Search tips
Search criteria

Results 1-17 (17)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
1.  Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei 
PLoS ONE  2014;9(12):e114628.
Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4–5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4–5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.
PMCID: PMC4262433  PMID: 25493940
2.  The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants 
PLoS Genetics  2014;10(2):e1004007.
Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.
Author Summary
Some plant trypanosomes, single-celled organisms living in phloem sap, are responsible for important palm diseases, inducing frequent expensive and toxic insecticide treatments against their insect vectors. Other trypanosomes multiply in latex tubes without detriment to their host. Despite the wide range of behaviors and impacts, these trypanosomes have been rather unceremoniously lumped into a single genus: Phytomonas. A battery of molecular probes has been used for their characterization but no clear phylogeny or classification has been established. We have sequenced the genomes of a pathogenic phloem-specific Phytomonas from a diseased South American coconut palm and a latex-specific isolate collected from an apparently healthy wild euphorb in the south of France. Upon comparison with each other and with human pathogenic trypanosomes, both Phytomonas revealed distinctive compact genomes, consisting essentially of single-copy genes, with the vast majority of genes shared by both isolates irrespective of their effect on the host. A strong cohort of enzymes in the sugar metabolism pathways was consistent with the nutritional environments found in plants. The genetic nuances may reveal the basis for the behavioral differences between these two unique plant parasites, and indicate the direction of our future studies in search of effective treatment of the crop disease parasites.
PMCID: PMC3916237  PMID: 24516393
3.  Revisiting the Central Metabolism of the Bloodstream Forms of Trypanosoma brucei: Production of Acetate in the Mitochondrion Is Essential for Parasite Viability 
The bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.
Methodology/Principal Findings
A comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway.
Acetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the “acetate shuttle” that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.
Author Summary
Many protists, including parasitic helminthes, trichomonads and trypanosomatids, produce acetate in their mitochondrion or mitochondrion-like organelle, which is excreted as a main metabolic end-product of their energy metabolism. We have recently demonstrated that mitochondrial production of acetate is essential for fatty acid biosynthesis and ATP production in the procyclic insect form of T. brucei. However, acetate metabolism has not been investigated in the long-slender bloodstream forms of the parasite, the proliferative forms responsible for the sleeping sickness. In contrast to the current view, we showed that the bloodstream forms produce almost as much acetate from glucose than the procyclic parasites. Acetate production from glucose and threonine is synthetically essential for growth and de novo synthesis of fatty acids of the bloodstream trypanosomes. These data highlight that the central metabolism of the bloodstream forms contains unexpected essential pathways, although minor in terms of metabolic flux, which could be targeted for the development of trypanocidal drugs.
PMCID: PMC3868518  PMID: 24367711
4.  The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets 
The FASEB Journal  2012;26(11):4650-4661.
The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9-Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.—Godel, C., Kumar, S., Koutsovoulos, G., Ludin, P., Nilsson, D., Comandatore, F., Wrobel, N., Thompson, M., Schmid, C. D., Goto, S., Bringaud, F., Wolstenholme, A., Bandi, C., Epe, C., Kaminsky, R., Blaxter, M., Mäser, P. The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets.
PMCID: PMC3475251  PMID: 22889830
comparative genomics; filaria; transposon; Wolbachia
5.  Flux Analysis of the Trypanosoma brucei Glycolysis Based on a Multiobjective-Criteria Bioinformatic Approach 
Advances in Bioinformatics  2012;2012:159423.
Trypanosoma brucei is a protozoan parasite of major of interest in discovering new genes for drug targets. This parasite alternates its life cycle between the mammal host(s) (bloodstream form) and the insect vector (procyclic form), with two divergent glucose metabolism amenable to in vitro culture. While the metabolic network of the bloodstream forms has been well characterized, the flux distribution between the different branches of the glucose metabolic network in the procyclic form has not been addressed so far. We present a computational analysis (called Metaboflux) that exploits the metabolic topology of the procyclic form, and allows the incorporation of multipurpose experimental data to increase the biological relevance of the model. The alternatives resulting from the structural complexity of networks are formulated as an optimization problem solved by a metaheuristic where experimental data are modeled in a multiobjective function. Our results show that the current metabolic model is in agreement with experimental data and confirms the observed high metabolic flexibility of glucose metabolism. In addition, Metaboflux offers a rational explanation for the high flexibility in the ratio between final products from glucose metabolism, thsat is, flux redistribution through the malic enzyme steps.
