Biomedical subjects
Publications and source records attributed to Julius Lukes.
The Sm-binding site of the kinetoplastid spliced leader RNA has been implicated in accurate spliced leader RNA maturation and trans-splicing competence. In Trypanosoma brucei, RNA interference-mediated knockdown of SmD1 caused defects in spliced leader RNA maturation, displaying aberrant 3'-end formation, partial formation of cap 4, and overaccumulation in the cytoplasm; U28 pseudouridylation was unaffected.
The Crithidia fasciculata KAP2 and KAP3 proteins are closely related kinetoplast-specific histone-like DNA-binding proteins. The KAP2 and KAP3 genes are 46% identical and are arranged in tandem on the chromosomal DNA. Disruption of both alleles of either gene alone shows no detectable phenotype. However, replacement of both copies of the sequence encoding the entire KAP2 and KAP3 locus increases maxicircle mRNA levels two- to fourfold. These double-knockout cells are viable but grow extremely slowly, have reduced respiration and very abnormal cell morphologies, and accumulate numerous large vacuoles. The extreme phenotype of these mutant cells suggests an important role for the KAP2 and KAP3 proteins in mitochondrial metabolism and cell growth.
Herein, we describe the first case of a natural infection of biting midges by a kinetoplastid protozoan. Flagellates from a female Culicoides kibunensis captured in a bird's nest were introduced into culture and characterized by light and electron microscopy. However, because the morphological data were inconclusive, the novel endosymbiont-free trypanosomatid was assigned into Herpetomonas primarily on the basis of the 18S and 5S ribosomal RNA (rRNA) gene sequences.
Random amplified polymorphic DNA (RAPD) was used to detect intraspecific diversity for the Leishmania donovani complex. Fifty-two decameric to 21-meric primers of arbitrary sequence were applied to 15 strains that belong to nine zymodemes. Strains belonging to the species L. major and L. tropica were used as outgroups. A total of 902 amplicons generated by RAPD were scored. Most primers produced species-specific profiles, only 0.6% amplicons were shared by all species, while 4.3% amplicons were common for all 15 strains of the L. donovani complex. Well-supported trees have been constructed, which show a rather strong correlation between the genetic polymorphism of studied strains and their geographic origin. In all obtained trees, L. infantum was paraphyletic. The RAPD profiles suggest that MON-30 belongs to L. donovani. Moreover, the genetic distance between the L. archibaldi strain and other leishmanias does not warrant existence of a separate species.
We have studied topoisomerase II (topo II) in the cells of Bodo saltans, a free-living bodonid (Kinetoplastida). Phylogenetic analysis based on the sequence of the entire topo II gene, which is a single-copy gene, confirmed that B. saltans is a predecessor of parasitic trypanosomatids. Antibodies generated against either an overexpressed unique C-terminal region of topo II or a synthetic oligopeptide derived from the same region did not cross-react with cell lysates of related trypanosomatids, while they recognized a single specific band in the B. saltans lysate. Immunolocalization experiments using both antibodies showed that topo II is evenly dispersed throughout the kinetoplast. This is in striking difference from the localization of topo II in other flagellates, where it occurs in two antipodal centers flanking the kinetoplast disk. Moreover, the same topo II has a distinct localization in multiple loci at the periphery of the nucleus of B. saltans. With a minicircle probe derived from the conserved region we have shown that all relaxed non-catenated minicircles are confined to the globular kinetoplast DNA bundle. Therefore, in the mitochondrion of this primitive eukaryote topo II does not catenate relaxed DNA circles into a network in vivo, while a decatenating activity is present in partially purified cell lysates.
