The genome sequence of Trypanosoma brucei gambiense, causative agent of chronic human african trypanosomiasis

Abstract

Background: Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans.

Methods and findings: An improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.

Conclusions: This study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.

Figure 1. Frequency distribution of pairwise sequence divergence between 6929 single-copy gene orthologs in T. b. brucei and T. b. gambiense.

Divergence values to the right of the dashed line are statistically significant; the identities of selected divergent gene pairs are noted. An asterisk * denotes genes belonging to a T. b. brucei subspecies-specific tandem gene array (see Figure 2 and Supplementary Figure S2).

Figure 2. Segmental duplication on chromosome 9 in T. b. brucei.

A single segment in T. b. gambiense comprising three coding sequences (Tbg972.9.4160, 4140 and 4130) corresponds to a three-gene segmental duplication (5 repeats) on chromosome 9 in T. b. brucei. The first coding sequence (shaded red) is a conserved, hypothetical gene encoding a putative secretory protein and all copies are identical. The second (shaded yellow) and third (shaded orange) coding sequences are tandem-duplicate, conserved hypothetical genes encoding putative membrane-bound proteins. Both second and third genes contain substantial sequence variation in T. b. brucei; the upstream-most copies are orthologous to the T. b. gambiense genes, but none of the remaining variants were identified among T. b. gambiense sequence reads. The segmental duplication is preceded immediately upstream by an INGI-mediated insertion (shaded purple).

Figure 3. Analysis of sequence variation among T. brucei 65 kDa invariant surface glycoprotein genes.

Gene copies are numbered consecutively in positional order from left to right on the chromosome, beginning with Tbb1 (Tb927.2.3270) and Tbg1 (Tbg972.2.1130) respectively. a. Sequence variation (expressed as Shannon entropy score, left scale) along a multiple sequence alignment, combined with recombination breakpoints (red lines, right scale) inferred by GARD analysis. A dotted blue line marks the boundary between coding and 3′ UTR regions. Coloured bars above the chart indicate recombination tracts as identified by GARD. b. A phylogenetic network including sequences unique to one subspecies (marked with an asterisk *). Annotated pseudogenes are indicated by ψ. c. A chart showing the affinities of sequence Tbg7 only; all sequences are represented in a circle, coloured bars connect Tbg7 (or regions thereof) to its closest relative among other sequences. Different colours are used to denote different affinities. d. Affinity chart for Tbg1.

Figure 4. Variant surface glycoprotein (VSG) repertoire of T. b. brucei (927) represented as a three-dimensional network graph.

1258 T. b. brucei VSG protein sequences were compared using pairwise BLAST searches. BLAST scores were used to arrange VSG into a graph using BioLayout Express 3D 3.0. 968 individual VSG are represented as coloured spheres and are joined by edges to all other nodes with which they share >70% amino acid identity. The program minimizes the distance required to arrange all nodes such that related nodes are arranged closest to one another. Nodes are shaded by type: orthologous sequence in T. b. gambiense (blue), orthologous sequence in T. b. gambiense, but closest relative in T. b. brucei (green), no corresponding sequence in T. b. gambiense (red), metacyclic-stage VSG (purple) and VSG-related (VR) proteins (yellow).