Comparative Genomic Analysis of Multi-Subunit Tethering Complexes Demonstrates an Ancient Pan-Eukaryotic Complement and Sculpting in Apicomplexa

<div><p>Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs), factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for <i>Guillardia</i><i>theta</i> and <i>Bigelowiella</i><i>natans</i>. The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate <i>Perkinsus marinus</i>. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during, and possibly pre-dating, the move from free-living marine algae to deadly human parasites.</p> </div

Comparative genomic survey of selected MTC proteins across the diversity of eukaryotes.

<p>The analysis demonstrates the conserved nature of the newly described TRAPPII subunit Tca17/TRAPPC2L, the CORVET complex, and the Dsl1 complex. In this and subsequent Coulson plots, filled pie sectors indicate an identified homologue, while unfilled indicate that no homologue was identified. Asterisks indicate that Vps39 is also present in these taxa (See Table S2). Infinity symbols represent organisms with divergent peroxisomes, or in which peroxisomes have not been identified. Colour-coding is arbitrary and for visual purposes only.</p

Phylogenetic analysis of Vps3, Vps39, and Vps41 proteins from select taxa.

<p>This figure demonstrates the separation of select Vps proteins from taxa spanning the breadth of eukaryotic diversity into distinct clades. Colour-coding is arbitrary and for visual purposes only.</p

Phylogenetic analysis separating Vps3 family proteins from Vps39.

<p>This figure demonstrates that the duplication into Vps3 and 39 occurred early in eukaryotic evolution and that both are widely retained proteins. The best Bayesian topology is shown and values for critical nodes are shown in the order of Bayesian posterior probabilities, PhyML bootstrap and RAxML bootstrap values. All other nodes are symbolized as inset.</p

Peroxisomal machinery genes in selected organisms.

<p>Black circles indicate the gene is present. Grey circles indicate multiple isoforms.</p

Spliceosomal introns in <i>Blastocystis</i>.

<p>(A) Sequences flanking the predicted exon–intron junctions in subtype (ST) 1 were aligned separately for each intron category and visualized with WebLogo3 (<a href="http://weblogo.threeplusone.com/" target="_blank">http://weblogo.threeplusone.com/</a>). The category and number (<i>N</i>) of each spliceosomal intron type are shown on the right. (B) Distribution of intron size in 3 sequenced <i>Blastocystis</i> ST genomes. Data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003769#pbio.2003769.s032" target="_blank">S11 Data</a>.</p

Randomized Axelerated Maximum Likelihood (RAxML) tree of kinases identified from <i>Blastocystis</i> subtypes (STs) 1, 4, and 7.

<p>Blue lines correspond to proteins from ST1, black from ST4, and red from ST7. Kinase families are indicated on the periphery.</p

Conserved polyadenylation motif sites (TGTTTGTT) in the genomes of <i>Blastocystis</i> ST1, ST4, and ST7.

<p>Conserved polyadenylation motif sites (TGTTTGTT) in the genomes of <i>Blastocystis</i> ST1, ST4, and ST7.</p

A comparison of unique genes between <i>Blastocystis</i> subtype (ST) pairs to pairs of protistan pathogens.

<p>The percentage of an organism’s protein-coding gene set, which is unique when compared to another organism’s protein-coding gene set and vice versa, is denoted by the width of the ribbon between the 2 as well as being indicated on the ribbon. For example, in a comparison between ST7 and ST1, 20% of the genes in ST7 are not represented in the ST1 set, while 10% of ST1's genes are not found in ST7. Comparisons are based on BLASTp results with an expect value (e-value) threshold of 1e-30 and >50% coverage of the query. <b>Abbreviations:</b> <i>C</i>., <i>Cryptosporidium</i>; <i>L</i>., <i>Leishmania</i>; <i>T</i>., <i>Theileria</i>. Plots were generated using Circos.</p

Maximum likelihood phylogenetic tree of Miro protein sequences.

<p>The tree was calculated using Randomized Axelerated Maximum Likelihood (RAxML). Bootstrap support values above 50% are shown at branches. For simplicity, clades comprising sequences from major monophyletic groups of eukaryotes were collapsed, displaying in detail only the clade of sequences from stramenopiles. Note the 3 Miro paralogs that have apparently emerged from <i>Blastocystis</i>-specific gene duplications before the divergence of the ST1, ST4, and ST7 lineages (gene identifiers of the <i>Blastocystis</i> Miro sequences are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003769#pbio.2003769.s025" target="_blank">S4 Data</a>).</p