Limitation of Trypanosoma brucei parasitaemia results from a combination of density-dependent parasite differentiation and parasite killing by the host immune response

In the bloodstream of its mammalian host, the "slender" form of Trypanosoma brucei replicates extracellularly, producing a parasitaemia. At high density, the level of parasitaemia is limited at a sublethal level by differentiation to the non-replicative "stumpy" form and by the host immune response. Here, we derive continuous time equations to model the time-course, cell types and level of trypanosome parasitaemia, and compare the best fits with experimental data. The best fits that were obtained favour a model in which both density-dependent trypanosome differentiation and host immune response have a role in limiting the increase of parasites, much poorer fits being obtained when differentiation and immune response are considered independently of one another. Best fits also favour a model in which the slender-to-stumpy differentiation progresses in a manner that is essentially independent of the cell cycle. Finally, these models also make the prediction that the density-dependent trypanosome differentiation mechanism can give rise to oscillations in parasitaemia level. These oscillations are independent of the immune system and are not due to antigenic variation

Comparative genomics and concerted evolution of β-tubulin paralogs in Leishmania spp

BACKGROUND: Tubulin isotypes and expression patterns are highly regulated in diverse organisms. The genome sequence of the protozoan parasite Leishmania major contains three distinct β-tubulin loci. To investigate the diversity of β-tubulin genes, we have compared the published genome sequence to draft genome sequences of two further species L. infantum and L. braziliensis. Untranscribed regions and coding sequences for each isoform were compared within and between species in relation to the known diversity of β-tubulin transcripts in Leishmania spp. RESULTS: All three β-tubulin loci were present in L. infantum and L. braziliensis, showing conserved synteny with the L. major sequence, hence confirming that these loci are paralogous. Flanking regions suggested that the chromosome 21 locus is an amastigote-specific isoform and more closely related (either structurally or functionally) to the chromosome 33 'array' locus than the chromosome 8 locus. A phylogenetic network of all isoforms indicated that paralogs from L. braziliensis and L. mexicana were monophyletic, rather than clustering by locus. CONCLUSION: L. braziliensis and L. mexicana sequences appeared more similar to each other than each did to its closest relative in another species; this indicates that these sequences have evolved convergently in each species, perhaps through ectopic gene conversion; a process not yet evident among the more recently derived L. major and L. infantum isoforms. The distinctive non-coding regions of each β-tubulin locus showed that it is the regulatory regions of these loci that have evolved most during the diversification of these genes in Leishmania, while the coding regions have been conserved and concerted. The various loci in Leishmania satisfy a need for innovative expression of β-tubulin, rather than elaboration of its structural role

Patterns of kinesin evolution reveal a complex ancestral eukaryote with a multifunctional cytoskeleton

Background: The genesis of the eukaryotes was a pivotal event in evolution and was accompanied by the acquisition of numerous new cellular features including compartmentalization by cytoplasmic organelles, mitosis and meiosis, and ciliary motility. Essential for the development of these features was the tubulin cytoskeleton and associated motors. It is therefore possible to map ancient cell evolution by reconstructing the evolutionary history of motor proteins. Here, we have used the kinesin motor repertoire of 45 extant eukaryotes to infer the ancestral state of this superfamily in the last common eukaryotic ancestor (LCEA). Results: We bioinformatically identified 1624 putative kinesin proteins, determined their protein domain architectures and calculated a comprehensive Bayesian phylogeny for the kinesin superfamily with statistical support. These data enabled us to define 51 anciently-derived kinesin paralogs (including three new kinesin families) and 105 domain architectures. We then mapped these characters acros eukaryotes, accounting for secondary loss within established eukaryotic groupings, and alternative tree topologies. Conclusions: We show that a minimum of 11 kinesin families and 3 protein domain architectures were present in the LCEA. This demonstrates that the microtubule-based cytoskeleton of the LCEA was surprisingly highly developed in terms of kinesin motor types, but that domain architectures have been extensively modified during the diversification of the eukaryotes. Our analysis provides molecular evidence for the existence of several key cellular functions in the LCEA, and shows that a large proportion of motor family diversity and cellular complexity had already arisen in this ancient cell

Evolution of Tubulin Gene Arrays in Trypanosomatid parasites: genomic restructuring in Leishmania

