>T. brucei</i> are conserved in <i>T. congolense</i> but absent from <i>T. vivax</i> and include an expanded gene family of insect stage-specific surface glycoproteins that includes many currently uncharacterized genes. We also identify species-specific features and innovations and confirm that these include most expression site-associated genes (<i>ESAGs</i>) in <i>T. brucei</i>, which are absent from <i>T. congolense</i> and <i>T. vivax</i>. The CSP presents the first global picture of the origins and dynamics of cell surface architecture in African trypanosomes, representing the principal differences in genomic repertoire between African trypanosome species and provides a basis from which to explore the developmental and pathological differences in surface architectures. All data can be accessed at: <a href="http://www.genedb.org/Page/trypanosoma_surface_phylome" target="_blank">http://www.genedb.org/Page/trypanosoma_surface_phylome</a>.</p> </div

Phylogeny and expression of a <i>T. brucei</i>-specific, VSG-like hypothetical protein (Fam1).

<p><b>A</b>. Fam1 consists of five, non-identical tandem gene copies at a strand-switch region on chromosome 5, which is unique to <i>T. brucei</i>. <b>B</b>. A Bayesian phylogram estimated from a multiple nucleotide sequence alignment of 1068 characters. The tree is midpoint-rooted. Nodes are supported by posterior probability values and non-parametric bootstraps generated from a maximum likelihood analysis using a GTR+G model. <b>C</b>. Immunofluorescence analysis of Tb927.6.1310 expression in <i>T. brucei</i>. Tb927.6.1310 was N-terminally HA epitope-tagged and expressed in bloodstream-form cells. Cells expressing HA::Tb927.6.1310 were loaded with FITC-concanavalin-A in serum-free media and incubated at either 4°C (upper panel) or 12°C (lower panel). ConA is restricted to the flagellar pocket at the lower temperature, whereas it is transported to, and trapped within, Rab5A positive early endosomes at 12°C. Columns in each panel (from left to right); fluorescent stain of HA epitope tag (red); FITC-ConA fluorescence (green); merged images for fluorescence; merged images from phase and fluorescence. DAPI-stain of the nucleus and kinetoplast is shown in blue. HA::Tb927.6.1310 co-localizes with ConA at the flagellar pocket (indicated with arrow head) and early endosomes (indicated with a whole arrow).</p

Expression of a Fam50 gene (Tb927.7.380) in <i>T. brucei</i>.

<p><b>A</b>. Western blot; 2×10⁷ cells were sampled from either induced (+) or uninduced (−) cells after 1-day induction with 1 µg/mL tetracycline. Markers shown in kDa. <b>B</b>. Immunofluorescence analysis of Tb927.7.380 expression in <i>T. brucei</i>, showing co-staining of non-permeabilised cells with plasma membrane protein VSG 221 (<i>top</i>) and PFR2 (<i>bottom</i>). <i>Left</i>: Merged images with color combination for DAPI-stain of the nucleus and kinetoplast (blue), fluorescent stain of HA epitope tag (red) and VSG/PFR fluorescence (green). <i>Right</i>: merged pictures from phase and fluorescence. <i>Inset</i>: co-staining of non-permeabilised and permeabilised cells with Rab11 (intracellular marker). No fluorescence was seen from non-transfected cells (data not shown). GFP expressed from pDEX-577 vector localised to the cytoplasm (data not shown). Scale bar is 2 µm.</p

Bayesian phylogeny of transferrin receptor-like genes in <i>T. brucei</i> and <i>T. congolense</i> (Fam14/15).

<p>The phylogeny was estimated from an amino acid sequence alignment of 342 characters including all <i>ESAG6</i>-like proteins from <i>T. brucei</i> 927 (light blue) and <i>T. congolense</i> IL3000 (green), as well as <i>ESAG6/7</i> sensu stricto from <i>T. brucei</i> 927, 427 and <i>T. b. gambiense</i> 972 (dark blue). A mixed amino acid substitution strategy was applied with default settings using MrBayes v3.2.1. The phylogeny is rooted using an outgroup of two a-type VSG protein sequences from <i>T. brucei</i>. Bayesian posterior probability/non-parametric bootstrap values are provided for selected nodes. Terminal nodes that describe sequences derived from tandem pairs in <i>T. congolense</i> are labeled with common symbols. Terminal nodes representing Procyclin-Associated Genes (<i>PAG1</i>, <i>2</i>, <i>4</i> and <i>5</i>) are numbered. Throughout, the presence or absence of predicted GPI anchor signals is noted using + and − respectively.</p

The taxonomic distribution of gene families in the cell-surface phylome, displayed in a Venn diagram.

<p>Phylogenies for all families are available through GeneDB. Each circle represents a family (i.e. >3 gene copies). The label in each circle refers to the description key, while size reflects the number of genes it contains; for large families the absolute number is shown in parentheses. For families present in multiple species, a pie chart is shown indicating relative gene numbers. The three tabs attending each species domain show the number of single-copy genes, pairs and triplets also predicted to have cell surface roles and to be species-specific (e.g. 101 singletons in <i>T. brucei</i>).</p

Multidimensional scaling (ms) plots of the genotype data by ethnicity (upper plot) and phenotype (lower plot).

<p>The plots show no evidence of clustering either by ethnicity or phenotype.</p

Positions of SNP genotyped within IL4.

<p>SNP with suggestive associations (uncorrected p <0.05) are indicated with a star.</p

Loci with significant associations with HAT.

<p>Loci with significant associations with HAT.</p

Association tests between cases (n = 99) and controls (n = 100).

<p>Association tests between cases (n = 99) and controls (n = 100).</p