PMCID: PMC3477527  PMID: 23097667
6.  Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti† 
Nucleic Acids Research  2012;40(18):9102-9114.
We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.
PMCID: PMC3467087  PMID: 22833609
7.  TSIDER1, a short and non-autonomous Salivarian trypanosome-specific retroposon related to the ingi6 subclade 
Graphical abstract
Position of African trypanosome-specific TSIDER1 retroposons in the ingi and trypanosomatid phylogenetic trees.
► A new retroposon family of the ingi clade, named TSIDER1, was identified. ► TSIDER1 are short degenerate retroposons. ► TSIDER1 is only present in the nuclear genome of African trypanosomes. ► In contrast to Leishmania, African trypanosomes have not expanded and domesticated SIDER.
Retroposons of the ingi clade are the most abundant transposable elements identified in the trypanosomatid genomes. Some are long autonomous elements (ingi, L1Tc) while others, such as RIME and NARTc, are short non-coding elements that parasitize the retrotransposition machinery of the active autonomous ones for their own mobilization. Here, we identified a new family of short non-autonomous retroposons of the ingi clade, called TSIDER1, which are present in the genome of Salivarian (African) trypanosomes, Trypanosoma brucei, T. congolense and T. vivax, but absent in the T. cruzi and Leishmania spp. genomes and, as such, TSIDER1 is the only retroposon subfamily conserved at the nucleotide level between African trypanosome species. We identified three TvSIDER1 families within the genome of T. vivax and the high level of sequence conservation within the TvSIDER1a and TvSIDER1b groups suggests that they are still active. We propose that TvSIDER1a/b elements are using the Tvingi retrotransposition machinery, as they are preceded by the same conserved pattern characteristic of the ingi6 subclade, which corresponds to the retroposon-encoded endonuclease binding site. In contrast, TcoSIDER1, TbSIDER1 and TvSIDER1c are too divergent to be considered as active retroposons. The relatively low number of SIDER elements identified in the T. congolense (70 copies), T. vivax (32 copies) and T. brucei (22 copies) genomes confirms that trypanosomes have not expanded short transposable elements, which is in contrast to Leishmania spp. (∼2000 copies), where SIDER play a role in the regulation of gene expression.
PMCID: PMC3820030  PMID: 21664383
SIDER, Short Interspersed DEgenerate Retroposons; DIRE, Degenerate Ingi/L1Tc-Related Element; African trypanosomes; Ingi; Retroposon; Non-LTR retrotransposon; Non-autonomous; SIDER
8.  Trypanosomatid Genomes Contain Several Subfamilies of ingi-Related Retroposons ▿ †  
Eukaryotic Cell  2009;8(10):1532-1542.
Retroposons are ubiquitous transposable elements found in the genomes of most eukaryotes, including trypanosomatids. The African and American trypanosomes (Trypanosoma brucei and Trypanosoma cruzi) contain long autonomous retroposons of the ingi clade (Tbingi and L1Tc, respectively) and short nonautonomous truncated versions (TbRIME and NARTc, respectively), as well as degenerate ingi-related retroposons devoid of coding capacity (DIREs). In contrast, Leishmania major contains only remnants of extinct retroposons (LmDIREs) and of short nonautonomous heterogeneous elements (LmSIDERs). We extend this comparative and evolutionary analysis of retroposons to the genomes of two other African trypanosomes (Trypanosoma congolense and Trypanosoma vivax) and another Leishmania sp. (Leishmania braziliensis). Three new potentially functional retroposons of the ingi clade have been identified: Tvingi in T. vivax and Tcoingi and L1Tco in T. congolense. T. congolense is the first trypanosomatid containing two classes of potentially active retroposons of the ingi clade. We analyzed sequences located upstream of these new long autonomous ingi-related elements, which code for the recognition site of the retroposon-encoded endonuclease. The closely related Tcoingi and Tvingi elements show the same conserved pattern, indicating that the Tcoingi- and Tvingi-encoded endonucleases share site specificity. Similarly, the conserved pattern previously identified upstream of L1Tc has also been detected at the same relative position upstream of L1Tco elements. A phylogenetic analysis of all ingi-related retroposons identified so far, including DIREs, clearly shows that several distinct subfamilies have emerged and coexisted, though in the course of trypanosomatid evolution, only a few have been maintained as active elements in modern trypanosomatid (sub)species.
