Research Article: Genome and Phylogenetic Analyses of Trypanosoma evansi Reveal Extensive Similarity to T. brucei and Multiple Independent Origins for Dyskinetoplasty

Date Published: January 8, 2015

Publisher: Public Library of Science

Author(s): Jason Carnes, Atashi Anupama, Oliver Balmer, Andrew Jackson, Michael Lewis, Rob Brown, Igor Cestari, Marc Desquesnes, Claire Gendrin, Christiane Hertz-Fowler, Hideo Imamura, Alasdair Ivens, Luděk Kořený, De-Hua Lai, Annette MacLeod, Suzanne M. McDermott, Chris Merritt, Severine Monnerat, Wonjong Moon, Peter Myler, Isabelle Phan, Gowthaman Ramasamy, Dhileep Sivam, Zhao-Rong Lun, Julius Lukeš, Ken Stuart, Achim Schnaufer, Christian Tschudi. http://doi.org/10.1371/journal.pntd.0003404

Abstract: Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

Partial Text: Trypanosomatid parasites Trypanosoma evansi and T. equiperdum are responsible for animal diseases with extensive pathological and economic impact and closely related to the T. brucei group [1], [2]. The latter includes three subspecies: the human parasite T. b. rhodesiense, the zoonotic parasite T. b. gambiense, and the animal parasite T. b. brucei. Together T. brucei, T. evansi, and T. equiperdum comprise the subgenus Trypanozoon. The exact nature of the phylogenetic relationship between these three species has been the subject of ongoing debate, with some evidence suggesting that T. evansi and T. equiperdum are monophyletic and other evidence suggesting that they are polyphyletic and have emerged multiple times from T. b. brucei[3]–[6]. Trypanosomatids are a family within the protist group Kinetoplastida, the eponymous feature of which is a large and complex network of circular DNAs (kinetoplast or kDNA) inside their single mitochondrion. Two key biological features distinguish T. evansi and T. equiperdum from the T. brucei group. Firstly, their transmission is independent from the tsetse fly as obligatory vector. T. evansi is predominantly transmitted by biting flies and causes surra in a wide variety of mammalian species (the name of the disease varies with geographical area), while T. equiperdum causes a sexually transmitted disease called dourine in horses [1], [3], [7]. The altered mode of transmission has enabled both parasites to escape from the sub-Saharan tsetse belt and become the pathogenic trypanosomes with the widest geographical distribution. Secondly, all strains of T. evansi and T. equiperdum investigated so far are dyskinetoplastic, i.e., lacking all (akinetoplastic) or critical parts of their kDNA [8]. The loss of kDNA is thought to lock T. evansi and T. equiperdum in the bloodstream life cycle stage, presumably because the absence of kDNA-encoded components of the oxidative phosphorylation system prevents ATP generation in the tsetse midgut [9]. Nonetheless, whether dyskinetoplasty preceded the switch to tsetse-independent transmission or vice versa is unresolved [8], [10], [11].

We sequenced and analyzed the genome of T. evansi strain STIB805, a representative of a large, clonal group of close relatives of T. brucei that are transmitted independently of tsetse flies. Our examination revealed a number of insights into the biology and phylogenetic origin of this akinetoplastic trypanosome and provides further support for the proposed classification of T. evansi and T. equiperdum as subspecies of T. brucei[4]. A comparison with the T. b. brucei TREU 927/4 reference shows broad similarity, with 94.6% of non-repetitive CDS having a nucleotide identity of 95% or greater and 78.9% having ≥99% identity (Fig. 2). This high degree of identity mirrors similarities previously observed between T. b. brucei TREU 927/4 and T. b. gambiense DAL972 (86.4% of CDS had ≥99% identity) [33]. Phylogenetic analyses of T. evansi STIB805 VSG sequences also show extensive similarity to T. b. brucei. A cladistic representation of VSG sequences from T. evansi and T. b. brucei shows these sequences broadly interspersed with little evidence for subspecies-specific grouping, which underscores the close evolutionary relationship of the two strains (S10 Fig.). Despite the lack of kDNA and the absence of the life cycle stages in the insect vector, where the T. b. brucei mitochondrion is exclusively active in oxidative phosphorylation, T. evansi has maintained the coding capacity for nearly all of the mitochondrial proteome found in T. b. brucei. To some extent this reflects the fact that many mitochondrial activities are expected to remain essential even in an akinetoplastic bloodstream trypanosome. Such activities include the glycine cleavage complex [65], the alternative oxidase [66] and ubiquinone biosynthesis [67], fatty acid metabolism [68], [69], the F1-ATP synthase and ADP/ATP carrier [20], iron sulfur cluster biosynthesis [70], and all activities required to maintain and duplicate the mitochondrion itself. Nonetheless, a focused analysis of those genes known to be involved in kDNA maintenance or expression, or in oxidative phosphorylation, identified not a single case of gene loss and very few cases of mutations with predicted consequences for protein function. Of the 35 kDNA replication and transcription CDS examined, only the mitochondrial DNA polymerase beta-PAK appeared to be functionally compromised (S6 Table). Similarly, all 133 of the CDS identified as the mitochondrial ribosome in T. b. brucei[62], [71] are maintained in T. evansi, with the largest difference observed being a relatively small region of TevSTIB805.11_01.4800 (Tb927.11.4650) that alters 24 amino acids in the middle of the coding sequence. The paucity of disruptions among such a large protein complex strongly suggests that the mitochondrial ribosomes in T. evansi STIB805 are fully functional despite the lack of known substrates. Editosome function also appears essentially intact in T. evansi. Previous experiments have shown that dyskinetoplastic T. b. brucei and another strain of T. evansi contain functional editing complexes, which is consistent with retention of proteins no longer required [72].

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http://doi.org/10.1371/journal.pntd.0003404

 

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