Search

Mitochondrial DNA replication is an essential process in most eukaryotes. Similar to the diversity in mitochondrial genome size and organization in the different eukaryotic supergroups, there is considerable diversity in the replication process of the mitochondrial DNA. In this review, we summarize the current knowledge of mitochondrial DNA replication and the associated factors in trypanosomes with a focus on Trypanosoma brucei , and provide a new model of minicircle replication for this protozoan parasite. The model assumes the mitochondrial DNA (kinetoplast DNA, kDNA) of T. brucei to be loosely diploid in nature and the replication of the genome to occur at two replication centers at the opposing ends of the kDNA disc (also known as antipodal sites, APS). The new model is consistent with the localization of most replication factors and in contrast to the current model, it does not require the assumption of an unknown sorting and transport complex moving freshly replicated DNA to the APS. In combination with the previously proposed sexual stages of the parasite in the insect vector, the new model provides a mechanism for maintenance of the mitochondrial genetic diversity. [ABSTRACT FROM AUTHOR]

Parasites of medical importance, such as Leishmania and Trypanosoma, are characterized by the presence of thousands of circular DNA molecules forming a structure known as kinetoplast, within the mitochondria. The maxicircles, which are equivalent to the mitochondrial genome in other eukaryotes, have been proposed as a promising phylogenetic marker. Using whole-DNA sequencing data, it is also possible to assemble maxicircle sequences as shown here and in previous works. In this study, based on data available in public databases and using a bioinformatics workflow previously reported by our group, we assembled the complete coding region of the maxicircles for 26 prototypical strains of trypanosomatid species. Phylogenetic analysis based on this dataset resulted in a robust tree showing an accurate taxonomy of kinetoplastids, which was also able to discern between closely related Leishmania species that are usually difficult to discriminate by classical methodologies. In addition, we provide a dataset of the maxicircle sequences of 60 Leishmania infantum field isolates from America, Western Europe, North Africa, and Eastern Europe. In agreement with previous studies, our data indicate that L. infantum parasites from Brazil are highly homogeneous and closely related to European strains, which were transferred there during the discovery of America. However, this study showed the existence of different L. infantum populations/clades within the Mediterranean region. A maxicircle signature for each clade has been established. Interestingly, two L. infantum clades were found coexisting in the same region of Spain, one similar to the American strains, represented by the Spanish JPCM5 reference strain, and the other, named "non-JPC like", may be related to an important leishmaniasis outbreak that occurred in Madrid a few years ago. In conclusion, the maxicircle sequence emerges as a robust molecular marker for phylogenetic analysis and species typing within the kinetoplastids, which also has the potential to discriminate intraspecific variability. [ABSTRACT FROM AUTHOR]

Rbp38 is a protein exclusively found in trypanosomatid parasites, including Leishmania amazonensis , the etiologic agent of tegumentar leishmaniasis in the Americas. The protein was first described as a Leishmania tarentolae mitochondrial RNA binding protein. Later, it was shown that the trypanosomes Rbp38 orthologues were exclusively found in the mitochondria and involved in the stabilization and replication of kinetoplast DNA (kDNA). In contrast, L. amazonensis Rbp38 (LaRbp38), co-purifies with telomerase activity and interacts not only with kDNA but also with telomeric DNA, although shares with its counterparts high sequence identity and a putative N-terminal mitochondrial targeting signal (MTS). To understand how LaRbp38 interacts both with nuclear and kDNA, we have first investigated its subcellular localization. Using hydroxy-urea synchronized L. amazonensis promastigotes we could show that LaRbp38 shuttles from mitochondria to the nucleus at late S and G2 phases. Further, we identified a non-classical nuclear localization signal (NLS) at LaRbp38 C-terminal that binds with importin alpha, a protein involved in the nuclear transport of several proteins. Also, we obtained LaRbp38 truncated forms among which, some of them also showed an affinity for both telomeric DNA and kDNA. Analysis of these truncated forms showed that LaRbp38 DNA-binding region is located between amino acid residues 95–235. Together, our findings strongly suggest that LaRbp38 is multifunctional with dual subcellular localization. • Rbp38 prior described as a mitochondrial protein, in Leishmania sp. is also found in the nucleus. • L. amazonensis Rbp38 (LaRbp38) dual subcellular localization is cell cycle-dependent. • LaRbp38 has a monopartite non-classical NLS that binds to importin-alpha. • LaRbp38 binds telomeric G-rich DNA and kDNA. • LaRbp38 DNA-binding region is located between amino acid residues 95–235. [ABSTRACT FROM AUTHOR]

