Search

The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.

To explore a new set of cytotoxic agents, β-carboline-combretastatin carboxamide conjugates were designed, synthesized and evaluated for their in vitro cytotoxicity potential, DNA binding affinity and Topoisomerase-II (topo-II) inhibition activity. Among the designed hybrids, 10v and 10af have shown significant cytotoxic effect against A549 (lung cancer) cell line having IC 50 value 1.01 µM and 1.17 µM respectively. Further, it was speculated that treatment with compound 10v may induce apoptosis among A549 cells, which was supported by Hoechst staining, DCFDA, Annexin V-FITC and morphological assays. Flow cytometric analysis revealed that the hybrid 10v arrests A549 cells in G2/M phase of cell cycle in a dose dependent manner. Amongst the active hybrids, most potent hybrid 10v was tested for DNA topo-II inhibition activity. Moreover, to further support the biological activity and to infer the mode of interaction between ligands and DNA, spectroscopy and molecular docking studies were carried out. The docking and spectroscopy results showed that the ligands exhibited an intercalative mode of binding with DNA and could efficiently bind to DNA and form topo-II ternary complex. Based on these experiments, the hybrids 10v and 10af were identified as proficient new scaffolds which need to be developed as hit molecules for therapeutic interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In Brazil, Leishmania amazonensis is one of the etiological agents of tegumentary leishmaniasis and can cause a wide spectrum of diseases in humans, resulting in cutaneous, mucosal, diffuse, and even visceral leishmaniasis. Besides, this species has also been reported to affect dogs, causing typical symptoms of visceral disease. Unfortunately, the diagnostic of the Leishmania species is not routinely performed due to the difficulties of the available methods. In view of this, different molecular methods have been used in an attempt to solve the problem of diagnosis. Loop-mediated isothermal amplification (LAMP) is a relatively new nucleic acid amplification method, which has been successfully applied in the diagnosis of Leishmania spp. infections. However, this is the first work that standardizes a specific LAMP reaction for L. amazonensis. The set of primers selected were designed from the kDNA minicircle sequence of the L. amazonensis (GenBank: U19810.1). The LAMP assay developed in the present study showed 100% specificity and 89% sensitivity when compared with conventional PCR and was more sensitive than qPCR. In addition, the LAMP reaction developed here was able to amplify a qPCR sample with a parasite load of only 28 parasites in 50 ng of DNA. Consequently, considering the LAMP reaction specific to L. amazonensis and several advantages of the method (such as high efficiency, sensitivity and specificity), we believe that this reaction can be used as a promising diagnostic tool in clinical practice, field studies, and research., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Leishmania is a parasitic protozoan which is transmitted to humans through the bite of an infected female Phlebotomus and Lutzomyia sand flies. Cutaneous leishmaniasis (CL), caused by Leishmania major and L. tropica, is an endemic disease in many areas of Jordan and considered as a major public health problem. The political instability in the Syrian Arab Republic has resulted in the immigration of large number of refugees into Jordan where most of them resided in camps near the Syrian borders. Therefore, the main objective of the present study was to inspect Leishmania species/genotypes which are responsible for CL infections among Syrian refugees and compare them with the recovered species/genotypes isolated from Jordanian patients. Three molecular-based assays (ITS1-PCR-RFLP, Nested ITS1-5.8S rDNA PCR, and Kinetoplast DNA PCR) followed by sequencing and phylogenetic analysis were undertaken and compared for their efficiency to confirm CL diagnosis and genotype the infecting Leishmania species. Thereafter, the evolutionary relationships among various Leishmania isolates from Syrian and Jordanian CL patients were elucidated. Results from the present study indicated that 20 and 9 out of the inspected 66 patients (39 Jordanian and 27 Syrian) were infected with L. major and L. tropica respectively. ITS1-PCR RFLP typing proved to be more sensitive in the detection of Leishmania species (positive in 44% of the isolates) compared to both ITS1-5.8S rDNA gene and Kinetoplast DNA PCR which were successful in identifying Leishmania species only in 23% and 33% of the isolates respectively. Sequencing and phylogenetic analysis of ITS1 and ITS1-5.8S rDNA genes revealed high levels of heterogeneity among the sequenced isolates. One sample typed as L. tropica from Jordanian patient showed high similarity with L. tropica sample isolated from a Syrian patient in a Lebanon refugee camp; therefore, the need for comprehensive studies to confirm if any new L. tropica strains might be introduced to Jordan by Syrian refugees is urgently indicated. These observations highlighted the need for further studies to clarify the risk status of species and strains which might be introduced from Syria to Jordan.

This study aimed to explore geographic distribution and molecular characterization of cutaneous leishmaniasis (CL) species by amplifying two popular markers in kinetoplast DNA and internal transcribed spacer 1 loci by nested-PCR, and characterized by sequencing and phylogenetic analyses. Findings demonstrated that two species co-existed in the province: L. tropica (88.5%) and L. major (11.5%). All gender and age groups were equally infected, although males, 21-30 years old, exhibited a significantly higher infection. Sequencing and phylogenetic analyses of 34 randomly selected samples showed that L. tropica isolates exhibited some degree of heterogeneity. Both anthroponotic CL and zoonotic CL are present in south-eastern Iran with predominance of L. tropica species. Some level of heterogeneity was observed in L. tropica isolates which possibly reflects different colonies in the area. Implementation of diagnostic tools directly from clinical samples could be an important strategic approach for exploration of spatial distribution, molecular characterization and phylogenetic analyses.