PMCID: PMC2756862  PMID: 19666780
9.  Organization and evolution of two SIDER retroposon subfamilies and their impact on the Leishmania genome 
BMC Genomics  2009;10:240.
We have recently identified two large families of extinct transposable elements termed Short Interspersed DEgenerated Retroposons (SIDERs) in the parasitic protozoan Leishmania major. The characterization of SIDER elements was limited to the SIDER2 subfamily, although members of both subfamilies have been shown to play a role in the regulation of gene expression at the post-transcriptional level. Apparent functional domestication of SIDERs prompted further investigation of their characterization, dissemination and evolution throughout the Leishmania genus, with particular attention to the disregarded SIDER1 subfamily.
Using optimized statistical profiles of both SIDER1 and SIDER2 subgroups, we report the first automated and highly sensitive annotation of SIDERs in the genomes of L. infantum, L. braziliensis and L. major. SIDER annotations were combined to in-silico mRNA extremity predictions to generate a detailed distribution map of the repeat family, hence uncovering an enrichment of antisense-oriented SIDER repeats between the polyadenylation and trans-splicing sites of intergenic regions, in contrast to the exclusive sense orientation of SIDER elements within 3'UTRs. Our data indicate that SIDER elements are quite uniformly dispersed throughout all three genomes and that their distribution is generally syntenic. However, only 47.4% of orthologous genes harbor a SIDER element in all three species. There is evidence for species-specific enrichment of SIDERs and for their preferential association, especially for SIDER2s, with different metabolic functions. Investigation of the sequence attributes and evolutionary relationship of SIDERs to other trypanosomatid retroposons reveals that SIDER1 is a truncated version of extinct autonomous ingi-like retroposons (DIREs), which were functional in the ancestral Leishmania genome.
A detailed characterization of the sequence traits for both SIDER subfamilies unveils major differences. The SIDER1 subfamily is more heterogeneous and shows an evolutionary link with vestigial DIRE retroposons as previously observed for the ingi/RIME and L1Tc/NARTc couples identified in the T. brucei and T. cruzi genomes, whereas no identified DIREs are related to SIDER2 sequences. Although SIDER1s and SIDER2s display equivalent genomic distribution globally, the varying degrees of sequence conservation, preferential genomic disposition, and differential association to orthologous genes allude to an intricate web of SIDER assimilation in these parasitic organisms.
PMCID: PMC2689281  PMID: 19463167
10.  Comparative genomic analysis of three Leishmania species that cause diverse human disease 
Nature genetics  2007;39(7):839-847.
Leishmania parasites cause a broad spectrum of clinical disease. Here we report the sequencing of the genomes of two species of Leishmania: Leishmania infantum and Leishmania braziliensis. The comparison of these sequences with the published genome of Leishmania major reveals marked conservation of synteny and identifies only ∼200 genes with a differential distribution between the three species. L. braziliensis, contrary to Leishmania species examined so far, possesses components of a putative RNA-mediated interference pathway, telomere-associated transposable elements and spliced leader–associated SLACS retrotransposons. We show that pseudogene formation and gene loss are the principal forces shaping the different genomes. Genes that are differentially distributed between the species encode proteins implicated in host-pathogen interactions and parasite survival in the macrophage.
PMCID: PMC2592530  PMID: 17572675
11.  Members of a Large Retroposon Family Are Determinants of Post-Transcriptional Gene Expression in Leishmania 
PLoS Pathogens  2007;3(9):e136.
Trypanosomatids are unicellular protists that include the human pathogens Leishmania spp. (leishmaniasis), Trypanosoma brucei (sleeping sickness), and Trypanosoma cruzi (Chagas disease). Analysis of their recently completed genomes confirmed the presence of non–long-terminal repeat retrotransposons, also called retroposons. Using the 79-bp signature sequence common to all trypanosomatid retroposons as bait, we identified in the Leishmania major genome two new large families of small elements—LmSIDER1 (785 copies) and LmSIDER2 (1,073 copies)—that fulfill all the characteristics of extinct trypanosomatid retroposons. LmSIDERs are ∼70 times more abundant in L. major compared to T. brucei and are found almost exclusively within the 3′-untranslated regions (3′UTRs) of L. major mRNAs. We provide experimental evidence that LmSIDER2 act as mRNA instability elements and that LmSIDER2-containing mRNAs are generally expressed at lower levels compared to the non-LmSIDER2 mRNAs. The considerable expansion of LmSIDERs within 3′UTRs in an organism lacking transcriptional control and their role in regulating mRNA stability indicate that Leishmania have probably recycled these short retroposons to globally modulate the expression of a number of genes. To our knowledge, this is the first example in eukaryotes of the domestication and expansion of a family of mobile elements that have evolved to fulfill a critical cellular function.