Inheritance of the single mitochondrial nucleoid (kinetoplast) in the trypanosome requires numerous proteins, many of whose precise roles are unclear. By considering kinetoplast DNA (kDNA) as a template for cleavage into two equal-size networks, we predicted sets of mutant kinetoplasts associated with defects in each of the five steps in the kinetoplast cycle. Comparison of these kinetoplasts with those obtained after gene knockdowns enabled assignment of proteins to five classes – kDNA synthesis, site of scission selection, scission, separation, and partitioning. These studies highlight how analysis of mutant kinetoplast phenotypes may be used to predict functional categories of proteins involved in the biogenesis of kinetoplasts. Highlights Kinetoplasts (mitochondrial genome nucleoids) are important in bloodstream trypanosomes for the establishment of mitochondrial membrane potential. Many proteins involved in segregation of kinetoplasts have been identified. A region between a kinetoplast and basal bodies is described as a tripartite attachment complex (TAC). A set of TAC-associated proteins (TACAPs) has been proposed as the machinery for kinetoplast segregation. Subcomplexes of TACAPs that form in vivo have been described. Several proteins that do not associate with TAC are involved in the maintenance of the kinetoplast. New kinetoplast-associated proteins have been identified. We are approaching an exciting period in the field when a molecular understanding of how all aspects of kinetoplast biogenesis are executed seems achievable. [ABSTRACT FROM AUTHOR]

Background: Leishmania contains a concatenated mitochondrial DNA, kDNA. Universal minicircle sequence binding protein (UMSBP), a mitochondrial protein, initiates kDNA replication by binding with a conserved universal minicircle sequence (UMS) of kDNA. Here, we describe first time in L. donovani the regulation of DNA binding activity of UMSBP and the role of UMSBP in virulence. Methods: Insilco and EMSA study were performed to show UMS-binding activity of UMSBP. Tryparedoxin(TXN)-tryparedoxin peroxidase(TXNPx) assay as well as co-overexpression of cytochrome-b5 reductase-like protein (CBRL) and tryparedoxin in L. donovani were done to know the regulation of DNA binding activity of UMSBP. Knockout and episomal-expression constructs of UMSBP were transfected in L. donovani. The cell viability assay and immunofluorescence study to know the status of kDNA were performed. Macrophages were infected with transfected parasites. mRNA level of cytochrome b, activity of complex-III, intracellular ATP level of both transfected promastigotes and amastigotes as well as ROS concentration and the level of apoptosis of transfected promastigotes were measured. Level of oxidative phosphorylation of both transfected and un-transfected amastigotes were compared. Burden of transfected amastigotes in both macrophages and BALB/c mice were measured. Results: L. donovani UMSBP is capable of binding with UMS, regulated by redox through mitochondrial enzymes, TXN, TXNPx and CBRL. Depletion of UMSBP (LdU-/-) caused kDNA loss, which decreased cytochrome-b expression [component of complex-III of electron transport chain (ETC)] and leads to the disruption of complex-III activity, decreased ATP generation, increased ROS level and promastigotes exhibited apoptosis like death. Interestingly, single knockout of UMSBP (LdU-/+) has no effect on promastigotes survival. However, single knockout in intracellular amastigotes demonstrate loss of mRNA level of cytochrome-b, disruption in the activity of complex-III and reduced production of ATP in amastigotes than wild type. This process interfere with the oxidative-phosphorylation and thereby completely inhibit the intracellular proliferation of LdU-/+ amastigotes in human macrophages and in BALB/c mice. Amastigotes proliferation was restored as wild type after episomal expression of LdUMSBP in LdU-/+ parasites (LdU-/+ AB). Conclusion: The LdUMSBP regulates leishmanial mitochondrial respiration and pathogenesis. So, LdUMSBP may be an attractive target for rational drug designing and LdU-/+ parasites could be considered as a live attenuated vaccine candidate against visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs. [ABSTRACT FROM AUTHOR]

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, class Kinetoplastea, family Trypanosomatidae. In Colombia it is estimated that there are between 700,000 and 1,200,000 inhabitants infected with T. cruzi and about 8,000,000 people at risk of acquiring the infection. Kinetoplastids are characterized by a single mitochondrion with an intricate organization, consisting of a network of dozens of maxicircles and thousands of concatenated minicircles representing up to 20-25% of the total amount of DNA per cell. Maxicircles contain the genes involved in the mitochondrial electron transport chain and their study may help to understand the phylogenetic and evolutionary relationships of this group of parasites and adaptive processes. The aim of the present work was to identify the genomic characteristics of the maxicircles and minicircles (kDNA) of T. cruzi circulating in Colombia, by using sequencing technologies based on long reads, to obtain complete genomes that encompass all complex regions that present a repetitive naturalness and low percentages of Guanine/Citokine, difficult to appreciate through studies with short reads. A strain from a rural area of the municipality of Coyaima-Tolima isolated from the natural reservoir Didelphis marsupialis was used, which was sequenced by PacBio Hifi 100x technology. As for the maxicircles, 10 molecules were assembled de novo, with a size range between 21,950-47,166 bp, whose heterogeneity in their final size is mainly due to the length of the divergent region which presents sizes ranging from 6,400 bp to 32,000 bp. Through bioinformatics tools, a final molecule with a size of 47,166 bp was obtained and its circular nature was verified by bioinformatics methods. Its annotation revealed a total of 2 ribosomal genes and 18 structural genes that showed perfect synteny with mitochondrial genomes of other trypanosomatids previously reported. However, partial and complete deletions in the ND5 and RPS12 genes were found in some assembled molecules, suggesting possible heteroplasmy phenomena within the sequenced strain. Additionally, it was possible to obtain the complete divergent region, finding that its size was being underestimated. For this reason, a deeper analysis of this region, which has been the least studied of this genome, was performed, distinguishing specific characteristics such as its repetitive nature. A representative repertoire of sequences was obtained for the minicircles, which presented genomic and size heterogeneity, finding different numbers of conserved regions (Conserved Sequence Blocks-CSB) and hypervariables. In the present work, the first report of mitochondrial genomes of a strain isolated from southern Tolima was carried out. The usefulness of sequencing based on long reads for the analysis of complex genomes, such as the kinetoplast (kDNA), which presents low percentages of Guanine/Citokine and very repetitive regions, was demonstrated, thus contributing to the study of unexplored regions such as the divergent region, laying the foundations for future studies that can evaluate the biological implications that this region has in molecular processes in trypanosomatids. [ABSTRACT FROM AUTHOR]