While the vast majority of cellular DNA in eukaryotes is contained in long linear strands in chromosomes, we have long recognized some exceptions like mitochondrial DNA, plasmids in yeasts, and double minutes (DMs) in cancer cells where the DNA is present in extrachromosomal circles. In addition, specialized extrachromosomal circles of DNA (eccDNA) have been noted to arise from repetitive genomic sequences like telomeric DNA or rDNA. Recently eccDNA arising from unique (nonrepetitive) DNA have been discovered in normal and malignant cells, raising interesting questions about their biogenesis, function and clinical utility. Here, we review recent results and future directions of inquiry on these new forms of eccDNA., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

Trypanosoma brucei, the causative agent of sleeping sickness (Human African Trypanosomiasis, HAT), contains a kinetoplast with the mitochondrial DNA (kDNA), comprising of >70% AT base pairs. This has prompted studies of drugs interacting with AT-rich DNA, such as the N-phenylbenzamide bis(2-aminoimidazoline) derivatives 1 [4-((4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide dihydrochloride] and 2 [N-(3-chloro-4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)-4-((4,5-dihydro-1H-imidazol-2-yl)amino)benzamide] as potential drugs for HAT. Both compounds show in vitro effects against T. brucei and in vivo curative activity in a mouse model of HAT. The main objective was to identify their cellular target inside the parasite. We were able to demonstrate that the compounds have a clear effect on the S-phase of T. brucei cell cycle by inflicting specific damage on the kinetoplast. Surface plasmon resonance (SPR)-biosensor experiments show that the drug can displace HMG box-containing proteins essential for kDNA function from their kDNA binding sites. The crystal structure of the complex of the oligonucleotide d[AAATTT]2 with compound 1 solved at 1.25 Å (PDB-ID: 5LIT) shows that the drug covers the minor groove of DNA, displaces bound water and interacts with neighbouring DNA molecules as a cross-linking agent. We conclude that 1 and 2 are powerful trypanocides that act directly on the kinetoplast, a structure unique to the order Kinetoplastida., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Non-coding RNA (ncRNA) genes make transcripts as same as the encoding genes, and ncRNAs directly function as RNAs rather than serve as blueprints for proteins. As the function of ncRNA is closely related to organelle genomes, it is desirable to explore ncRNA function by confirming its provenance. In this paper, the topology secondary structure, motif and the triplets under three reading frames are considered as parameters of ncRNAs. A method of SVM combining the increment of diversity (ID) algorithm is applied to construct the classifier. When the method is applied to the ncRNA dataset less than 80% sequence identity, the overall accuracies reach 95.57%, 96.40% in the five-fold cross-validation and the jackknife test, respectively. Further, for the independent testing dataset, the average prediction success rate of our method achieved 93.24%. The higher predictive success rates indicate that our method is very helpful for distinguishing ncRNAs from various organelle genomes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The haemoflagellate Trypanosoma lewisi is a kinetoplastid parasite which, as it has been recently reported to cause human disease, deserves increased attention. Characteristic features of all kinetoplastid flagellates are a uniquely structured mitochondrial DNA or kinetoplast, comprised of a network of catenated DNA circles, and RNA editing of mitochondrial transcripts. The aim of this study was to describe the kinetoplast DNA of T. lewisi., Methods/results: In this study, purified kinetoplast DNA from T. lewisi was sequenced using high-throughput sequencing in combination with sequencing of PCR amplicons. This allowed the assembly of the T. lewisi kinetoplast maxicircle DNA, which is a homologue of the mitochondrial genome in other eukaryotes. The assembly of 23,745 bp comprises the non-coding and coding regions. Comparative analysis of the maxicircle sequence of T. lewisi with Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma brucei and Leishmania tarentolae revealed that it shares 78%, 77%, 74% and 66% sequence identity with these parasites, respectively. The high GC content in at least 9 maxicircle genes of T. lewisi (ATPase6; NADH dehydrogenase subunits ND3, ND7, ND8 and ND9; G-rich regions GR3 and GR4; cytochrome oxidase subunit COIII and ribosomal protein RPS12) implies that their products may be extensively edited. A detailed analysis of the non-coding region revealed that it contains numerous repeat motifs and palindromes., Conclusions: We have sequenced and comprehensively annotated the kinetoplast maxicircle of T. lewisi. Our analysis reveals that T. lewisi is closely related to T. cruzi and T. brucei, and may share similar RNA editing patterns with them rather than with L. tarentolae. These findings provide novel insight into the biological features of this emerging human pathogen.

Background: The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking., Methods: We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed., Principal Findings: Persistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus., Conclusions: Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.

In this work we present a computational study of the kinetoplast genome, modelled as a large number of semiflexible unknotted loops, which are allowed to link with each other. As the DNA density increases, the systems shows a percolation transition between a gas of unlinked rings and a network of linked loops which spans the whole system. Close to the percolation transition, we find that the mean valency of the network, i.e. the average number of loops which are linked to any one loop, is around three, as found experimentally for the kinetoplast DNA (kDNA). Even more importantly, by simulating the digestion of the network by a restriction enzyme, we show that the distribution of oligomers, i.e. structures formed by a few loops which remain linked after digestion, quantitatively matches experimental data obtained from gel electrophoresis, provided that the density is, once again, close to the percolation transition. With respect to previous work, our analysis builds on a reduced number of assumptions, yet can still fully explain the experimental data. Our findings suggest that the kDNA can be viewed as a network of linked loops positioned very close to the percolation transition, and we discuss the possible biological implications of this remarkable fact.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major parasitic disease that affects millions of people in America. However, despite the high impact of this disease on human health, no effective and safe treatment has been found that eliminates the infecting parasite from human patients. Among the possible chemotherapeutic targets that could be considered for study in T. cruzi are the DNA polymerases, in particular DNA polymerase beta (polß), which previous studies have shown to be involved in kinetoplast DNA replication and repair. In this paper, we describe the expression, purification, and biochemical characterization of the Miranda clone polß, corresponding to lineage T. cruzi I (TcI). The recombinant enzyme purified to homogeneity displayed specific activity in the range described for a highly purified mammalian polß. However, the trypanosome enzyme exhibited important differences in biochemical properties compared to the mammalian enzymes, specifically an almost absolute dependency on KCl, high sensitivity to N-ethylmaleimide (NEM), and low sensitivity to ddTTP. Immuno-affinity purification of T. cruzi polymerase beta (Tcpolß) from epimastigote extracts showed that the native enzyme was phosphorylated. In addition, it was demonstrated that Tcpolß interacts with some proteins in a group of about 15 proteins which are required to repair 1-6 bases of gaps of a double strand damaged DNA. It is possible that these proteins form part of a DNA repair complex, analogous to that described in mammals and some trypanosomatids.

