Background: Although thousands of clinical isolates of Plasmodium falciparum are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15 P. falciparum isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal var genes clusters.   Results: The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates.  We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa. Conclusions: These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions.

The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen Pf EMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against Pf EMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized Pf EMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P . falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding Pf EMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P . falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express Pf EMP1 variants unrecognized by the host’s immune system, thus promoting overall parasite survival in the face of host immunity.

All mitochondrial tRNAs in Trypanosoma brucei derive from cytosolic tRNAs that are in part imported into mitochondria. Some trypanosomal tRNAs are thiolated in a compartment-specific manner. We have identified three proteins required for the thio modification of cytosolic tRNA Gln , tRNA Glu , and tRNA Lys . RNA interference-mediated ablation of these proteins results in the cytosolic accumulation non-thio-modified tRNAs but does not increase their import. Moreover, in vitro import experiments showed that both thio-modified and non-thio-modified tRNA Glu can efficiently be imported into mitochondria. These results indicate that unlike previously suggested the cytosolspecific thio modifications do not function as antideterminants for mitochondrial tRNA import. Consistent with these results we showed by using inducible expression of a tagged tRNA Glu that it is mainly the thiolated form that is imported in vivo. Unexpectedly, the imported tRNA becomes dethiolated after import, which explains why the non-thiolated form is enriched in mitochondria. Finally, we have identified two genes required for thiolation of imported tRNA Trp whose wobble nucleotide is subject to mitochondrial C to U editing. Interestingly, downregulation of thiolation resulted in an increase of edited tRNA Trp but did not affect growth.Most protozoa, many fungi, plants, and a few animals lack a variable number of mitochondrial tRNA genes. It has been shown in these organisms that the missing genes are compensated for by import of a small fraction of the corresponding cytosolic tRNAs (1, 2). Among all organisms that import tRNAs trypanosomatids such as Trypanosoma brucei and Leishmania are extreme in that their mitochondrial genomes have lost all tRNA genes. Trypanosomatids therefore need to import the entire set of mitochondrial tRNAs. As a consequence all mitochondrial tRNAs in trypanosomatids derive from eukaryotic-type cytosolic tRNAs that need to function in the context of the bacterial-type translation system of mitochondria (3). Two tRNAs, the initiator tRNA Met and the tRNA Sec , are cytosol-specific. It has been shown that in T. brucei the tRNA import specificity is mediated by binding to cytosolic translation elongation factor 1a (4). The fact that initiator tRNA Met and tRNA Sec do not bind to elongation factor 1a therefore explains their exclusive cytosolic localization.The extent of mitochondrial localization of different trypanosomal tRNAs varies by at least an order of magnitude (5, 6). The same is true for other organisms that import tRNAs (7). Cells therefore need to determine both the tRNA import specificity as well as the extent of mitochondrial localization of each imported tRNA species. How this is achieved is not known in any species.In trypanosomatids mitochondrial tRNAs and their cytosolic counterparts derive from the same nuclear genes. However, due to compartment-specific post-transcriptional nucleotide modifications, the cytosolic and the corresponding imported tRNAs are often physically different. Mitochondria-specifi...

The duration of intrahost replication influences in vitro var gene transcript patterns. Differences between premosquito and postpatient cultures decrease with prolonged in vitro growth.