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Background One of the most intriguing and unusual features of trypanosomatids is their mitochondrial DNA, known as kinetoplast DNA (kDNA), which is organized into a network of concatenated circles. The kDNA is contained within the mitochondrial matrix and can exhibit distinct arrangements across different species and during cell differentiation. In addition to kDNA, the kinetoplast contains multiple proteins, including those involved in mitochondrial DNA topology and metabolism, such as the kinetoplast-associated proteins (KAPs). In this work, we obtained mutant cells to investigates the role of KAP7 in two trypanosomatid species, Trypanosoma cruzi and Angomonas deanei , which have distinct kinetoplast shapes and kDNA arrangements. Methods For this purpose, the kDNA replication process and cell morphology and ultrastructure were evaluated using microscopy methods. Furthermore, the proliferation of cells treated with genotoxic agents, such as cisplatin and ultraviolet radiation, was analyzed. Results In A. deanei , which contains a symbiotic bacterium, KAP7 seems to be essential, since the deletion of one KAP7 allele generated mutants with a decay in cell proliferation, as well as changes in kDNA structure and replication. In T. cruzi , null mutants exhibited disturbances in kDNA replication, although the overall topology remained unaltered. The use of cisplatin and ultraviolet (UV) radiation affected the ultrastructure of A. deanei and T. cruzi . Cisplatin promoted increased kDNA compaction in both KAP7 mutants, but only in T. cruzi did the proliferative capacity fail to recover after treatment, as was also observed following UV radiation exposure. Conclusions Proteins associated with DNA are evolutionarily conserved and usually perform similar functions in different organisms. Our findings reveal that KAP7 is involved in kDNA replication, but its roles differ in trypanosomatid species: in A. deanei , KAP7 is associated with kDNA arrangement, while in T. cruzi , it is related to mitochondrial metabolism, such as kDNA replication and damage response. Graphical Abstract

Viability of the tsetse fly-transmitted African trypanosome Trypanosoma brucei depends on maintenance and expression of its kinetoplast (kDNA), the mitochondrial genome of this parasite and a putative target for veterinary and human antitrypanosomatid drugs. However, the closely related animal pathogens T. evansi and T. equiperdum are transmitted independently of tsetse flies and survive without a functional kinetoplast for reasons that have remained unclear. Here, we provide definitive evidence that single amino acid changes in the nuclearly encoded F ₁F O–ATPase subunit γ can compensate for complete physical loss of kDNA in these parasites. Our results provide insight into the molecular mechanism of compensation for kDNA loss by showing F O-independent generation of the mitochondrial membrane potential with increased dependence on the ADP/ATP carrier. Our findings also suggest that, in the pathogenic bloodstream stage of T. brucei , the huge and energetically demanding apparatus required for kDNA maintenance and expression serves the production of a single F ₁F O–ATPase subunit. These results have important implications for drug discovery and our understanding of the evolution of these parasites.

Trypanosoma brucei is a tractable protist parasite for which many genetic tools have been developed to study novel biology. A striking feature of T. brucei is the catenated mitochondrial DNA network called the kinetoplast DNA (kDNA) that is essential for parasite survival and life cycle completion. Maintenance of kDNA requires three independently essential paralogs that have homology to bacterial DNA polymerase I (POLIB, POLIC and POLID). We previously demonstrated that POLIB has a divergent domain architecture that displayed enzymatic properties atypical for replicative DNA polymerases. To evaluate the functional domains required for kDNA replication in vivo , we pursued an RNAi complementation approach based on the widely used tetracycline (Tet) single inducer system. Tet induction of RNAi and complementation with wildtype POLIB (POLIBWT) resulted in a 93% knockdown of endogenous POLIB mRNA but insufficient ectopic POLIBWT expression. This incomplete rescue emphasized the need for a more versatile induction system that will allow independent, tunable, and temporal regulation of gene expression. Hence, we adapted a dual control vanillic acid (Van)-Tet system that can independently control gene expression for robust RNAi complementation. Dual induction with Van and Tet (RNAi + Overexpression) resulted in 91% endogenous POLIB knockdown accompanied by robust and sustained ectopic expression of POLIBWT, and a near complete rescue of the POLIB RNAi defects. To more precisely quantify changes in kDNA size during RNAi, we also developed a semi-automated 3D image analysis tool to measure kDNA volume. Here we provide proof of principle for a dual inducer system that allows more flexible control of gene expression to perform RNAi and overexpression independently or concurrently within a single cell line. This system overcomes limitations of the single inducer system and can be valuable for elegant mechanistic studies in the field.

