Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata

A type II DNA topoisomerase (topollmt), purified to near homogeneity from the trypanosomatid C. fasciculata has been shown to be localized to the single mitochondrion of these kinetoplastid protozoa. Immunoblots show at least a 10-fold higher level of topollmt (per milligram of protein) in preparations of partially purified mitochondria as compared with those from whole cells. Analyses of type I and type II topoisomerase activities in both mitochondrial and whole cell extracts show a 4- to 5-fold higher specific activity of topollmt in mitochondrial extracts while a nuclear type I topoisomerase has a 4- to 5-fold lower specific activity in the same extract. Immunolocalizations using anti-topollmt antibodies show the enzyme to be present in close association with the mitochondrial DNA networks (kinetoplast DNA or kDNA). This association appears at two distinct locations on opposite sides of the kDNA network.     

Cytochemical study of free-living flagellates of suborder Bodonina. I. Morphology and nucleic acids]

Nucleic acids (DNA and RNA) have been discovered in the kinetoplast of free-living Bodonina: Bodo caudatus, Pleuromonas jaculans, Rhynchomonas nasuta--by means of cytochemical methods. The kinetoplast has variable contents of nucleic acids whose chemoarchitectonics is due to their non-homogeneous distribution within the kinetoplast. The Feulgen reaction in the kinetoplast is more intensive than in the nucleus. Kinetoplast is closely connected with the cytoplasmic RNA metabolism. Many individuals of R. nasuta were found to have two kinetoplasts, no other signs of cell division being observed. P. jaculans has up to 45% of dyskinetoplastic forms.     

Cryptobia cataractae from the blood of Semotilus atromaculatus: structure and division in the fish

A cryptobiid was found in the blood of 2 of 9 Semotilus atromaculatus from a tributary of the Saugeen River in Ontario, Canada. Blood inoculation produced an infection in 2 uninfected S. atromaculatus but not in any Oncorhynchus mykiss, Catostomus commersoni, or Carassius auratus. The flagellate was identified as Cryptobia cataractae, based on host restriction. Cryptobia cataractae occurred as slender and broad forms (body width 3.0-8.7 microns). The length of the anterior flagellum was equal to body length, whereas that of the free recurrent flagellum was half body length. Cryptobia cataractae divided by equal binary fission that produced elongate, slender daughter cells.     

Expression, purification and characterization of recombinant mitochondrial topoisomerase II of kinetoplastid Crithidia fasciculata in High-five insect cells

Topoisomerase II of kinetoplastid parasites plays an important role in the replication of unusual networks of kinetoplast DNA (kDNA) and is a very useful target for antiparasitic drugs. In this study, we cloned full-length Crithidia fasciculata mitochondrial topoisomerase II gene into pFastBac-HTc vector and successfully expressed an active recombinant full-length mitochondrial topoisomerase II in Bac-to-Bac baculovirus expression system. A rapid and simple purification strategy was established by incorporating a FLAG-tag at the C-terminus of the protein. The purified recombinant topoisomerase II showed a major single band on SDS-PAGE (>96% purity) and was verified through Western blot analysis. The recombinant full-length mitochondrial topoisomerase II exhibited decatenation, catenation and relaxation activity of type II topoisomerase as well as various sensitivities to a series of known topoisomerase inhibitors. These studies explore new way and lay groundwork to express all other similar full-length kinetoplastid topoisomerases, it will also facilitate further elucidation of X-ray structure, catalysis mechanism of kinetoplastid topoisomerases and design of new antiparasitic drugs targeting kinetoplastid topoisomerases.     

Distinct genes encode type II Topoisomerases for the nucleus and mitochondrion in the protozoan parasite Trypanosoma brucei

Topoisomerases are essential for orderly nucleic acid metabolism and cell survival and are proven targets for clinically useful antimicrobial and anticancer drugs. Interest in the topologically intricate mitochondrial DNA (kinetoplast or kDNA) of Trypanosoma brucei brucei and related kinetoplastid protozoan parasites has led to many reports of type II topoisomerases that participate in kDNA metabolism (we term the T. brucei brucei gene TbTOP2mt). We have now identified and characterized two new genes for type II topoisomerases in T. brucei brucei, termed TbTOP2alpha and TbTOP2beta. Phylogenetically, they share a common node with other nuclear topoisomerases, clearly distinct from a clade that includes the previously reported kinetoplastid genes, all of which are homologs of TbTOP2mt. Southern blot analysis reveals the new genes are single copy and positioned approximately 1.7 kb apart. Cognate mRNAs are expressed in African trypanosomes, but only a single message is detected in Leishmania or Crithidia. TbTOP2alpha encodes an ATP-dependent topoisomerase that appears as a single approximately 170-kDa band on immunoblots and localizes to the nucleus; RNA interference leads to pleomorphic nuclear (but not kDNA) abnormalities and early growth arrest. The role of TbTOP2beta is unclear. Although transcribed in trypanosomes, TbTOP2beta is not detected by beta-specific antiserum, and RNAi silencing results in no obvious phenotype. These studies indicate that African trypanosomes and related kinetoplastid human pathogens are unusual in having independent topoisomerase II genes to service their nuclear and mitochondrial genomes, and they highlight TbTOP2alpha as a promising target for the development of much-needed new therapies.     

