Signatures of hybridization in Trypanosoma brucei

Genetic exchange among disease-causing micro-organisms can generate progeny that combine different pathogenic traits. Though sexual reproduction has been described in trypanosomes, its impact on the epidemiology of Human African Trypanosomiasis (HAT) remains controversial. However, human infective and non-human infective strains of Trypanosoma brucei circulate in the same transmission cycles in HAT endemic areas in subsaharan Africa, providing the opportunity for mating during the developmental cycle in the tsetse fly vector. Here we investigated inheritance among progeny from a laboratory cross of T. brucei and then applied these insights to genomic analysis of field-collected isolates to identify signatures of past genetic exchange. Genomes of two parental and four hybrid progeny clones with a range of DNA contents were assembled and analysed by k-mer and single nucleotide polymorphism (SNP) frequencies to determine heterozygosity and chromosomal inheritance. Variant surface glycoprotein (VSG) genes and kinetoplast (mitochondrial) DNA maxi- and minicircles were extracted from each genome to examine how each of these components was inherited in the hybrid progeny. The same bioinformatic approaches were applied to an additional 37 genomes representing the diversity of T. brucei in subsaharan Africa and T. evansi. SNP analysis provided evidence of crossover events affecting all 11 pairs of megabase chromosomes and demonstrated that polyploid hybrids were formed post-meiotically and not by fusion of the parental diploid cells. VSGs and kinetoplast DNA minicircles were inherited biparentally, with approximately equal numbers from each parent, whereas maxicircles were inherited uniparentally. Extrapolation of these findings to field isolates allowed us to distinguish clonal descent from hybridization by comparing maxicircle genotype to VSG and minicircle repertoires. Discordance between maxicircle genotype and VSG and minicircle repertoires indicated inter-lineage hybridization. Significantly, some of the hybridization events we identified involved human infective and non-human infective trypanosomes circulating in the same geographic areas.     

Trypanosoma cruzi in the chicken model: Chagas-like heart disease in the absence of parasitism

Background

The administration of anti-trypanosome nitroderivatives curtails Trypanosoma cruzi infection in Chagas disease patients, but does not prevent destructive lesions in the heart. This observation suggests that an effective treatment for the disease requires understanding its pathogenesis.

Methodology/Principal Findings

To understand the origin of clinical manifestations of the heart disease we used a chicken model system in which infection can be initiated in the egg, but parasite persistence is precluded. T. cruzi inoculation into the air chamber of embryonated chicken eggs generated chicks that retained only the parasite mitochondrial kinetoplast DNA minicircle in their genome after eight days of gestation. Crossbreeding showed that minicircles were transferred vertically via the germ line to chicken progeny. Minicircle integration in coding regions was shown by targeted-primer thermal asymmetric interlaced PCR, and detected by direct genomic analysis. The kDNA-mutated chickens died with arrhythmias, shortness of breath, cyanosis and heart failure. These chickens with cardiomyopathy had rupture of the dystrophin and other genes that regulate cell growth and differentiation. Tissue pathology revealed inflammatory dilated cardiomegaly whereby immune system mononuclear cells lyse parasite-free target heart fibers. The heart cell destruction implicated a thymus-dependent, autoimmune; self-tissue rejection carried out by CD45⁺, CD8γδ⁺, and CD8α lymphocytes.

Conclusions/Significance

These results suggest that genetic alterations resulting from kDNA integration in the host genome lead to autoimmune-mediated destruction of heart tissue in the absence of T. cruzi parasites.     