Leishmania donovani is a kinetoplastid protozoan parasite which causes the fatal disease visceral leishmaniasis in humans. Genome sequencing of L. donovani revealed information about the arrangement of genes and genome architecture. After curation of the genome sequence, many genes in L. donovani were assigned as truncated or "partial" genes by the genome sequencing group. In the present study, we have carried out an extensive analysis and attempted to improve the gene models of these partial genes. Our analysis resulted in the identification of 308 partial genes in L. donovani, which were further categorized as C-terminal extensions, joining of genes, tandemly repeated paralogs and wrong chromosomal assignments. We have analyzed each of these genes from these categories and have improved the annotation of existing gene models in L. donovani. Some of these corrections have been confirmed by mass spectrometry derived peptide data from our previous comparative proteogenomics study in L. donovani., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We previously identified salicylate as a novel catalytic inhibitor of human DNA topoisomerase II (topo II; EC 5.99.1.3) that preferentially targets the alpha isoform by interfering with topo II-mediated DNA cleavage. Many pharmaceuticals and compounds found in foods are salicylate-based. We have now investigated whether these are also catalytic inhibitors of topo II and the structural determinants modulating these effects. We have determined that a number of hydroxylated benzoic acids attenuate doxorubicin-induced DNA damage signaling mediated by the ATM protein kinase and inhibit topo II decatenation activity in vitro with varying potencies. Based on the chemical structures of these and other derivatives, we identified unique properties influencing topo II inhibition, including the importance of substitutions at the 2'- and 5'-positions. We extended our findings to a number of salicylate-based pharmaceuticals including sulfasalazine and diflunisal and found that both were effective at attenuating doxorubicin-induced DNA damage signaling, topo II DNA decatenation and they blocked stabilization of doxorubicin-induced topo II cleavable complexes in cells. In a manner similar to salicylate, we determined that these agents inhibit topo II-mediated DNA cleavage. This was accompanied by a concomitant decrease in topo II-mediated ATP-hydrolysis. Taken together, these findings reveal a novel function for the broader class of salicylate-related compounds and highlight the need for additional studies into whether they may impact the efficacy of chemotherapy regimens that include topo II poisons., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Leishmania species belong to the family Trypanosomatidae and cause leishmaniasis, a geographically widespread disease that infects humans and other vertebrates. This disease remains endemic in China. Due to the large geographic area and complex ecological environment, the taxonomic position and phylogenetic relationship of Chinese Leishmania isolates remain uncertain. A recent internal transcribed spacer 1 and cytochrome oxidase II phylogeny of Chinese Leishmania isolates has challenged some aspects of their traditional taxonomy as well as cladistics hypotheses of their phylogeny. The current study was designed to provide further disease background and sequence analysis., Methods: We systematically analyzed 50 cytochrome b (cyt b) gene sequences of 19 isolates (16 from China, 3 from other countries) sequenced after polymerase chain reaction (PCR) using a special primer for cyt b as well as 31 sequences downloaded from GenBank. After alignment, the data were analyzed using the maximum parsimony, Bayesian and netwok methods., Results: Sequences of six haplotypes representing 10 Chinese isolates formed a monophyletic group and clustered with Leishmania tarentolae. The isolates GS1, GS7, XJ771 of this study from China clustered with other isolates of Leishmania donovani complex. The isolate JS1 was a sister to Leishmania tropica, which represented an L. tropica complex instead of clustering with L. donovani complex or with the other 10 Chinese isolates. The isolates KXG-2 and GS-GER20 formed a monophyletic group with Leishmania turanica from central Asia. In the different phylogenetic trees, all of the Chinese isolates occurred in at least four groups regardless of geographic distribution., Conclusions: The undescribed Leishmania species of China, which are clearly causative agents of canine leishmaniasis and human visceral leishmaniasis and are related to Sauroleishmania, may have evolved from a common ancestral parasite that came from the Americas and may have split off earlier than the other old world Leishmania. Our results also suggest the following: the isolates GS7, GS1 and XJ771 occur as part of the L. donovani complex; the JS1 isolate is L. tropica; and the isolate GS-GER20 identified as Leishmania gerbilli is close to KXG-2 which is L. turanica.

Little is known about the sylvatic transmission cycle of Trypanosoma cruzi in the Gran Chaco ecoregion. We conducted surveys to identify the main sylvatic hosts of T. cruzi, parasite discrete typing units and vector species involved in Pampa del Indio, a rural area in the humid Argentinean Chaco. A total of 44 mammals from 14 species were captured and examined for infection by xenodiagnosis and polymerase chain reaction amplification of the hyper-variable region of kinetoplast DNA minicircles of T. cruzi (kDNA-PCR). Ten (22.7%) mammals were positive by xenodiagnosis or kDNA-PCR. Four of 11 (36%) Didelphis albiventris (white-eared opossums) and six of nine (67%) Dasypus novemcinctus (nine-banded armadillos) were positive by xenodiagnosis and or kDNA-PCR. Rodents, other armadillo species, felids, crab-eating raccoons, hares and rabbits were not infected. Positive animals were highly infectious to the bugs that fed upon them as determined by xenodiagnosis. All positive opossums were infected with T. cruzi I and all positive nine-banded armadillos with T. cruzi III. Extensive searches in sylvatic habitats using 718 Noireau trap-nights only yielded Triatoma sordida whereas no bug was collected in 26 light-trap nights. Four armadillos or opossums fitted with a spool-and-line device were successfully tracked to their refuges; only one Panstrongylus geniculatus was found in an armadillo burrow. No sylvatic triatomine was infected with T. cruzi by microscopical examination or kDNA-PCR. Our results indicate that two independent sylvatic transmission cycles of T. cruzi occur in the humid Chaco. The putative vectors of both cycles need to be identified conclusively., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Fifty-six Trypanosoma cruzi stocks from Chile and neighboring countries and different hosts, humans, and Triatoma infestans and Mepraia sp., vectors of domiciliary and natural environments were characterized by using three molecular markers. These were cytochrome b (Cyt b) gene sequencing, minicircle DNA blotting, and hybridization with five genotype-specific DNA probes and nuclear analysis of 1f8 and gp72 by polymerase chain reaction-restriction fragment length polymorphism. The results with all three molecular markers are concordant, with minor limitations, grouping T. cruzi stocks into four discrete typing units (DTUs) (TcI, TcII, TcV, and TcVI). TcI and TcII stocks were heterogeneous. TcI and TcII stocks were clustered in two main subgroups determined by Cyt b gene sequencing and minicircle hybridization. However, TcV and TcVI stocks were homogeneous and not differentiated by Cyt b gene sequencing or minicircle DNA hybridization. The discriminatory power and limitations of the molecular markers are discussed, as well as the distribution of the four DTUs in the domiciliary and sylvatic transmission cycles of Chile and the limitations of each marker for molecular epidemiological studies performed with T. cruzi stocks rather than the analysis of direct T. cruzi samples from natural hosts.

