Mitochondrial genomics of human pathogenic parasite Leishmania (Viannia) panamensis.
ABSTRACT: Background:The human parasite Leishmania (V.) panamensis is one of the pathogenic species responsible for cutaneous leishmaniasis in Central and South America. Despite its importance in molecular parasitology, its mitochondrial genome, divided into minicircles and maxicircles, haven't been described so far. Methods:Using NGS-based sequencing (454 and ILLUMINA), and combining de novo genome assembly and mapping strategies, we report the maxicircle kDNA annotated genome of L. (V.) panamensis, the first reference of this molecule for the subgenus Viannia. A comparative genomics approach is performed against other Leishmania and Trypanosoma species. Results:The results show synteny of mitochondrial genes of L. (V.) panamensis with other kinetoplastids. It was also possible to identify nucleotide variants within the coding regions of the maxicircle, shared among some of them and others specific to each strain. Furthermore, we compared the minicircles kDNA sequences of two strains and the results show that the conserved and divergent regions of the minicircles exhibit strain-specific associations.
Project description:The mitochondrial DNA (mtDNA), which is present in almost all eukaryotic organisms, is a useful marker for phylogenetic studies due to its relative high conservation and its inheritance manner. In Leishmania and other trypanosomatids, the mtDNA (also referred to as kinetoplast DNA or kDNA) is composed of thousands of minicircles and a few maxicircles, catenated together into a complex network. Maxicircles are functionally similar to other eukaryotic mtDNAs, whereas minicircles are involved in RNA editing of some maxicircle-encoded transcripts. Next-generation sequencing (NGS) is increasingly used for assembling nuclear genomes and, currently, a large number of genomic sequences are available. However, most of the time, the mitochondrial genome is ignored in the genome assembly processes. The aim of this study was to develop a pipeline to assemble Leishmania minicircles and maxicircle DNA molecules, exploiting the raw data generated in the NGS projects. As a result, the maxicircle molecules and the plethora of minicircle classes for Leishmania major, Leishmania infantum and Leishmania braziliensis have been characterized. We have observed that whereas the heterogeneity of minicircle sequences existing in a single cell hampers their use for Leishmania typing and classification, maxicircles emerge as an extremely robust genetic marker for taxonomic studies within the clade of kinetoplastids.
Project description:Kinetoplast DNA (kDNA), the trypanosome mitochondrial DNA, contains thousands of minicircles and dozens of maxicircles interlocked in a giant network. Remarkably, Trypanosoma brucei's genome encodes 8 PIF1-like helicases, 6 of which are mitochondrial. We now show that TbPIF2 is essential for maxicircle replication. Maxicircle abundance is controlled by TbPIF2 level, as RNAi of this helicase caused maxicircle loss, and its overexpression caused a 3- to 6-fold increase in maxicircle abundance. This regulation of maxicircle level is mediated by the TbHslVU protease. Previous experiments demonstrated that RNAi knockdown of TbHslVU dramatically increased abundance of minicircles and maxicircles, presumably because a positive regulator of their synthesis escaped proteolysis and allowed synthesis to continue. Here, we found that TbPIF2 level increases following RNAi of the protease. Therefore, this helicase is a TbHslVU substrate and an example of a positive regulator, thus providing a molecular mechanism for controlling maxicircle replication.
Project description:Leishmania (Viannia) panamensis is one of the most important Leishmania species associated with cutaneous leishmaniasis (CL) in Latin America. Despite its wide geographic distribution and pathogenic potential in humans and animals, the genomic variability of this species is low compared with other Leishmania species circulating in the same geographical area. No studies have reported a detailed analysis of the whole genome of L. panamensis from clinical isolates using DNA high-throughput sequencing to clarify its intraspecific genomic variability or plausible divergence. Therefore, this study aimed to evaluate the intraspecific genomic variability of L. panamensis from Colombia and Panama. A total of 22 genomes were analyzed, 19 from Colombian patients with CL and three genomes from Panama obtained from public databases. The phylogenomic analysis revealed the potential existence of three well-supported clades as evidence of intraspecific divergence. Additionally, the whole-genome analysis showed low structural variations in terms of ploidy, copy number variations, and single-nucleotide polymorphisms (SNPs). SNPs shared among all clades were identified, revealing their importance in different biological processes of L. panamensis. The findings not only expand our knowledge of intraspecific genomic variability of one of the most important Leishmania species in South America but also highlights the possible existence of different clades/lineages/subpopulations across a geographic scale.