Author Summary
Transposable elements (TEs) are DNA sequences capable of moving from one chromosomal region to another. A considerable fraction of higher eukaryote genomes is comprised of TEs, as exemplified in human (over 40% of the genome) and maize (over 50% of the genome). There is now a growing body of evidence to suggest that TEs can be functionally important and not just “junk,” “selfish,” or “parasitic” DNA sequences that make as many copies of themselves as possible. Indeed, during the past ten years, a considerable number of TE copies have been described as domesticated or exapted elements playing a cellular function, such as transcriptional regulation and contribution to protein-coding regions. TE domestication has been described for only a few copies of TE families, and exaption of a whole TE family has not been reported so far. We provide evidence that Leishmania spp., unicellular protists responsible for human diseases, have recycled and expanded a whole family of short and extinct TEs (retroposons) that have evolved to fulfill an important biological pathway, i.e., regulation of gene expression. We also observed that Trypanosoma brucei (a close relative of Leishmania spp.) developed other approaches to maintain the same cellular function.
PMCID: PMC2323293  PMID: 17907803
12.  Evolution of energy metabolism and its compartmentation in Kinetoplastida 
Kinetoplastida are protozoan organisms that probably diverged early in evolution from other eukaryotes. They are characterized by a number of unique features with respect to their energy and carbohydrate metabolism. These organisms possess peculiar peroxisomes, called glycosomes, which play a central role in this metabolism; the organelles harbour enzymes of several catabolic and anabolic routes, including major parts of the glycolytic and pentosephosphate pathways. The kinetoplastid mitochondrion is also unusual with regard to both its structural and functional properties.
In this review, we describe the unique compartmentation of metabolism in Kinetoplastida and the metabolic properties resulting from this compartmentation. We discuss the evidence for our recently proposed hypothesis that a common ancestor of Kinetoplastida and Euglenida acquired a photosynthetic alga as an endosymbiont, contrary to the earlier notion that this event occurred at a later stage of evolution, in the Euglenida lineage alone. The endosymbiont was subsequently lost from the kinetoplastid lineage but, during that process, some of its pathways of energy and carbohydrate metabolism were sequestered in the kinetoplastid peroxisomes, which consequently became glycosomes. The evolution of the kinetoplastid glycosomes and the possible selective advantages of these organelles for Kinetoplastida are discussed. We propose that the possession of glycosomes provided metabolic flexibility that has been important for the organisms to adapt easily to changing environmental conditions. It is likely that metabolic flexibility has been an important selective advantage for many kinetoplastid species during their evolution into the highly successful parasites today found in many divergent taxonomic groups.
Also addressed is the evolution of the kinetoplastid mitochondrion, from a supposedly pluripotent organelle, attributed to a single endosymbiotic event that resulted in all mitochondria and hydrogenosomes of extant eukaryotes. Furthermore, indications are presented that Kinetoplastida may have acquired other enzymes of energy and carbohydrate metabolism by various lateral gene transfer events different from those that involved the algal- and α-proteobacterial-like endosymbionts responsible for the respective formation of the glycosomes and mitochondria.
PMCID: PMC317351  PMID: 14613499
13.  The DNA sequence of chromosome I of an African trypanosome: gene content, chromosome organisation, recombination and polymorphism 
Nucleic Acids Research  2003;31(16):4864-4873.
The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99–100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters.
PMCID: PMC169939  PMID: 12907729
14.  Two Related Subpellicular Cytoskeleton-associated Proteins in Trypanosoma brucei Stabilize Microtubules 
Molecular Biology of the Cell  2002;13(3):1058-1070.