To determine the infestation and trypanosome infection of triatomines captured in Mauritia flexuosa palm trees across its geographic distribution in the Brazilian savanna (Cerrado), we sampled 42 localities in eight states and in the Federal District, Brazil, between July 2005 and January 2010. Overall, 2154 specimens of the species Rhodnius neglectus, Psammolestes tertius, Triatoma sordida, and Microtriatoma borbai, were collected. Among the 341 palms sampled, 182 (53.3%) were infested with R. neglectus, which resulted in the capture of 1639 specimens (9.0 insects per infested palm). P. tertius occurred in 26 palms (8%), which resulted in the capture of 484 specimens (19 insects per infested palm). T. sordida (n=30) and M. borbai (n=1) occurred in only one location. From 537 R. neglectus examined, 44 were infected (8%) with Trypanosoma rangeli and/or Trypanosoma cruzi (Tc Id). M. flexuosa was previously recognized as a suitable breeding ecotope for R. neglectus in the Brazilian states of Minas Gerais, Goiás, Tocantins and the Federal District. Our results expand this distribution to other states (São Paulo, Bahia, Mato Grosso, Maranhão and Piauí), and also show that this particular palm tree harbors other triatomine species. Finally, we show that R. neglectus plays an important role in maintaining the enzootic circulation of T. cruzi and T. rangeli in the Brazilian savanna., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Leishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between Leishmania isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect Leishmania infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk., Results: A SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between Leishmania subgenera. A panel of reference strains representing subgenera Leishmania and Viannia was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95 °C ± 0.01 and 77.36 °C ± 0.02 for Leishmania and Viannia, respectively (p < 0.05). Using the Neighbor-Joining method and Kimura 2-parameters, the alignment of 12 sequences from the amplified conserved minicircles segment grouped together L. (V.) braziliensis and L. (V.) shawii with a bootstrap value of 100%; while for L. (L.) infantum and L. (L.) amazonensis, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus Viannia amplicons could be due to a lower proportion of guanine/cytosine sites (43.6%) when compared to species from subgenus Leishmania (average of 48.4%). The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected Lutzomyia spp. captured in two Brazilian localities., Conclusions: For all tested samples, a characteristic amplicon melting profile was evidenced for each Leishmania subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast DNA minicircles region is able to provide a rapid and reliable method to identify the main etiologic agents of cutaneous and visceral leishmaniases in endemic regions of Brazil., (© 2012 Pita-Pereira et al; licensee BioMed Central Ltd.)

Background: Visceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region., Principal Findings: KDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World., Conclusions: LSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains.

Due to limited efficacy and considerable toxicity, the therapy for Chagas' disease is far from being ideal, and thus new compounds are desirable. Diamidines and related compounds such as arylimidamides have promising trypanocidal activity against Trypanosoma cruzi. To better understand the mechanism of action of these heterocyclic cations, we investigated the kinetoplast DNA (kDNA) binding properties and trypanocidal efficacy against T. cruzi of 13 compounds. Four diamidines (DB75, DB569, DB1345, and DB829), eight arylimidamides (DB766, DB749, DB889, DB709, DB613, DB1831, DB1852, and DB2002), and one guanylhydrazone (DB1080) were assayed in thermal denaturation (T(m)) and circular dichroism (CD) studies using whole purified T. cruzi kDNA and a conserved synthetic parasite sequence. The overall CD spectra using the whole kDNA were similar to those found for the conserved sequence and were indicative of minor groove binding. Our findings showed that some of the compounds that exhibited the highest trypanocidal activities (e.g., DB766) caused low or no change in the T(m) measurements. However, while some active compounds, such as DB766, induced profound alterations of kDNA topology, others, like DB1831, although effective, did not result in altered T(m) and CD measurements. Our data suggest that the strong affinity of amidines with kDNA per se is not sufficient to generate and trigger their trypanocidal activity. Cell uptake differences and possibly distinct cellular targets need to be considered in the final evaluation of the mechanisms of action of these compounds.

DNA is the biopolymer most studied by scanning probe methods, and it is now possible to obtain reliable and reproducible images of DNA using atomic force microscopy (AFM). AFM has been extensively used to elucidate morphological changes to DNA structure, such as the formation of knots, nicks, supercoiling and bends. The mitochondrial or kinetoplast DNA (kDNA) of trypanosomatids is the most unusual DNA found in nature, being unique in organization and replication. The kDNA is composed of thousands of topologically interlocked DNA circles that form a giant network. To understand the biological significance of the kinetoplast DNA, it is necessary to learn more about its structure. In the present work, we used two procedures to prepare kDNA networks of Crithidia fasciculata for observation by AFM. Because AFM allows for the examination of kDNA at high resolution, we were able to identify regions of overlapping kDNA molecules and sites where several molecules cross. This found support the earlier described kDNA structural organization as composed by interlocked circles. We also observed an intricate high-density height pattern around the periphery of the network of C. fasciculata, which appears to be a bundle of DNA fibers that organizes the border of the network. Our present data confirm that AFM is a powerful tool to study the structural organization of biological samples, including complex arrays of DNA such as kDNA, and can be useful in revealing new details of structures previously visualized by other means., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Leishmaniasis is a geographically widespread disease caused by protozoan parasites belonging to the genus Leishmania and transmitted by certain species of sand fly. This disease still remains endemic in China, especially in the west and northwest frontier regions. A recent ITS1 phylogeny of Chinese Leishmania isolates has challenged some aspects for their traditional taxonomy and cladistic hypotheses of their phylogeny. However, disagreement with respect to relationships within Chinese Leishmania isolates highlights the need for additional data and analyses. Here, we test the phylogenetic relationships among Chinese isolates and their relatives by analyzing kinetoplast cytochrome oxidase II (COII) gene sequences, including 14 Chinese isolates and three isolates from other countries plus 17 sequences retrieved from GenBank. The COII gene might have experienced little substitution saturation, and its evolutionary process was likely to have been stationary, reversible, and homogeneous. Both neighbor-joining and Bayesian analyses reveal a moderately supported group comprising ten newly determined isolates, which is closely related to Leishmania tarentolae and Endotrypanum monterogeii. In combination with genetic distance analysis as well as Bayesian hypothesis testing, this further corroborates the occurrence of an undescribed species of Leishmania. Our results also suggest that (1) isolate MHOM/CN/93/GS7 and isolate IPHL/CN/77/XJ771 are Leishmania donovani; (2) isolate MHOM/CN/84/JS1 is Leishmania tropica; (3) the status referring to an isolate MRHO/CN/62/GS-GER20 from a great gerbil in Gansu, China, as Leishmania gerbilli, formerly based on multilocus enzyme electrophoresis, is recognized; and (4) E. monterogeii is nested within the genus Leishmania, resulting in a paraphyletic Leishmania. In addition, the results of this study enrich our understanding of the heterogeneity and relationships of Chinese Leishmania isolates.