Leishmania kinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitive Leishmania detection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization of Leishmania minicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18 Leishmania strains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis of Leishmania relationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications of Leishmania spp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications in Leishmania research.

Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.

Background Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). Methods Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. Results Haplotype diversity of Sri Lankan isolates were high ( H d  = 0.757) with strong inter-geographical genetic differentiation ( F ST  > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. Conclusion This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis.

Extensive divergence of protein sequences from those of model eukaryotes makes recognition of well-studied signaling pathways impossible in Trypanosoma brucei . Consequently, substrates and effectors of most protein kinases are unknown. We addressed these general problems by developing workflows to (i) identify effectors for protein kinases, (ii) discover new effectors and substrates of CK1.2 as proof of principle, and (iii) decode linear motifs in CK1.2 substrates . Casein kinase CK1.2 promotes cytokinesis and division of mitochondrial DNA (kinetoplast) (kDNA). Applying our principles to CK1.2 we identified four new substrates, BBP59, LRRP1, CAP64, and BBP268, that harbored two unique linear motifs, S-x(1,3)-T-x(1,4)-S-x(2)-A-x(2)-[AIV] and S-x(1,3)-T-x(1,4)-S. Ectopic expression of BBP59 inhibited cytokinesis and kinetoplast DNA division, like knockdown of CK1.2. Phosphorylation of BBP59 was blocked after knockdown of CK1.2 or treatment with SB-431542 an inhibitor of CK1.2 (IC 50 49.2 nM). Highlighting phospho-regulation of its activities, Ser72 substitution with Ala72 or Asp72 in BBP59 caused preferential blockage of cytokinesis or kDNA division. The sequence-agnostic workflow used to identify effectors of CK1.2 can be deployed for other protein kinases, and to assist functional annotation of hypothetical proteins.

Kinetoplast (k) DNA, a specialized form of mitochondrial DNA in trypanosomes, is composed of catenated maxicircles and minicircles, and is essential for the survival of these parasitic protists. In this study, we provide a detailed description of the near-complete minicircle complement of Trypanosoma musculi , a globally distributed, mouse-specific haemoflagellate. We have identified 39 minicircle classes, which can be grouped via their structural organization into five distinct categories. Using RNA-seq, we have reconstructed mitochondrial transcripts of ATP6 , RPS12 , CYTB , COX2 and COX3 , all of which undergo canonical editing that renders them translatable, as well as abnormally edited transcripts of four respiratory Complex I subunits. Additionally, through a combined analysis of the kDNA minicircles, edited mRNAs and small RNAs, we identified 108 guide (g)RNA genes and further categorized their conservation in comparison with Trypanosoma lewisi , an opportunistic human parasite. Moreover, we demonstrate a small respiratory Complex I in T. musculi , highlighting the functional consequences of missing gRNAs. Finally, we propose a model for the evolutionary maintenance of gRNAs, contributing to our understanding of kDNA dynamics in trypanosomes and other kinetoplastid flagellates. Mitochondrial kDNA and RNA editing in Trypanosoma musculi are associated with a small Complex I, providing insights into mitochondrial metabolism and kDNA dynamics in trypanosomes.

Genetic exchange has not been shown to be a mechanism underlying the extensive diversity of Leishmania parasites. We report here evidence that the invertebrate stages of Leishmania are capable of having a sexual cycle consistent with a meiotic process like that described for African trypanosomes. Hybrid progeny were generated that bore full genomic complements from both parents, but kinetoplast DNA maxicircles from one parent. Mating occurred only in the sand fly vector, and hybrids were transmitted to the mammalian host by sand fly bite. Genetic exchange likely contributes to phenotypic diversity in natural populations, and analysis of hybrid progeny will be useful for positional cloning of the genes controlling traits such as virulence, tissue tropism, and drug resistance.

Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania) infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1), whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2). The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania) and Leishmania (Viannia) using the qPCR2 assay followed by melting or High Resolution Melt (HRM) analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania) and Leishmania (Viannia) subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L.) infantum WHO international reference strain (MHOM/TN/80/IPT1), highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical samples, must be taken into account in quantitative PCR-based applications; however, it might also be used to differentiate between Leishmania subgenera.