BACKGROUND: α- and β-tubulin are fundamental components of the eukaryotic cytoskeleton and cell division machinery. While overall tubulin expression is carefully controlled, most eukaryotes express multiple tubulin genes in specific regulatory or developmental contexts. The genomes of the human parasites Trypanosoma brucei and Leishmania major reveal that these unicellular kinetoplastids possess arrays of tandem-duplicated tubulin genes, but with differences in organisation. While L. major possesses monotypic α and β arrays in trans, an array of alternating α- and β tubulin genes occurs in T. brucei. Polycistronic transcription in these organisms makes the chromosomal arrangement of tubulin genes important with respect to gene expression. RESULTS: We investigated the genomic architecture of tubulin tandem arrays among these parasites, establishing which character state is derived, and the timing of character transition. Tubulin loci in T. brucei and L. major were compared to examine the relationship between the two character states. Intergenic regions between tubulin genes were sequenced from several trypanosomatids and related, non-parasitic bodonids to identify the ancestral state. Evidence of alternating arrays was found among non-parasitic kinetoplastids and all Trypanosoma spp.; monotypic arrays were confirmed in all Leishmania spp. and close relatives. CONCLUSION: Alternating and monotypic tubulin arrays were found to be mutually exclusive through comparison of genome sequences. The presence of alternating gene arrays in non-parasitic kinetoplastids confirmed that separate, monotypic arrays are the derived state and evolved through genomic restructuring in the lineage leading to Leishmania. This fundamental reorganisation accounted for the dissimilar genomic architectures of T. brucei and L. major tubulin repertoires

A new generation of T7 RNA polymerase-independent inducible expression plasmids for Trypanosoma brucei.

Expression of transgenes is central to forward and reverse genetic analysis in Trypanosoma brucei. The inducible expression of transgenes in trypanosomes is based on the tetracycline repressor binding to a tetracycline operator to prevent transcription in the absence of tetracycline. The same inducible system is used to produce double-stranded RNA for RNAi knockdown of target genes. This study describes a new plasmid pSPR2.1 that drives consistent high-level expression of tetracycline repressor in procyclic form trypanosomes. A complementary expression plasmid, p3227, was constructed. The major difference between this and current plasmids is the separation of the inducible transgene and selectable marker promoters by the plasmid backbone. The plasmid p3227 was able to support inducible expression in cell lines containing pSPR2.1 as well as the established Lister 427 29-13 cell line. p3666, a derivative of p3227, was made for inducible expression of stem loop RNAi constructs and was effective for knockdown of DRBD3, which had proved problematic using existing RNAi plasmids with head-to-head promoters. The plasmid system was also able to support inducible transgene expression and DRBD3 RNAi knockdown in bloodstream form cells expressing tetracycline repressor from an integrated copy of the plasmid pHD1313

Shape, form, function and Leishmania pathogenicity: from textbook descriptions to biological understanding

The shape and form of protozoan parasites are inextricably linked to their pathogenicity. The evolutionary pressure associated with establishing and maintaining an infection and transmission to vector or host has shaped parasite morphology. However, there is not a ‘one size fits all’ morphological solution to these different pressures, and parasites exhibit a range of different morphologies, reflecting the diversity of their complex life cycles. In this review, we will focus on the shape and form of Leishmania spp., a group of very successful protozoan parasites that cause a range of diseases from self-healing cutaneous leishmaniasis to visceral leishmaniasis, which is fatal if left untreated

Conservation of ciliary proteins in plants with no cilia

<p>Abstract</p> <p>Background</p> <p>Eukaryotic cilia are complex, highly conserved microtubule-based organelles with a broad phylogenetic distribution. Cilia were present in the last eukaryotic common ancestor and many proteins involved in cilia function have been conserved through eukaryotic diversification. However, cilia have also been lost multiple times in different lineages, with at least two losses occurring within the land plants. Whereas all non-seed plants produce cilia for motility of male gametes, some gymnosperms and all angiosperms lack cilia. During these evolutionary losses, proteins with ancestral ciliary functions may be lost or co-opted into different functions.</p> <p>Results</p> <p>Here we identify a core set of proteins with an inferred ciliary function that are conserved in ciliated eukaryotic species. We interrogate this genomic dataset to identify proteins with a predicted ancestral ciliary role that have been maintained in non-ciliated land plants. In support of our prediction, we demonstrate that several of these proteins have a flagellar localisation in protozoan trypanosomes. The phylogenetic distribution of these genes within the land plants indicates evolutionary scenarios of either sub- or neo-functionalisation and expression data analysis shows that these genes are highly expressed in <it>Arabidopsis thaliana </it>pollen cells.</p> <p>Conclusions</p> <p>A large number of proteins possess a phylogenetic ciliary profile indicative of ciliary function. Remarkably, many genes with an ancestral ciliary role are maintained in non-ciliated land plants. These proteins have been co-opted to perform novel functions, most likely before the loss of cilia, some of which appear related to the formation of the male gametes.</p