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Leishmaniasis caused by Leishmania (Leishmania) infantum is a zoonotic disease endemic in South Europe, from Portugal to the Middle East. The aim of the present study was to investigate the genetic diversity of L. infantum parasites in Iberian Peninsula. Twenty-four L. infantum strains isolated from immunocompetent patients with leishmaniasis from several localities of Portugal and Spain were studied. The use of kinetoplast DNA-PCR-restriction fragment length polymorphism as a molecular marker revealed intra-specific variation. No association was found between genotype and clinical form of the disease or patients age group. Two main clusters were identified with this marker: (i) zymodeme MON-1 strains and (ii) non-MON-1 strains. However, no association was found between strains variability and geographical distribution suggesting that parasite populations of different regions in the Iberian Peninsula are homogenous., (© 2010 Blackwell Verlag GmbH.)

Background: Canine leishmaniasis (CanL) is endemic in western China, resulting in important public health problem. It is essential to evaluate the prevalence of canine Leishmania infantum infection for designing control policy. In the present study we report for the first time prevalence of Leishmania infection in dogs living in Jiuzhaigou County (Sichuan Provence, China), which is not only an important endemic area of CanL but also a tourism scenic spot, detected by PCR, ELISA and dipstick test. The results could provide key information for designing control programs against canine and human leishmaniasis. In addition, the complete sequence of the Leishmania isolate from Sichuan Province has not been reported to date and we present the sequences of 116 base-pair (bp) fragment of the conserved region in the minicircle kinetoplast DNA (kDNA) and the results of phylogenetic analyses based on the sequence of the amplified fragment., Results: The proportion of dogs infected with Leishmania in Jiuzhaigou County was 36.79%, 9.43%, and 51.88% detected by ELISA, dipstick test, and PCR, respectively. The ELISA and PCR tests were more sensitive than dipstick test. The PCR method is the most sensitive way to detect dogs infected with Leishmania parasites. The total positive rate for infected dogs in the area was 59.43% by the three methods. The PCR products of 116-bp fragment amplified from the kDNA conserved region of dog blood samples and laboratory maintained L. infantum were DNA sequenced and the variation of the sequences was observed. The phylogenetic tree based on the sequences of 116-bp fragment reveals that L. infantum is more genetically related to visceralizing species L. donovani than to the Leishmania species associated with cutaneous disease., Conclusions: More than half of dogs living in the endemic Jiuzhaigou County were infected by L. infantum. Control measures, such as treatment or eradication of infected dogs, or prohibition of maintaining dogs, must be taken against these infected dogs due to their role in the transmission of the infection to vectors. The phylogenetic tree based on the sequences of conserved region in kDNA of Leishmania can effectively distinguish species of Leishmania.

Shiraz district in south of Iran is a classical focus of Cutaneous leishmaniasis (CL) and previous research has consistently documented the etiologic agent to be Leishmania tropica and Leishmania major in urban and rural areas, respectively. However, none of the Phlebotomus sergenti, a known vector for L. tropica, of the region has been found infected. We report the first isolation of L. tropica from sandflies in urban community of southern part of Shiraz city. Parasite polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and gene sequencing analyses indicate CL cases in this community were caused by either L. major or L. tropica. Sandflies of P. sergenti were infrequent, however, three out of 10 (30.0%) females captured in urban area were found infected with L. tropica. But, no human cases were found to be infected with L. tropica. Phlebotomus papatasi were found the most dominant and infected species where 41 out of 207 (20%) tested individuals harboring L. major in suburb area of the city. Patients have been lived in the suburb area of the city where people keep normally domestic animals in their houses which provide appropriate environment for completion of sandfly life cycle and expansion of CL disease in the region.

Drug unresponsiveness in patients with visceral leishmaniasis (VL) is a problem in many endemic areas. This study aimed to determine genetic diversity of Leishmania donovani isolates from a VL endemic area in Sudan as a possible explanation for drug unresponsiveness in some patients. Thirty clinically stibogluconate (SSG)-sensitive isolates were made SSG-unresponsive in vitro by gradually increasing SSG concentrations. The sensitive isolates and their SSG-unresponsive counterparts were typed using mini-circle kDNA and categorized using PCR-RAPD. All the isolates were typed as L. donovani, the resulting PCR-RAPD characterization of the SSG-sensitive isolates gave three distinct primary genotypes while, the SSG-unresponsive isolates showed only a single band. L. donovani isolates from eastern Sudan are diverse; this probably resulted from emergence of new L. donovani strains during epidemics due to the pressure of widespread use of antimonials. In this communication the possible role of isolates diversity in antimonial unresponsiveness and the in vitro changing PCR-RAPD band pattern in SSG-unresponsive strains were discussed., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Cutaneous leishmaniasis, caused by Leishmania tropica, has recently emerged in urban and rural foci of central and northern Israel, and constitutes a major public health concern. Rock hyraxes (Procavia capensis), the suspected natural reservoir, were trapped in the cutaneous leishmaniasis urban focus of Maale Adumim in central Israel and evaluated for L. tropica infection by real-time kinetoplast DNA (kDNA) polymerase chain reaction (PCR) and serology. Real-time PCR on blood and computerized western blot serology analysis was positive for L. tropica in 58% and 80%, respectively, of the hyraxes tested. Phylogenetic analysis of the ribosomal internal transcribed spacer 1 region indicated that similar genotypes were present in humans and hyraxes from the same habitat. The high rates of infection and exposure to L. tropica among hyraxes supports their involvement in the transmission cycle of this parasite, and their potential role as a reservoir for human disease.