The mitochondrial genome of kinetoplastids, called kinetoplast DNA (kDNA) is a complex structure that must be faithfully duplicated exactly once per cell cycle. Despite many years of thorough investigation into the kDNA replication mechanism, many of the molecular details of the later stages of the process, particularly kDNA division and segregation, remain mysterious. In addition, perturbation of several cellular activities, some only indirectly related to kDNA, can lead to asymmetric kDNA division and other segregation defects. This review will examine unifying features and possible explanations for these phenotypes in the context of current models for kDNA division and segregation. [ABSTRACT FROM AUTHOR]

Abstract: Trypanosomatid mitochondrial DNA (kDNA) possesses thousands of copies of small circular molecules called minicircles. Due to a high level of nucleotide polymorphism among copies, sequence alignment for species or strain characterization is not appropriate. In this work we report dinucleotide absolute frequency as a method to analyze minicircle sequences heterogeneity in trypanosomatids. Using Trypanosoma rangeli and Leishmania guyanensis minicircles as example of sequence length heterogeneity, we show that dinucleotide frequency of minicircles whose length variation is less than to 10% is relatively constant. Dinucleotide frequencies in Leishmania genus point out three clusters of predominant dinucleotide profiles: GG/TT/TG for Old World species; ii) TT/AA/TA for New World species and iii) TT/GG(AA) TA(AT) for Sauroleishmania. Trypanosoma species displayed broad range composition and the highest frequency values. Their dinucleotide profile appears to be species specific, except for African trypanosomes which exhibit similar composition. The low number of sequences from Crithidia, Herpetomonas, Phytomonas and Wallaceina did not allow a generalized analysis, however some species present highly similar compositional profile, e.g., Wallaceina species. Distinct signatures for Trypanosomatidae family members can be generated by using values of absolute frequencies, range and composition of most/least frequent dinucleotides from minicircles. Each species can be graphically represented by a diagram of frequencies along with a box plot of summary statistics. [Copyright &y& Elsevier]

Abstract: DNA replication mechanisms are poorly understood in most of trypanosomatids, in particular the replication of the peculiar mitochondrial DNA, the kinetoplast DNA (kDNA). To contribute to the knowledge on the mechanism of kDNA replication in Trypanosoma cruzi, we have previously characterized the Universal Minicircle Sequence Binding Protein of this parasite (TcUMSBP), which was first called PDZ5 [E.R. Coelho, T.P. Urmenyi, J. Franco da Silveira, E. Rondinelli, R. Silva, Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi, Int. J. Parasitol. 33 (2003) 853–858]. In this work, we describe two highly polymorphic alleles of the TcUMSBP locus in the T. cruzi reference clone CL Brener and the differential expression pattern of these alleles. A 62bp sequence in the TcUMSBP upstream intergenic region in one of its alleles affects the efficiency of polycistronic RNA processing and the polyadenylation sites, and therefore regulates the differential expression of TcUMSBP alleles of this locus. [Copyright &y& Elsevier]

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 ∼8400 minicircles per pankinetoplast­mitochondrion, with the pan-kDNA representing ∼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. [ABSTRACT FROM AUTHOR]

The parasite protozoan Leishmania, the causative agent of leishmaniasis, includes two subgenera of medical interest: Leishmania (Leishmania) and Leishmania (Viannia). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the Leishmania genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish L. (L.) infantum and L. (L.) amazonensis from L. Viannia species. Successively, this assay has been integrated with other qPCR assays, to differentiate L. (L.) infantum, L. (L.) amazonensis and L. (L.) mexicana. In this work, we tested the applicability of our qPCR-ML assay on L. (L.) donovani, L. (L.) major, L. (L.) tropica and L. (L.) aethiopica, showing that the qPCR-ML assay can also amplify Old World species, different from L. (L.) infantum, with good quantification limits (1 × 10−4–1 × 10−6 ng/pcr tube). Moreover, we evaluated 11 L. (L.) infantum strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-Leishmania assay. [ABSTRACT FROM AUTHOR]