Multiple and frequent trypanosomatid co-infections of insects: the Cuban case study.

Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas/Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri, which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.

Unexpectedly high diversity of trypanosomes in small sub-Saharan mammals.

The extremely species-rich genus Trypanosoma has recently been divided into 16 subgenera, most of which show fairly high host specificity, including the subgenus Herpetosoma parasitizing mainly rodents. Although most Herpetosoma spp. are highly host-specific, the best-known representative, Trypanosoma lewisi, has a cosmopolitan distribution and low host specificity. The present study investigates the general diversity of small mammal trypanosomes in East and Central Africa and the penetration of invasive T. lewisi into communities of native rodents. An extensive study of blood and tissue samples from Afrotropical micromammals (1528 rodents, 135 shrews, and five sengis belonging to 37 genera and 133 species) captured in the Central African Republic, Ethiopia, Kenya, Malawi, Mozambique, Tanzania, and Zambia revealed 187 (11.2%) trypanosome-positive individuals. The prevalence of trypanosomes in host genera ranged from 2.1% in Aethomys to 37.1% in Lemniscomys. The only previously known trypanosome detected in our dataset was T. lewisi, newly found in Ethiopia, Kenya, and Tanzania in a wide range of native rodent hosts. Besides T. lewisi, 18S rRNA sequencing revealed 48 additional unique Herpetosoma genotypes representing at least 15 putative new species, which doubles the known sequence-based diversity of this subgenus, and approaches the true species richness in the study area. The other two genotypes represent two new species belonging to the subgenera Ornithotrypanum and Squamatrypanum. The trypanosomes of white-toothed shrews (Crocidura spp.) form a new phylogroup of Herpetosoma, unrelated to flagellates previously detected in insectivores. With 13 documented species, Ethiopia was the richest region for trypanosome diversity, which corresponds to the very diverse environments and generally high biodiversity of this country. We conclude that besides T. lewisi, the subgenus Herpetosoma is highly host-specific (e.g., species parasitizing the rodent genera Acomys and Gerbilliscus). Furthermore, several newly detected trypanosome species are specific to their endemic hosts, such as brush-furred mice (Lophuromys), dormice (Graphiurus), and white-toothed shrews (Crocidura).

Dynamin-like proteins in Trypanosoma brucei: A division of labour between two paralogs?

Dynamins and dynamin-like proteins (DLPs) belong to a family of large GTPases involved in membrane remodelling events. These include both fusion and fission processes with different dynamin proteins often having a specialised function within the same organism. Trypanosoma brucei is thought to have only one multifunctional DLP (TbDLP). While this was initially reported to function in mitochondrial division only, an additional role in endocytosis and cytokinesis was later also proposed. Since there are two copies of TbDLP present in the trypanosome genome, we investigated potential functional differences between these two paralogs by re-expressing either protein in a TbDLP RNAi background. These paralogs, called TbDLP1 and TbDLP2, are almost identical bar a few amino acid substitutions. Our results, based on cell lines carrying tagged and RNAi-resistant versions of each protein, show that overexpression of TbDLP1 alone is able to rescue the observed endocytosis and growth defects in the mammalian bloodstream form (BSF) of the parasite. While TbDLP2 shows no rescue in our experiments in BSF, this might also be due to lower expression levels of the protein in this life stage. In contrast, both TbDLP proteins apparently play more complementary roles in the insect procyclic form (PCF) since neither TbDLP1 nor TbDLP2 alone can fully restore wildtype growth and morphology in TbDLP-depleted parasites.

Trypanosome Letm1 protein is essential for mitochondrial potassium homeostasis.

Letm1 is a conserved protein in eukaryotes bearing energized mitochondria. Hemizygous deletion of its gene has been implicated in symptoms of the human disease Wolf-Hirschhorn syndrome. Studies almost exclusively performed in opisthokonts have attributed several roles to Letm1, including maintaining mitochondrial morphology, mediating either calcium or potassium/proton antiport, and facilitating mitochondrial translation. We address the ancestral function of Letm1 in the highly diverged protist and significant pathogen, Trypanosoma brucei. We demonstrate that Letm1 is involved in maintaining mitochondrial volume via potassium/proton exchange across the inner membrane. This role is essential in the vector-dwelling procyclic and mammal-infecting bloodstream stages as well as in Trypanosoma brucei evansi, a form of the latter stage lacking an organellar genome. In the pathogenic bloodstream stage, the mitochondrion consumes ATP to maintain an energized state, whereas that of T. brucei evansi also lacks a conventional proton-driven membrane potential. Thus, Letm1 performs its function in different physiological states, suggesting that ion homeostasis is among the few characterized essential pathways of the mitochondrion at this T. brucei life stage. Interestingly, Letm1 depletion in the procyclic stage can be complemented by exogenous expression of its human counterpart, highlighting the conservation of protein function between highly divergent species. Furthermore, although mitochondrial translation is affected upon Letm1 ablation, it is an indirect consequence of K(+) accumulation in the matrix.

Both human ferredoxins equally efficiently rescue ferredoxin deficiency in Trypanosoma brucei.

Ferredoxins are highly conserved proteins that function universally as electron transporters. They not only require Fe-S clusters for their own activity, but are also involved in Fe-S formation itself. We identified two homologues of ferredoxin in the genome of the parasitic protist Trypanosoma brucei and named them TbFdxA and TbFdxB. TbFdxA protein, which is homologous to other eukaryotic mitochondrial ferredoxins, is essential in both the procyclic (= insect-transmitted) and bloodstream (mammalian) stage, but is more abundant in the active mitochondrion of the former stage. Depletion of TbFdxA caused disruption of Fe-S cluster biogenesis and lowered the level of intracellular haem. However, TbFdxB, which is present exclusively within kinetoplastid flagellates, was non-essential for the procyclic stage, and double knock-down with TbFdxA showed this was not due to functional redundancy between the two homologues. Heterologous expressions of human orthologues HsFdx1 and HsFdx2 fully rescued the growth and Fe-S-dependent enzymatic activities of TbFdxA knock-down. In both cases, the genuine human import signals allowed efficient import into the T. brucei mitochondrion. Given the huge evolutionary distance between trypanosomes and humans, ferredoxins clearly have ancestral and highly conserved function in eukaryotes and both human orthologues have retained the capacity to participate in Fe-S cluster assembly.

Morphology, ultrastructure and life cycle of Vitrella brassicaformis n. sp., n. gen., a novel chromerid from the Great Barrier Reef.

Chromerida are photoautotrophic alveolates so far only isolated from corals in Australia. It has been shown that these secondary plastid-containing algae are closely related to apicomplexan parasites and share various morphological and molecular characters with both Apicomplexa and Dinophyta. So far, the only known representative of the phylum was Chromera velia. Here we provide a formal description of another chromerid, Vitrella brassicaformis gen. et sp. nov., complemented with a detailed study on its ultrastructure, allowing insight into its life cycle. The novel alga differs significantly from the related chromerid C. velia in life cycle, morphology as well as the plastid genome. Analysis of photosynthetic pigments on the other hand demonstrate that both chromerids lack chlorophyll c, the hallmark of phototrophic chromalveolates. Based on the relatively high divergence between C. velia and V. brassicaformis, we propose their classification into distinct families Chromeraceae and Vitrellaceae. Moreover, we predict a hidden and unexplored diversity of the chromerid algae.