A photolyase-like protein gene found in the Trypanosoma cruzi genome database was cloned and expressed in Escherichia coli resulting in the formation of inclusion bodies. Antibodies against this protein were used to determine expression of the protein in the different forms of the parasite. It was visualized in the epimastigote form but not in amastigote or trypomastigote forms obtained from culture in Vero cells. In epimastigotes, this protein is located at the level of the mitochondrion associated to both sides of the kinetoplast. Sequence analyses indicated that this protein, as well as other photolyases from Leishmania spp. and Trypanosoma brucei are related to single-stranded photolyases or cryptochromes DASH., (Copyright 2009 Elsevier Inc. All rights reserved.)

Canine leishmaniosis is a widespread disease caused by Leishmania parasites, which are transmitted by phlebotomine sand flies. However, in some areas where canine leishmaniosis is endemic, but the primary vectors have not been found, ticks have been suspected to play a role in transmitting the infection. Herewith, we report the detection of Leishmania infantum kinetoplast minicircle DNA (kDNA) in ticks collected from naturally infected dogs living in rural areas of Southern Italy (site A) and Northeastern Brazil (site B). Between March and October 2007, ticks were collected from 26 dogs positive to anti-Leishmania antibodies (one from site A and 25 from site B) and either placed directly into vials containing 70% ethanol or maintained alive for identification and subsequent dissection. All the 95 ticks collected were morphologically identified as Rhipicephalus sanguineus. After identification, their genomic DNA was extracted (either individually or in pools) and processed by polymerase chain reaction (PCR) for the detection of L. infantum kDNA. Two pools of salivary glands from ticks (one from five females and other from five males) found on a dog from site A and tested by a conventional PCR were positive. Amplicon sequencing confirmed the identity of the parasite. In addition, nine (12.3%) out of the 73 ticks found on dogs from site B and tested by a real-time PCR were positive, with a low parasite load (less than 1 parasite/ml). The retrieval of L. infantum kDNA in salivary glands of R. sanguineus ticks has been here reported for the first time. Therefore, further studies are needed to assess the competence of ticks as vectors of Leishmania parasites from dog to dog.

Trypanosoma brucei's mitochondrial genome, kinetoplast DNA (kDNA), is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei's six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5' to 3' DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb), are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments.

To determine what topological changes antiparasitic heterocyclic dications can have on kinetoplast DNA, we have constructed ligation ladders, with phased A5 and ATATA sequences in the same flanking sequence context, as models. Bending by the A5 tract is observed, as expected, while the ATATA sequence bends DNA very little. Complexes of these DNAs with three diamidines containing either furan, thiophene or selenophene groups flanked by phenylamidines were investigated along with netropsin. With the bent A5 ladder the compounds caused either a slight increase or decrease in the bending angle. Surprisingly, however, with ATATA all of the compounds caused significant bending, to values close to or even greater than the A5 bend angle. Results with a mixed cis sequence, which has one A5 and one ATATA, show that the compounds bend ATATA in the same direction as a reference A5 tract, that is, into the minor groove. These results are interpreted in terms of a groove structure for A5 which is largely pre-organized for a fit to the heterocyclic amidines. With ATATA the groove is intrinsically wider and must close to bind the compounds tightly. The conformational change at the binding site then leads to significant bending of the alternating DNA sequence.

Natural populations of Trypanosoma cruzi are structured into five genetic lineages, T. cruzi I and T. cruzi II a to e, as the result of clonal evolution with rare genetic recombination events. To explore more in depth these phenomenons, a multigene sequencing approach was used, for the first time in the case of T. cruzi. Three nuclear loci and a maxicircle locus were sequenced on 18 T. cruzi stocks. Sequences were used to build phylogenetic trees from each locus and from concatenated sequences of all loci. The data confirmed the hybrid origin of DTUs IId and IIe, as the result of an ancient genetic recombination between strains pertaining to IIb and IIc. The data confirmed also a hybrid origin of DTUs IIa and IIc. Contrary to previous reports, we failed to detect mosaic genes. The phylogenetic relationship between DTUs and the respective roles of recombination and selection were tested.

Kinetoplast maxicircle DNA sequence organisation was investigated in Leishmania donovani, strain 1S LdBob. Gene arrangement in the coding (conserved) region of the maxicircle is collinear with that of most trypanosomatids, with individual genes showing 80-90% nucleotide identity to Leishmania tarentolae, strain UC. The notable exception was an integration of a full-size minicircle sequence in the ND1 gene coding region found in L. donovani. Editing patterns of the mitochondrial mRNAs investigated also followed L. tarentolae UC patterns, including productive editing of the components of respiratory complexes III-V, and ribosomal protein S12 (RPS12), as well as the lack of productive editing in five out of six pan-edited cryptogenes (ND3, ND8, ND9, G3, G4) found in these species. Several guide RNAs for the editing events were localised in minicircles and maxicircles in the locations that are conserved between the species. Mitochondrial activity, including rates of oxygen consumption, the presence and the levels of respiratory complexes and their individual subunits and the steady-state levels of several mitochondrial-encoded mRNAs were essentially the same in axenically grown amastigotes and in promastigotes of L. donovani. However, some modulation of mitochondrial activity between these developmental stages was suggested by the finding of an amastigote-specific component in complex IV, a down-regulation of mitochondrial RNA-binding proteins (MRP) and MRP-associated protein (MRP-AP) in amastigotes, and by variations in the levels of RPS12, ND3, ND9, G3 and G4 pre-edited transcripts.