Project description:The mitochondrial DNA of trypanosomatid protozoa consists of a complex, intercatenated network of tens of maxicircles and thousands of minicircles. This structure, called kinetoplast DNA (kDNA), requires numerous proteins and multiprotein complexes for replication, segregation, and transcription. In this study, we used a proteomic approach to identify proteins that are associated with the kDNA network. We identified a novel protein encoded by Tb927.2.6100 that was present in a fraction enriched for kDNA and colocalized the protein with kDNA by fluorescence microscopy. RNA interference (RNAi) knockdown of its expression resulted in a growth defect and changes in the proportion of kinetoplasts and nuclei in the cell population. RNAi also resulted in shrinkage and loss of the kinetoplasts, loss of maxicircle and minicircle components of kDNA at similar rates, and (perhaps secondarily) loss of edited and pre-edited mRNA. These results indicate that the Tb927.2.6100 protein is essential for the maintenance of kDNA.
Project description:The unicellular parasite Trypanosoma brucei harbors one mitochondrial organelle with a singular genome called the kinetoplast DNA (kDNA). The kDNA consists of a network of concatenated minicircles and a few maxicircles that form the kDNA disc. More than 30 proteins involved in kDNA replication have been described. However, several mechanistic questions are only poorly understood. Here, we describe and characterize minicircle replication factor 172 (MiRF172), a novel mitochondrial genome replication factor that is essential for cell growth and kDNA maintenance. By performing super-resolution microscopy, we show that MiRF172 is localized to the kDNA disc, facing the region between the genome and the mitochondrial membranes. We demonstrate that depletion of MiRF172 leads to a loss of minicircles and maxicircles. Detailed analysis suggests that MiRF172 is involved in the reattachment of replicated minicircles to the kDNA disc. Furthermore, we provide evidence that the localization of the replication factor MiRF172 not only depends on the kDNA itself, but also on the mitochondrial genome segregation machinery, suggesting an interaction between the two essential entities.This article has an associated First Person interview with the first author of the paper.
Project description:Trypanosoma brucei is a kinetoplastid flagellate, the agent of human sleeping sickness and ruminant nagana in Africa. Kinetoplastid flagellates contain their eponym kinetoplast DNA (kDNA), consisting of two types of interlocked circular DNA molecules: scores of maxicircles and thousands of minicircles. Maxicircles have typical mitochondrial genes, most of which are translatable only after RNA editing. Minicircles encode guide RNAs, required for decrypting the maxicircle transcripts. The life cycle of T. brucei involves a bloodstream stage (BS) in vertebrates and a procyclic stage (PS) in the tsetse fly vector. Partial [dyskinetoplastidy (Dk)] or total [akinetoplastidy (Ak)] loss of kDNA locks the trypanosome in the BS form. Transmission between vertebrates becomes mechanical without PS and tsetse mediation, allowing the parasite to spread outside the African tsetse belt. Trypanosoma equiperdum and Trypanosoma evansi are agents of dourine and surra, diseases of horses, camels, and water buffaloes. We have characterized representative strains of T. equiperdum and T. evansi by numerous molecular and classical parasitological approaches. We show that both species are actually strains of T. brucei, which lost part (Dk) or all (Ak) of their kDNA. These trypanosomes are not monophyletic clades and do not qualify for species status. They should be considered two subspecies, respectively T. brucei equiperdum and T. brucei evansi, which spontaneously arose recently. Dk/Ak trypanosomes may potentially emerge repeatedly from T. brucei.
Project description:Mitochondrial chaperones have multiple functions that are essential for proper functioning of mitochondria. In the human-pathogenic protist Trypanosoma brucei, we demonstrate a novel function of the highly conserved machinery composed of mitochondrial heat shock proteins 70 and 40 (mtHsp70/mtHsp40) and the ATP exchange factor Mge1. The mitochondrial DNA of T. brucei, also known as kinetoplast DNA (kDNA), is represented by a single catenated network composed of thousands of minicircles and dozens of maxicircles packed into an electron-dense kDNA disk. The chaperones mtHsp70 and mtHsp40 and their cofactor Mge1 are uniformly distributed throughout the single mitochondrial network and are all essential for the parasite. Following RNA interference (RNAi)-mediated depletion of each of these proteins, the kDNA network shrinks and eventually disappears. Ultrastructural analysis of cells depleted for mtHsp70 or mtHsp40 revealed that the otherwise compact kDNA network becomes severely compromised, a consequence of decreased maxicircle and minicircle copy numbers. Moreover, we show that the replication of minicircles is impaired, although the lack of these proteins has a bigger impact on the less abundant maxicircles. We provide additional evidence that these chaperones are indispensable for the maintenance and replication of kDNA, in addition to their already known functions in Fe-S cluster synthesis and protein import.Impairment or loss of mitochondrial DNA is associated with mitochondrial dysfunction and a wide range of neural, muscular, and other diseases. We present the first evidence showing that the entire mtHsp70/mtHsp40 machinery plays an important role in mitochondrial DNA replication and maintenance, a function likely retained from prokaryotes. These abundant, ubiquitous, and multifunctional chaperones share phenotypes with enzymes engaged in the initial stages of replication of the mitochondrial DNA in T. brucei.