Cyst-forming coccidia of the genus Sarcocystis (Alveolata: Apicomplexa: Coccidea) parasitize vertebrates worldwide. Data from the small subunit rRNA genes (SSU) and the D2 domain of the large subunit rRNA genes were used to reconstruct phylogeny for all species in the Sarcocystidae for which sequences are currently available. We have focused on the evolutionary history of species that circulate between snakes as definitive hosts and rodents as intermediate hosts. Trees were reconstructed using maximum parsimony, minimum evolution, maximum likelihood and the bayesian phylogenetics. Our reconstructions support monophyly of Sarcocystidae but fail to robustly resolve the relationship within clades. Using a concatenated dataset of available rDNAs, the "isosporoid" coccidia Neospora, Toxoplasma, Besnoitia, Isospora and Hyaloklossia form a sister group to the monophyletic Sarcocystis. Moreover, we show that Sarcocystis from arboreal vipers of the genus Atheris, which are endemic to the mountain rain forests of the Equatorial Africa, are monophyletic, with sister species parasitizing the desert viper Pseudocerastes persicus from the Near East. We report the co-evolution of Sarcocystis spp. with their final snake hosts. The geological history of the African continent, mountain ranges, forests and general SSU rDNA rates were used to construct a linearized tree. Possible origin of the heteroxenous life cycle of Sarcocystis is discussed.
Results of molecular determination of a dog isolate of Neospora caninum in the Czech Republic are presented. Colorless bisporocystic oocysts measuring 10-13 micro m x 10-11 micro m were recovered from feces and used for DNA isolation. A diagnostic PCR procedure using previously described molecular methods was performed to determine the species. The N. caninum species-specific primers based on the Nc 5 region produced a positive result, while primers specific for Hammondia heydorni rDNA internal transcribed spacer 1 (ITS1) was negative. Sequencing and phylogenetic comparison of ITS1 rDNA and the D2 domain of the large subunit rDNA (D2 LSU) determined our isolate to be N. caninum. Phylogenetic analysis of closely related genera Toxoplasma, Neospora and Hammondia based on ITS1 and D2 LSU robustly distinguished three clades: (i). Toxoplasma gondii + Hammondia hammondi, (ii). N. caninum + Neospora hughesi, and (iii). H. heydorni. Based on phylogenetic relationships we propose three acceptable suggestions to solve the problem of taxonomy of these genera.
The coccidium Sarcocystis singaporensis (Apicomplexa: Sarcocystidae) is a cyst-forming parasite with potential as a biological agent for the control of wild populations of rodents in non-native environments. Phylogenetic analysis based on the ssrDNA supports S. singaporensisisolates as a sister species to sarcosporidians transmitted between snakes and rodents but an association with the carnivore-ruminant Sarcocystis spp. could not be rejected by likelihood ratio tests. Four complete and six partial ssrDNA sequences representing this species are monophyletic in any tree reconstruction method; however, they possess very high pairwise distances of up to 0.053. The obtained sequences suggest the probable existence of at least two divergent paralogous ssrDNAs. Moreover, our results support the co-evolution of lsrDNA and ssrDNA in S. singaporensis. The utility of coccidian lsrDNA and ssrDNA for evolutionary studies and their abundance in the primary nucleotide databases is discussed.
Apicomplexan protozoa contain a single mitochondrion and a multimembranous plastid-like organelle termed apicoplast. The size of the apicomplexan plastid genome is extremely small (35 kb) thus offering a limited number of genes for phylogenetic analysis. Moreover, the sequences of apicoplast genes are highly adenosine+thymidine-rich and rapidly evolving. Due to these facts, phylogenetic analyses based on different genes or the structure of the ribosomal operon show conflicting results and the evolutionary history of this exciting organelle remains unclear. Although it is evident that the apicoplast and its genome is plastid-derived, our detailed phylogenetic analysis of amino acid and nucleotide sequences of selected apicoplast ribosomal protein genes rpl2, rpl14 and rps12 show their possible mitochondrial origin. The affinity of apicoplast ribosomal proteins to their mitochondrial homologs is very stable and well supported. Based on our results we propose that apicoplasts might contain both plastid and mitochondrial genes, thus constituting a hybrid assembly.