UMSBP is a CCHC-type zinc finger protein, which functions during replication initiation of kinetoplast DNA minicircles and the segregation of kinetoplast DNA networks. Interactions of UMSBP with origin sequences, as well as the protein oligomerization, are affected by its redox state. Reduction yields UMSBP monomers and activates its binding to DNA, while oxidation drives UMSBP oligomerization and impairs its DNA-binding activity. Kinetics analyses of UMSBP-DNA interactions revealed that redox affects the association of free UMSBP with the DNA, but has little effect on its dissociation from the nucleoprotein complex. A previously proposed model, suggesting that binding of DNA is regulated via the reversible interconversions of active UMSBP monomers and inactive oligomers, was challenged here, revealing that the two redox-driven processes are not interrelated. No correlation could be observed between DNA-binding inhibition and UMSBP oligomerization, upon oxidation of UMSBP. Moreover, while the presence of zinc ions was found to be essential for the interaction of UMSBP with DNA, UMSBP oligomerization occurred through zinc-depleted, unfolded zinc finger domains. Site directed mutagenesis analysis of UMSBP suggested that its unique methionine residue, which can be oxidized into methionine sulfoxide, is not involved in the redox-mediated regulation of UMSBP-DNA interactions.

The objective of this study was to develop specific primers for Leishmania (Viannia) braziliensis species identification using PCR. The designed primers (LBF1 and LBR1) were evaluated for sensitivity and specificity using various L. (V.) braziliensis serodemes and various Leishmania species and also using Trypanosoma cruzi. A specific fragment of 536bp was detected from 50ng of DNA in a crude extract derived from L. (V.) braziliensis. The DNA fragment was not detected when DNA from other Leishmania species or from T. cruzi was used as template in the PCR. Furthermore, when tested with DNA from cutaneous leishmaniasis the designed primers and reaction gave positive results. Taking into consideration that the primers LBF1 and LBR1 could specifically identify L. (V.) braziliensis, they could be considered for use in L. (V.) braziliensis diagnosis and epidemiological studies.

The current intraspecific nomenclature in Trypanosoma cruzi describes two major lineages, named T. cruzi I and T. cruzi II, and five sublineages within T. cruzi II, named IIa, IIb, IIc, IId and IIe. The polymorphism of minicircle hypervariable regions (mHVRs) of T. cruzi has been used in many studies for the molecular characterization of parasite populations directly from biological samples. However, the molecular bases that allow strain typing by these markers are still unclear. In this work we examined forty cloned mHVRs sequences of CL-Brener reference strain (IIe sublineage), and we found a predominant group of sequences, with 40% of frequency in this strain, with a 97% of identity among them. Out of the forty clones analyzed, we identified other less representative types, and a few unique ones. This predominant sequence is also present in different reference strains belonging to the other main T. cruzi lineages and sublineages (TcI, IIa, IIb, IIc and IId) although in a many thousand times lower frequency than in the CL-Brener strain, as shown by semiquantitative PCR. Similarly, predominant mHVR sequences previously described for TcIId strains, were clearly more frequent (many thousand times higher) in the IId reference strain analyzed by us (Mncl2) than within the reference strains belonging to the other lineages and sublineages. The analysis of the cloned sequences shows that more sequences than just the major one contribute to define the global pattern of mHVRs RFLP in the CL-Brener strain. The possible usefulness of these predominant sequences for typing TcIId and TcIIe sublineages by semiquantitative PCR, as well as the possible role of these sequences in genotype identification by mHVR probes are discussed.

In the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, RNA editing inserts and/or deletes uridines from pre-mRNAs to produce mature, translatable mRNAs. RNA editing is carried out by several related multiprotein complexes known as editosomes, which contain all of the enzymatic components required for catalysis of editing. In addition, noneditosome accessory factors necessary for editing of specific RNAs have also been described. Here, we report the in vitro and in vivo characterization of the mitochondrial TbRGG2 protein (originally termed TbRGGm) and demonstrate that it acts as an editing accessory factor. TbRGG2 is an RNA-binding protein with a preference for poly(U). TbRGG2 protein levels are up-regulated 10-fold in procyclic form T. brucei compared with bloodstream forms. Nevertheless, the protein is essential for growth in both life cycle stages. TbRGG2 associates with RNase-sensitive and RNase-insensitive mitochondrial complexes, and a small fraction of the protein co-immunoprecipitates with editosomes. RNA interference-mediated depletion of TbRGG2 in both procyclic and bloodstream form T. brucei leads to a dramatic decrease in pan-edited RNAs and in some cases a corresponding increase in the pre-edited RNA. TbRGG2 down-regulation also results in moderate stabilization of never-edited and minimally edited RNAs. Thus, our data are consistent with a model in which TbRGG2 is multifunctional, strongly facilitating the editing of pan-edited RNAs and modestly destabilizing minimally edited and never-edited RNAs. This is the first example of an RNA editing accessory factor that functions in the mammalian infective T. brucei life cycle stage.

Kinetoplast DNA (kDNA) is a novel form of mitochondrial DNA consisting of thousands of interlocked minicircles and 20-30 maxicircles. The minicircles replicate free of the kDNA network but nicks and gaps in the newly synthesized strands remain at the time of reattachment to the kDNA network. We show here that the steady-state population of replicated, network-associated minicircles only becomes repaired to the point of having nicks with a 3'OH and 5'deoxyribonucleoside monophosphate during S phase. These nicks represent the origin/terminus of the strand and occur within the replication origins (oriA and oriB) located 180 degrees apart on the minicircle. Minicircles containing a new L strand have a single nick within either oriA or oriB but not in both origins in the same molecule. The discontinuously synthesized H strand contains single nicks within both oriA and oriB in the same molecule implying that discontinuities between the H-strand Okazaki fragments become repaired except for the fragments initiated within the two origins. Nicks in L and H strands at the origins persist throughout S phase and only become ligated as a prelude to network division. The failure to ligate these nicks until just prior to network division is not due to inappropriate termini for ligation.