Morphology and Ultrastructure of Cryptobia eilatica n. sp. (Bodonidae: Kinetoplastida), an Ectoparasite from the Gills of Marine Fish

ABSTRACT. A marine kinetoplastid flagellate, Cryptobia eilatica n. sp., is described from the gills of cultured gilt-head sea bream Sparus aurata L. and wild black-spot sea bream Diplodus noct (Valenciennes) in the Red Sea. The trophozoite is elongated and lacks a contractile vacuole and undulating membrane. The body averages 13.5 × 4.1 μm, anterior flagellum 9.7 μm, and free portion of recurrent flagellum 15.2 μm. The ultrastructural features of the species exhibit great similarity to various previously studied Cryptobiids. Cryptobia eilatica trophozoites feed on bacteria, show a preference for the branc hial interlamellar crypts, and attach to the host epithelium by means of the recurrent flagellum. Neither penetration into the epithelial cells, nor any direct damage to host tissue was observed. Cryptobia eilatica inhabits a purely marine habitat, but its trophozoite tolerates salinities as low as 10 ppt.     

Molecular taxonomy of the suborder Bodonina (Order Kinetoplastida), including the important fish parasite,Ichthyobodo necator

Ichthyobodo necator is an important fish ectoparasite with a broad host and ecological range. A novel method, involving the use of an anesthetic, allowed the collection of large numbers of parasites from the skin and gills of hybrid striped bass (Morone saxatilis male x M. chrysops female). Genomic DNA from these samples was used to amplify and clone the 18S rRNA gene. The 18S rRNA gene was similarly cloned from Bodo caudatus, Bodo edax, Bodo saltans, an unidentified Bodo species, and Dimastigella trypaniformis. The resulting sequences were aligned with other representative kinetoplastid species using pileup and similarities in secondary structure. Phylogenetic relationships within the suborder Bodonina and representatives of the suborder Trypanosomatina were determined using maximum-likelihood statistics. The phylogenetic analyses strongly supported the order Kinetoplastida as a monophyletic assemblage consisting of at least two major lineages. One lineage consisted exclusively of L. necator, indicating that it may represent a new suborder. The second lineage consisted of all other kinetoplastid species. This second lineage appeared to contain at least 8 bodonine sublineages, none of which correlated with currently recognized families. For three sublineages, there was a close correspondence between the 18S phylogeny and the classical taxonomy of Dimastigella, Rhynchobodo, and Rhynchomonas. In contrast, Bodo and Cryptobia were polyphyletic, containing species in two or more sublineages that may represent separate genera.     

Topoisomerases in kinetoplastids

Topoisomerases are enzymes that mediate topological changes in DNA that are essential for nucleic acid biosynthesis and for cell survival. The kinetoplastid protozoa, which include pathogenic trypanosomes and Leishmania, have yielded an interesting variety of purified topoisomerase activities as well as several topoisomerase genes. In these parasites, topoisomerases are involved in the metabolism of both nuclear and mitochondrial (kinetoplast) DNA. In this review, Christian Burri, Armette Bodley and Theresa Shapiro summarize what is known about topoisomerases in kinetoplastids, and consider the intriguing possibility that these enzymes may act as valuable antiparasite drug targets.     

Molecular Comparison of the Mitochondrial and Cytoplasmic hsp 70 of Trypanosoma cruzi, Trypanosoma brucei and Leishmania major

ABSTRACT. We compared the expression and localization of the mitochondrial and cytoplasmic hsp70 of the protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major. The mitochondrial protein is encoded by multiple mRNA in all species, while the cytoplasmic protein is encoded by a single mRNA. In all three species, the mitochondrial hsp70 is concentrated in the kinetoplast, a submitochondrial structure that houses the unusual DNA (kDNA) that characterizes this group of organisms, while the cytoplasmic protein is distributed throughout the cell. These results suggest that, in all kinetoplastid species, mt-hsp70 has a specific function in kDNA biology, possibly in the processes of kDNA replication, RNA editing or kinetoplast structure.     