We performed a phylogenetic analysis, based on the partial small subunit rRNA gene (SSU rRNA) sequences from 13 apicoplasts (including new sequences of Sarcocystis muris and Hyaloklossia lieberkuehni) and 16 other plastids, with cyanobacteria as an outgroup. The apicoplast sequences formed a highly supported monophyletic clade with two distinct clades, representing coccidia and haemosporidia, with coccidia divided into Eimeriidae and Sarcocystidae subclades. We confirmed the phylogenetic position of H. lieberkuehni within the Sarcocistidae, as a sister to the Toxoplasma/Neospora group. The coccidian plastid sequences appear to evolve slowly, while their homologues from haemosporidians are more rapidly evolving. We suggest that the higher evolutionary rate is reflected by the increase in the AT content and the possible reduction of the outer apicoplast membrane in some haemosporidians. Since the apicoplast SSU rRNA gene sequences, when compared with their nuclear homologues, offer a higher number of informative positions, they can be used for phylogenetic inference within the Apicomplexa.
The phylogenetic relationships of Goussia janae and Choleoeimeria sp. were analyzed using the small subunit ribosomal RNA gene (SSU rDNA). This is a first attempt to study the molecular phylogeny of coccidian genera parasitizing strictly poikilotherm hosts. The biliary Eimeria-like coccidia of reptiles classified into the genus Choleoeimeria form a sister clade to the family Eimeriidae, which confirms the separate generic status of the genus Choleoeimeria. The position of Goussia is less robustly resolved, since it forms a trichotomy with the Eimeriidae and Sarcocystidae, or alternatively constitutes the earliest branch of the coccidian lineage. Morphological similarities, namely the extracytoplasmic location of the endogenous stages, and the presence of sutures in the sporocyst wall are discussed in the context of the traditional classification of eimeriids. In contrast to the morphology-based systematics, the monophyly of Goussia and Choleoeimeria is not supported by the SSU rDNA data.
Despite extensive phylogenetic analysis of small subunit ribosomal RNA (SSUrRNA) genes, the deep-level relationships among kinetoplastids remain poorly understood, limiting our grasp of their evolutionary history, especially the origins of their bizarre mitochondrial genome organizations. In this study we examine the SSUrRNA data in the light of a new marker--cytoplasmic heat shock protein 90 (hsp90) sequences. Our phylogenetic analyses divide kinetoplastids into four main clades. Clades 1-3 include the various bodonid kinetoplastids. Trypanosomatids comprise the fourth clade. SSUrRNA analyses give vastly different and poorly supported positions for the root of the kinetoplastid tree, depending on the out-group and analysis method. This is probably due to the extraordinary length of the branch between kinetoplastids and any out-group. In contrast, almost all hsp90 analyses place the root between clade 1 (including Dimastigella, Rhynchomonas, several Bodo spp., and probably Rhynchobodo) and all other kinetoplastids. Maximum likelihood and maximum likelihood distance analyses of hsp90 protein and second codon-position nucleotides place trypanosomatids adjacent to Bodo saltans and Bodo cf. uncinatus (clade 3), as (weakly) do SSUrRNA analyses. Hsp90 first codon- plus second codon-position nucleotide analyses return a slightly different topology. We show that this may be an artifact caused, in part, by the different evolutionary behavior of first- and second-codon positions. This study provides the most robust evidence to date that trypanosomatids are descended from within bodonids and that B. saltans is a close relative of trypanosomatids. A total reevaluation of the high-level systematics within kinetoplastids is needed. We confirm that the interlocking network organization of kinetoplast DNA seen in trypanosomatids is a derived condition within kinetoplastids but suggest that open-conformation minicircles may have arisen early in kinetoplastid evolution. Further understanding of the evolution of kinetoplast structure and RNA editing is hampered by a paucity of data from basal (i.e., clade 1) bodonids.