Project description:The mitochondrial DNA of Trypanosoma brucei, termed kinetoplast DNA or kDNA, consists of thousands of minicircles and a small number of maxicircles catenated into a single network organized as a nucleoprotein disk at the base of the flagellum. Minicircles are replicated free of the network but still contain nicks and gaps after rejoining to the network. Covalent closure of remaining discontinuities in newly replicated minicircles after their rejoining to the network is delayed until all minicircles have been replicated. The DNA ligase involved in this terminal step in minicircle replication has not been identified. A search of kinetoplastid genome databases has identified two putative DNA ligase genes in tandem. These genes (LIG k alpha and LIG k beta) are highly diverged from mitochondrial and nuclear DNA ligase genes of higher eukaryotes. Expression of epitope-tagged versions of these genes shows that both LIG k alpha and LIG k beta are mitochondrial DNA ligases. Epitope-tagged LIG k alpha localizes throughout the kDNA, whereas LIG k beta shows an antipodal localization close to, but not overlapping, that of topoisomerase II, suggesting that these proteins may be contained in distinct structures or protein complexes. Knockdown of the LIG k alpha mRNA by RNA interference led to a cessation of the release of minicircles from the network and resulted in a reduction in size of the kDNA networks and rapid loss of the kDNA from the cell. Closely related pairs of mitochondrial DNA ligase genes were also identified in Leishmania major and Crithidia fasciculata.
Project description:Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomatids, is a giant planar network of catenated minicircles and maxicircles. In vivo kDNA is organized as a highly condensed nucleoprotein disk. So far, in Trypanosoma brucei, proteins involved in the maintenance of the kDNA condensed structure remain poorly characterized. In Crithidia fasciculata, some small basic histone H1-like kinetoplast-associated proteins (CfKAP) have been shown to condense isolated kDNA networks in vitro. High-mobility group (HMG) box-containing proteins, such as mitochondrial transcription factor A (TFAM) in mammalian cells and Abf2 in the budding yeast, have been shown essential for the packaging of mitochondrial DNA (mtDNA) into mitochondrial nucleoids, remodeling of mitochondrial nucleoids, gene expression, and maintenance of mtDNA. Here, we report that TbKAP6, a mitochondrial HMG box-containing protein, is essential for parasite cell viability and involved in kDNA replication and maintenance. The RNA interference (RNAi) depletion of TbKAP6 stopped cell growth. Replication of both minicircles and maxicircles was inhibited. RNAi or overexpression of TbKAP6 resulted in the disorganization, shrinkage, and loss of kDNA. Minicircle release, the first step in kDNA replication, was inhibited immediately after induction of RNAi, but it quickly increased 3-fold upon overexpression of TbKAP6. Since the release of covalently closed minicircles is mediated by a type II topoisomerase (topo II), we examined the potential interactions between TbKAP6 and topo II. Recombinant TbKAP6 (rTbKAP6) promotes the topo II-mediated decatenation of kDNA. rTbKAP6 can condense isolated kDNA networks in vitro. These results indicate that TbKAP6 is involved in the replication and maintenance of kDNA.
Project description:The mitochondrial DNA (mtDNA) of a primitive kinetoplastid flagellate Cryptobia helicis is composed of 4.2 kb minicircles and 43 kb maxicircles. 85% and 6% of the minicircles are in the form of supercoiled (SC) and relaxed (OC) monomers, respectively. The remaining minicircles (9%) constitute catenated oligomers composed of both the SC and OC molecules. Minicircles contain bent helix and sequences homologous to the minicircle conserved sequence blocks. Maxicircles encode typical mitochondrial genes and are not catenated. The mtDNA, which we describe with the term 'pankinetoplast DNA', is spread throughout the mitochondrial lumen, where it is associated with multiple electron-lucent loci. There are approximately 8400 minicircles per pankinetoplast-mitochondrion, with the pan-kDNA representing approximately 36% of the total cellular DNA. Based on the similarity of the C.helicis minicircles to plasmids, we present a theory on the formation of the kDNA network.