Minicircular Kinetoplast DNA of Trypanosomatidae

Analysis of primary structure and organization of mitochondrial (kinetoplast) DNA of flagellates occupies a prominent place in the studies of eukaryote mitochondrial genomes, owing to its unusual organization and functioning as well as to the epidemiological role of the Trypanosomatidae family. According to contemporary notions, living zooflagellates are direct descendants of the ancestral forms that gave rise to all eukaryotic kingdoms. Hence, comparative mtDNA studies of recent Trypanosomatidae open broad prospects for phylogenetic reconstructions and analysis of presumable routes of eukaryote evolution. The structure, characteristics, and functions of Trypanosomatidae minicircular kinetoplast DNA are discussed here.     

A tyrosyl DNA phosphodiesterase 1 from kinetoplastid parasite Leishmania donovani (LdTdp1) capable of removing topo I–DNA covalent complexes

Tyrosyl DNA phosphodiesterase 1 (Tdp1) is a member of phospholipase D superfamily, which cleaves a broad range of 3′‐DNA adducts, the best characterized of which is the phosphodiester bond formed between DNA and topoisomerase IB. This study describes cloning and functional characterization of the enzyme, termed as LdTdp1 in the kinetoplastid parasite Leishmania donovani. Sequence analysis confirmed conservation of the active site motifs typical for all Tdp1 proteins. LdTdp1 activity was detected in the parasite nucleus as well as in the kinetoplast. The enzyme harbours a nuclear localization signal at its C‐terminus. Overexpression of the active enzyme protected the parasites against topoisomerase IB inhibitor camptothecin (CPT) and oxidative agent H₂O₂‐mediated cytotoxicity and its downregulation rendered the parasites hypersensitive to CPT. Trapping of mutant LdTdp1 on DNA takes place following CPT treatment in L. donovani cells. The expression level and associated activity of LdTdp1 were found to be higher in CPT‐resistant L. donovani parasites. Altogether, this is the first report of Tdp1 from the kinetoplastid parasite L. donovani, which actively participates in topoisomerase I‐mediated DNA damage repair process and thereby counteracts the cytotoxic effect of topoisomerase I inhibitors.     

Minicircular kinetoplast DNA from Trypanosomatidae

Analysis of primary structure and organization of mitochondrial (kinetoplast) DNA of flagellates occupies a prominent place in the studies of eukaryote mitochondrial genomes, owing to its unusual organization and functioning as well as to the epidemiological role of the Trypanosomatidae family. According to contemporary notions, living zooflagellates are direct descendants of the ancestral forms that gave rise to all eukaryotic kingdoms. Hence, comparative mtDNA studies of recent Trypanosomatidae open broad prospects for phylogenetic reconstructions and analysis of presumable routes of eukaryote evolution. The structure, characteristics, and functions of Trypanosomatidae minicircular kinetoplast DNA are discussed here.     

Phylogeny of the kinetoplastida: taxonomic problems and insights into the evolution of parasitism

To further investigate phylogeny of kinetoplastid protozoa, the sequences of small subunit (18S) ribosomal RNA of nine bodonid isolates and ten isolates of insect trypanosomatids have been determined. The root of the kinetoplastid tree was attached to the branch of Bodo designis and/or Cruzella marina. The suborder Trypanosomatina appeared as a monophyletic group, while the suborder Bodonina was paraphyletic. Among bodonid lineages, parasitic organisms were intermingled with free-living ones, implying multiple transitions to parasitism and supporting the 'vertebrate-first hypothesis'. The tree indicated that the genera Cryptobia and Bodo are artificial taxa. Separation of fish cryptobias and Trypanoplasma borreli as different genera was not supported. In trypanosomatids, the genera Leptomonas and Blastocrithidia were polyphyletic, similar to the genera Herpetomonas and Crithidia and in contrast to the monophyletic genera Trypanosoma and Phytomonas. This analysis has shown that the morphological classification of kinetoplastids does not in general reflect their genetic affinities and needs a revision.     

Sex and evolution in trypanosomes

Trypanosoma brucei is still the only kinetoplastid known to undergo genetic exchange, but it seems unreasonable to suppose that it evolved this process all by itself. The position of T. brucei on a molecular phylogenetic tree constructed from 18S ribosomal RNA gene sequences offers no clues to the likely existence of genetic exchange in trypanosome species other than the Salivaria, because this group of trypanosomes appears to have diverged from the rest a very long time ago. Antigenic variation is one characteristic shared by the Salivaria, which has been particularly well-studied in T. brucei. The large proportion of the genome devoted to variant antigen genes and related sequences in T. brucei, suggests a possible role for genetic exchange in enhancing the diversity of the repertoire. Alternatively, genetic exchange may counter potential excessive double-strand DNA damage brought about by the DNA rearrangements associated with antigenic variation. The remarkable biparental inheritance of organelle DNA (=kinetoplast DNA) in T. brucei is without precedent in other eukaryotes. The result of genetic exchange is to enhance the heterogeneity of the kinetoplast DNA minicircles.     