Dependency relationships between IFT-dependent flagellum elongation and cell morphogenesis in Leishmania

Flagella have multiple functions that are associated with different axonemal structures. Motile flagella typically have a 9+2 arrangement of microtubules, whereas sensory flagella normally have a 9+0 arrangement. Leishmania exhibits both of these flagellum forms and differentiation between these two flagellum forms is associated with cytoskeletal and cell shape changes. We disrupted flagellum elongation in Leishmania by deleting the intraflagellar transport (IFT) protein IFT140, and examined the effects on cell morphogenesis. Δift140 cells have no external flagellum, having only a very short flagellum within the flagellar pocket. This short flagellum had a collapsed 9+0 (9v) axoneme configuration reminiscent of that in the amastigote, and was not attached to the pocket membrane. Although amastigote-like changes occurred in the flagellar cytoskeleton, the cytoskeletal structures of Δift140 cells retained their promastigote configurations, as examined by fluorescence microscopy of tagged proteins and serial electron tomography. Thus, Leishmania promastigote cell morphogenesis does not depend on the formation of a long flagellum attached at the neck. Furthermore, our data show that disruption of the IFT system is sufficient to produce a switch from the 9+2 to the collapsed 9+0 (9v) axonemal structure; echoing the process that occurs during the promastigote to amastigote differentiation

Ab Initio Identification of Novel Regulatory Elements in the Genome of Trypanosoma brucei by Bayesian Inference on Sequence Segmentation

Abstract Background: The rapid increase in the availability of genome information has created considerable demand for both comparative and ab initio predictive bioinformatic analyses. The biology laid bare in the genomes of many organisms is often novel, presenting new challenges for bioinformatic interrogation. A paradigm for this is the collected genomes of the kinetoplastid parasites, a group which includes Trypanosoma brucei the causative agent of human African trypanosomiasis. These genomes, though outwardly simple in organisation and gene content, have historically challenged many theories for gene expression regulation in eukaryotes

Blocking variant surface glycoprotein synthesis in Trypanosoma brucei triggers a general arrest in translation initiation.

BACKGROUND: The African trypanosome Trypanosoma brucei is covered with a dense layer of Variant Surface Glycoprotein (VSG), which protects it from lysis by host complement via the alternative pathway in the mammalian bloodstream. Blocking VSG synthesis by the induction of VSG RNAi triggers an unusually precise precytokinesis cell-cycle arrest. METHODOLOGY/PRINCIPAL FINDINGS: Here, we characterise the cells arrested after the induction of VSG RNAi. We were able to rescue the VSG221 RNAi induced cell-cycle arrest through expression of a second different VSG (VSG117 which is not recognised by the VSG221 RNAi) from the VSG221 expression site. Metabolic labeling of the arrested cells showed that blocking VSG synthesis triggered a global translation arrest, with total protein synthesis reduced to less than 1-4% normal levels within 24 hours of induction of VSG RNAi. Analysis by electron microscopy showed that the translation arrest was coupled with rapid disassociation of ribosomes from the endoplasmic reticulum. Polysome analysis showed a drastic decrease in polysomes in the arrested cells. No major changes were found in levels of transcription, total RNA transcript levels or global amino acid concentrations in the arrested cells. CONCLUSIONS: The cell-cycle arrest phenotype triggered by the induction of VSG221 RNAi is not caused by siRNA toxicity, as this arrest can be alleviated if a second different VSG is inserted downstream of the active VSG221 expression site promoter. Analysis of polysomes in the stalled cells showed that the translation arrest is mediated at the level of translation initiation rather than elongation. The cell-cycle arrest induced in the presence of a VSG synthesis block is reversible, suggesting that VSG synthesis and/or trafficking to the cell surface could be monitored during the cell-cycle as part of a specific cell-cycle checkpoint