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The de novo synthesis of cytochrome c oxidase subunits I, II (COI and COII), and apocytochrome b (Cyb) was investigated in kinetoplast-mitochondria of Leishmania. The organelles were isolated after breaking whole cells with nitrogen cavitation. Individual COI, COII, and Cyb polypeptides were identified by fractionation of the kinetoplast membranes, labeled with [(35)S]methionine and cysteine, using two-dimensional (9 versus 14% and 20 versus 11%) denaturing gel electrophoresis. The reaction did not require exogenous energy sources or amino acids. On the contrary, the presence of amino acids other than methionine somewhat inhibited the labeling reaction probably by competing with the uptake of labeled amino acids. The synthesis reaction was insensitive to 100 microg/ml chloramphenicol, gentamycin, paromomycin, lincomycin, hygromycin, and tetracycline, as well as cycloheximide. The process showed a linear increase in the amount of synthesized polypeptides during the first 2 h of incubation, followed by a slower accumulation of products for up to 4 h. The de novo synthesized polypeptides were stable for several additional hours. Their assembly into respiratory complexes, investigated using two-dimensional Blue Native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-SDS gels, began early during the incubation and continued throughout the course of the synthesis. This work represents the first unequivocal identification of the polypeptide synthesis in kinetoplasts.
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A relatively large nuclear-encoded polypeptide, designated trCOIV, is found in the cytochrome c oxidase (CO) complex of trypanosomatids. In order to determine if this polypeptide represents a bona fide subunit of the complex, we have characterized the cDNA and the gene for this polypeptide in Leishmania tarentolae. Its nuclear gene has no sequence similarity to mammalian COIV. The trCOIV preprotein has a long mitochondrial targeting sequence of 31 residues. The mature polypeptide cofractionates with kinetoplast-mitochondria and its preferential mitochondrial localization was confirmed by immunofluorescence and immunoelectron microscopy. Based on the hydropathy plot analysis, the protein lacks pronounced transmembrane domains and likely occupies a peripheral position within the CO complex. The corresponding genes are also present in the sequenced portions of the Trypanosoma cruzi, Trypanosoma brucei and Leishmania major genomes, and the same polypeptide is found in cytochrome oxidase isolated from procyclic T. brucei and promastigote Leishmania mexicana amazonensis. However, the trCOIV gene, the mRNA and the polypeptide could not be detected in a respiration-deficient trypanosomatid Phytomonas serpens.
We have determined the sequences of 5S rRNA and spliced leader (SL) RNA genes, and adjacent intergenic regions for representatives of all known trypanosomatid genera parasitizing insects. The genetic loci have been analyzed separately as well as by a combined approach. Several isolates, assigned by morphology to different genera (Leptomonas spp., Blastocrithidia spp.), seem to belong to a single species with an unexpectedly wide host and geographical range. An unnamed trypanosomatid isolated from rats in Egypt was found to belong to the genus Herpetomonas, so far associated with insect hosts only. It is closely related to Herpetomonas ztiplika, a parasite of a blood-sucking biting midge. Apparently several different trypanosomatid species can infect one insect species, as exemplified by Leptomonas sp. PL and Wallaceina sp. Wsd, which were isolated from different specimens of Salda littoralis on the same locality and day. However, since the same species of Leptomonas was obtained from insect hosts belonging to different genera, some insect trypanosomatids may have low host specificity. Our data revealed additional discrepancies between molecular phylogenetic data and cell morphology, rendering current trypanosomatid taxonomy unreliable.
Leptomonas podlipaevi n. sp., a new trypanosomatid species, is described herein based on light microscopic, ultrastructural, and molecular phylogenetic data. The organism is pleomorphic both in host and culture, with two predominant forms-a typical promastigote with a long flagellum and a shorter promastigote with a small or barely extending flagellum. Several spliced leader RNA repeat sequences obtained from the original cultures and the clonal lines representing two types of cells were all nearly identical. These sequences formed a tight cluster in the neighbor-joining tree well separated from other trypanosomatid species. Glyceraldehyde phosphate dehydrogenase gene sequences were determined for L. podlipaevi and 10 previously described trypanosomatid species. Molecular phylogenetic analysis has demonstrated that the new species is most closely related to Leptomonas seymouri and Leptomonas pyrrhocoris. The analysis has also highlighted the polyphyly of the genus Leptomonas.