Trypanosoma carassii is a fish kinetoplastid parasite that belongs to the family Trypanosomatida. In the present study we cloned a cathepsin L-like proteinase from T. carassii. The nucleotide sequence of 1371bp translated into a preproprotein of 456 amino acids. The preproprotein contained the oxyanion hole (Gln), the active triad formed by Cys, His and Asn and the conserved ERFNIN-like, GNFD and GCNGG motifs, characteristic for cathepsin L proteinases. Phylogenetic analysis showed that the T. carassii cysteine proteinase clustered with other cathepsin L-like proteinases from the Trypanosomatida. We produced a recombinant T. carassii cysteine proteinase in Escherichia coli and demonstrated that it has cathepsin L activity. Immunization of common carp (Cyprinus carpio L.) with the recombinant protein induced a very high increase in proteinase-specific antibodies but only slightly lowered parasitaemia. Our findings suggest that the T. carassii cysteine proteinase is highly conserved within the Trypanosomatida with respect to structure and activity but is not a major protective antigen in carp.

Trypanosomes and Leishmania, protozoans that cause major human diseases, have a topologically intricate mitochondrial DNA (kinetoplast or kDNA) in the form of a network of thousands of interlocked circles. This unusual system provides a useful reporter for studying topoisomerase functions in vivo. We now find that these organisms have three type IA topoisomerases, one of which is phylogenetically distinctive and which we designate topoisomerase IA(mt). In Trypanosoma brucei topoisomerase IA(mt) immunolocalizes within the mitochondrion close to the kDNA disk in patterns that vary with the cell cycle. When expression of TOPIA(mt) is silenced by RNAi there is a striking accumulation of kDNA late theta structure replication intermediates, with subsequent loss of kDNA networks and halt in cell growth. This essential enzyme provides clear molecular evidence for the obligatory role of a type IA enzyme in the resolution of late theta structures in vivo. With no close orthologue in humans it also offers a target for the rational development of selectively toxic new antiprotozoal therapies.

A real-time polymerase chain reaction (PCR) assay was used for quantifying Leishmania infantum DNA in urine samples from naturally infected dogs. Forty-one infected dogs were divided into 3 groups: 22 dogs showing only cutaneous signs (group 1), 12 dogs showing hematuria (group 2), and 7 dogs affected by severe nephropathy (group 3). Groups 2 and 3 dogs showed altered laboratory parameters related to an impairment of renal function. The real-time PCR analysis showed higher levels of Leishmania DNA in the lymph node aspirates from all groups of infected dogs versus those measured in their blood or urine. Interestingly, urine samples from dogs belonging to groups 2 and 3 contained a higher Leishmania DNA load than that detected in their blood. This finding suggests that a real-time PCR analysis of urine from infected dogs could be a useful and noninvasive tool for monitoring the severity of leishmaniasis.

Kinetoplastid parasites are responsible for the potentially fatal diseases leishmaniasis, African sleeping sickness and Chagas disease. The current treatments for these diseases are far from ideal and new compounds are needed as antiparasitic drug candidates. Tubulin is the accepted target for treatments against cancer and helminths, suggesting that kinetoplastid tubulin is also a suitable target for antiprotozoal compounds. Selective lead compounds against kinetoplastid tubulin have been identified that could represent a starting point for the development of new drug candidates against these parasites.

This study presents the first genetic characterization of five Trypanosoma rangeli isolates from Minas Gerais, in the southeast of Brazil and their comparison with Colombian populations by minicircle classification, RAPD-PCR and LSSP-PCR analyses. Our results demonstrated a homogenous T. rangeli population circulating among Didelphis albiventris as reservoir host in Brazil while heterogeneous populations were found in different regions of Colombia. KP1(+) minicircles were found in 100% isolates from Brazil and in 36.4% of the Colombian samples, whereas the KP2 and KP3 minicircles were detected in both groups. RAPD-PCR and LSSP-PCR profiles revealed a polymorphism within KP1(+) and KP1(-) T. rangeli populations and allowed the division of T. rangeli in two branches. The Brazilian KP1(+) isolates were more homogenous than the KP1(+) isolates from Colombia. The RAPD-PCR were entirely consistent with the distribution of KP1 minicircles while those obtained by LSSP-PCR were associated in 88.9% and 71.4% with KP1(+) and KP1(-) populations, respectively.

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.

The mitochondrial genome of Trypanosoma brucei is contained in a specialized structure termed the kinetoplast. Kinetoplast DNA (kDNA) is organized into a concatenated network of mini and maxicircles, positioned at the base of the flagellum, to which it is physically attached. Here we have used electron microscope cytochemistry to determine structural and functional domains involved in replication and segregation of the kinetoplast. We identified two distinct subdomains within the kinetoflagellar zone (KFZ) and show that the unilateral filaments are composed of distinct inner and outer filaments. Ethanolic phosphotungstic acid (E-PTA) and EDTA regressive staining indicate that basic proteins and DNA are major constituents of the inner unilateral filaments adjoining the kDNA disc. This evidence for an intimate connection of the unilateral filaments in the KFZ with DNA provides support for models of minicircle replication involving vectorial export of free minicircles into the KFZ. Unexpectedly however, detection of DNA in the KFZ throughout the cell cycle suggests that other processes involving kDNA occur in this domain. We also describe a hitherto unrecognized, intramitochondrial, filamentous structure rich in basic proteins that links the kDNA discs during their segregation and is maintained between them for an extended period of the cell cycle.

The conventional methods for identification and typing of Leishmania species depend on previous culture isolation of the parasites. Not infrequently, culture is unsuccessful and may result in misrepresentation of the heterogeneity of the original isolate. Thus, more reliable and precise identification of genotypes of Leishmania spp. is important for a better clinical and epidemiological understanding of the disease. We evaluated the potential of LSSP-PCR targeting kDNA minicircles in discriminating different variants of the parasite with the use of clinical samples directly or cultivated parasites. The 1st step of this procedure consists of the amplification of the minicircles by conventional PCR; the 2nd step is low-stringency amplification of the minicircles previously amplified, with the use of 1 of the primers. Although LSSP-PCR produced complex and distinct kDNA signatures for isolates representing different species, further experiments demonstrated that the approach had the potential for discriminating intraspecific variants of L. braziliensis. Thus, the generated profiles were too variable to be useful as markers for species identification. Moreover, we demonstrated that the approach can be directly applied to clinical samples. In conclusion, LSSP-PCR targeting kDNA minicircles produces profiles that reflect polymorphisms of the predominant classes of minicircles, and can be useful for studies aimed at discriminating Leishmania braziliensis genotypes without the need for previous cultivation of the parasite.