The subpellicular microtubules of the trypanosome cytoskeleton are cross-linked to each other and the plasma membrane, creating a cage-like structure. We have isolated, from Trypanosoma brucei, two related low-molecular-weight cytoskeleton-associated proteins (15- and 17-kDa), called CAP15 and CAP17, which are differentially expressed during the life cycle. Immunolabeling shows a corset-like colocalization of both CAPs and tubulin. Western blot and electron microscope analyses show CAP15 and CAP17 labeling on detergent-extracted cytoskeletons. However, the localization of both proteins is restricted to the anterior, microtubule minus, and less dynamic half of the corset. CAP15 and CAP17 share properties of microtubule-associated proteins when expressed in heterologous cells (Chinese hamster ovary and HeLa), colocalization with their microtubules, induction of microtubule bundle formation, cold resistance, and insensitivity to nocodazole. When overexpressed in T. brucei, both CAP15 and CAP17 cover the whole subpellicular corset and induce morphological disorders, cell cycle-based abnormalities, and subsequent asymmetric cytokinesis.
PMCID: PMC99619  PMID: 11907282
16.  A New, Expressed Multigene Family Containing a Hot Spot for Insertion of Retroelements Is Associated with Polymorphic Subtelomeric Regions of Trypanosoma brucei 
Eukaryotic Cell  2002;1(1):137-151.
We describe a novel gene family that forms clusters in subtelomeric regions of Trypanosoma brucei chromosomes and partially accounts for the observed clustering of retrotransposons. The ingi and ribosomal inserted mobile element (RIME) non-LTR retrotransposons share 250 bp at both extremities and are the most abundant putatively mobile elements, with about 500 copies per haploid genome. From cDNA clones and subsequently in the T. brucei genomic DNA databases, we identified 52 homologous gene and pseudogene sequences, 16 of which contain a RIME and/or ingi retrotransposon inserted at exactly the same relative position. Here these genes are called the RHS family, for retrotransposon hot spot. Comparison of the protein sequences encoded by RHS genes (21 copies) and pseudogenes (24 copies) revealed a conserved central region containing an ATP/GTP-binding motif and the RIME/ingi insertion site. The RHS proteins share between 13 and 96% identity, and six subfamilies, RHS1 to RHS6, can be defined on the basis of their divergent C-terminal domains. Immunofluorescence and Western blot analyses using RHS subfamily-specific immune sera show that RHS proteins are constitutively expressed and occur mainly in the nucleus. Analysis of Genome Survey Sequence databases indicated that the Trypanosoma brucei diploid genome contains about 280 RHS (pseudo)-genes. Among the 52 identified RHS (pseudo)genes, 48 copies are in three RHS clusters located in subtelomeric regions of chromosomes Ia and II and adjacent to the active bloodstream form expression site in T. brucei strain TREU927/4 GUTat10.1. RHS genes comprise the remaining sequence of the size-polymorphic “repetitive region” described for T. brucei chromosome I, and a homologous gene family is present in the Trypanosoma cruzi genome.
PMCID: PMC118050  PMID: 12455980
17.  The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control 
Molecular Microbiology  2013;90(1):114-129.
The Trypanosoma brucei procyclic form resides within the digestive tract of its insect vector, where it exploits amino acids as carbon sources. Threonine is the amino acid most rapidly consumed by this parasite, however its role is poorly understood. Here, we show that the procyclic trypanosomes grown in rich medium only use glucose and threonine for lipid biosynthesis, with threonine's contribution being ∼ 2.5 times higher than that of glucose. A combination of reverse genetics and NMR analysis of excreted end-products from threonine and glucose metabolism, shows that acetate, which feeds lipid biosynthesis, is also produced primarily from threonine. Interestingly, the first enzymatic step of the threonine degradation pathway, threonine dehydrogenase (TDH, EC, is under metabolic control and plays a key role in the rate of catabolism. Indeed, a trypanosome mutant deleted for the phosphoenolpyruvate decarboxylase gene (PEPCK, EC shows a 1.7-fold and twofold decrease of TDH protein level and activity, respectively, associated with a 1.8-fold reduction in threonine-derived acetate production. We conclude that TDH expression is under control and can be downregulated in response to metabolic perturbations, such as in the PEPCK mutant in which the glycolytic metabolic flux was redirected towards acetate production.
PMCID: PMC4034587  PMID: 23899193

Results 1-17 (17)