Leishmania actin binds and nicks kDNA as well as inhibits decatenation activity of type II topoisomerase

Leishmania actin (LdACT) is an unconventional form of eukaryotic actin in that it markedly differs from other actins in terms of its filament forming as well as toxin and DNase-1-binding properties. Besides being present in the cytoplasm, cortical regions, flagellum and nucleus, it is also present in the kinetoplast where it appears to associate with the kinetoplast DNA (kDNA). However, nothing is known about its role in this organelle. Here, we show that LdACT is indeed associated with the kDNA disc in Leishmania kinetoplast, and under in vitro conditions, it specifically binds DNA primarily through electrostatic interactions involving its unique DNase-1-binding region and the DNA major groove. We further reveal that this protein exhibits DNA-nicking activity which requires its polymeric state as well as ATP hydrolysis and through this activity it converts catenated kDNA minicircles into open form. In addition, we show that LdACT specifically binds bacterial type II topoisomerase and inhibits its decatenation activity. Together, these results strongly indicate that LdACT could play a critical role in kDNA remodeling.     

Unique poly(dA).poly(dT) B'-conformation in cellular and synthetic DNAs

Poly(dA).poly(dT), but not B-form DNA, is specifically recognized by experimentally induced anti-kinetoplast or anti-poly(dA).poly(dT) immunoglobulins. Antibody binding is completely competed by poly(dA).poly(dT) and poly(dA).poly(dU) but not by other single- or double-stranded DNA sequences in a right-handed B-form. Antibody interaction with poly(dA).poly(dT) depends on immunoglobulin concentration, incubation time and temperature, and is sensitive to elevated ionic strengths. Similar conformations, for example, (dA)4-6 X (dT)4-6, in the kinetoplast DNA of the parasite Leishmania tarentolae are also immunogenic and induce specific anti-poly(dA).poly(dT) antibodies. These antibody probes specifically recognize nuclear and kinetoplast DNA in fixed flagellated kinetoplastid cells as evidenced by immunofluorescence microscopy. Anti-poly(dA).poly(dT) immunofluorescence is DNase-sensitive and competed by poly(dA).poly(dT), but not other classical double-stranded B-DNAs. Thus, these unique cellular B'-DNA helices are immunogenic and structurally similar to synthetic poly(dA).poly(dT) helices in solution.     

Characterization by sedimentation analysis of kinetoplast DNA from Trypanosoma cruzi at different stages of culture.

The kinetoplast DNA networks of Trypanosoma cruzi exist under two forms which have been studied by equilibrium density centrifugation in CsCl gradients containing ethidium bromide and by band sedimentation analysis. The relative proportion of the two forms has been measured and varies significantly between the exponential and stationary phase of growth, suggesting that one of these forms is a replicative intermediate. Both forms exhibit very high sedimentation coefficients. The sedimentation velocity ethidium titration was used to measure the superhelix density of the kinetoplast DNA after having established the validity of the method with in vitro closed DNA networks. The superhelix density of the native form of the kinetoplast DNA minicircles is very low and varies according to the physiological state of the trypanosomes. Furthermore, we observed a significant increase of the superhelix density of the kinetoplast DNA of trypanosomes grown in the presence of ethidium.     

In vitro attenuation of Cryptobia salmositica and its use as a live vaccine against cryptobiosis in Oncorhynchus mykiss

The present communication reports on the attenuation of a pathogenic hemoflagellate, Cryptobia salmositica Katz (Sarcomastigophora: Kinetoplastida) and its use as a live vaccine against cryptobiosis. The parasite was attenuated by continuous in vitro culture (at 10 C for 55 wk) in minimum essential medium. Attenuated (culture) forms are morphologically similar to virulent (blood) forms. They are however more slender and have a shorter anterior flagellum and a smaller nucleus and kinetoplast. The attenuated form returned to its normal size and multiplied when inoculated into naive Oncorhynchus mykiss. It produced a low parasitemia but did not cause disease (e.g., no exophthalmia or anemia) in fish. At four wk after infection, the vaccinated fish were challenged with the virulent parasite. They were protected from the disease, whereas the control (naive) fish, infected with only the virulent parasite, had the usual clinical signs (e.g., anemia, exophthalmia). No parasite was detected in any of 10 vaccinated fish at 22 wk after challenge with the virulent parasite. However, 5 of 9 fish infected with culture forms and 6 of 9 fish infected with blood forms still had detectable parasites at 26 and 22 wk after infection, respectively.