Trypanosoma cruzi: variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis

Nineteen Trypanosoma cruzi stocks, most of them of wild origin, and four Trypanosoma rangeli stocks from Colombia were analysed by molecular karyotype analysis with cloned DNA cruzipain as the probe. Another 27 cloned stocks of T. cruzi from different geographic areas of South America were used as reference for T. cruzi lineages. Phenetic analysis of chromosome size polymorphism demonstrated a great variability of Colombian T. cruzi stocks, suggesting that most belong to lineage I, although two of them belong to lineage II. The 2 lineage II T. cruzi, 17 T. cruzi lineage I, and 3 T. rangeli stocks from Colombia were studied further by Southern blot analysis with a panel of kinetoplast DNA minicircle probes. Hybridisation results indicate that the two T. cruzi II stocks are genetically distant from each other and from T. cruzi lineages IIb, IId, and IIe from Chile. Finally, T. cruzi minicircle probes do not cross-hybridise in any stringency condition tested with T. rangeli minicircles, a clear indication that these parasites can be easily distinguished by this method.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Role of p38 in replication of Trypanosoma brucei kinetoplast DNA

Trypanosomes have an unusual mitochondrial genome, called kinetoplast DNA, that is a giant network containing thousands of interlocked minicircles. During kinetoplast DNA synthesis, minicircles are released from the network for replication as theta-structures, and then the free minicircle progeny reattach to the network. We report that a mitochondrial protein, which we term p38, functions in kinetoplast DNA replication. RNA interference (RNAi) of p38 resulted in loss of kinetoplast DNA and accumulation of a novel free minicircle species named fraction S. Fraction S minicircles are so underwound that on isolation they become highly negatively supertwisted and develop a region of Z-DNA. p38 binds to minicircle sequences within the replication origin. We conclude that cells with RNAi-induced loss of p38 cannot initiate minicircle replication, although they can extensively unwind free minicircles.     

Molecular diversity of dinoflagellate symbionts of Cnidaria: the psbA minicircle of Symbiodinium

Dinoflagellate algae of the genus Symbiodinium are important symbionts within corals and other benthic marine animals. The molecular diversity of Symbiodinium has been described mainly by use of ribosomal DNA sequence data. We tested whether minicircle sequences, which appear to form the chloroplast genome in many dinoflagellates, could be used as a marker for molecular diversity among symbionts found in corals and sea anemones. Partial and full-length sequences for psbA were obtained from environmental samples of coral and sea anemones of wide-ranging geographical distribution. Phylogenetic trees constructed with partial psbA sequences were consistent with the known phylotypes of the isolates. Further sequencing suggested that the psbA gene is present on a minicircle in all Symbiodinium phylotypes. The length and DNA sequence of the non-coding portion of the minicircles varied considerably among Symbiodinium phylotypes. In two Symbiodinium isolates from different phylotypes an elaborate pattern of repeat sequences of unknown function was found in the non-coding region. Phylogenetic analysis of the non-coding region of the psbA minicircle indicates that minicircle sequences could be a useful chloroplast-derived marker for differentiating both closely related and distantly related Symbiodinium isolates.     

TbPIF1, a Trypanosoma brucei Mitochondrial DNA Helicase, Is Essential for Kinetoplast Minicircle Replication

Kinetoplast DNA, the trypanosome mitochondrial genome, is a network of interlocked DNA rings including several thousand minicircles and a few dozen maxicircles. Minicircles replicate after release from the network, and their progeny reattach. Remarkably, trypanosomes have six mitochondrial DNA helicases related to yeast PIF1 helicase. Here we report that one of the six, TbPIF1, functions in minicircle replication. RNA interference (RNAi) of TbPIF1 causes a growth defect and kinetoplast DNA loss. Minicircle replication intermediates decrease during RNAi, and there is an accumulation of multiply interlocked, covalently closed minicircle dimers (fraction U). In studying the significance of fraction U, we found that this species also accumulates during RNAi of mitochondrial topoisomerase II. These data indicate that one function of TbPIF1 is an involvement, together with topoisomerase II, in the segregation of minicircle progeny.     

The kinetoplast DNA of the Australian trypanosome,Trypanosoma copemani,shares features with Trypanosoma cruzi and Trypanosoma lewisi

Kinetoplast DNA (kDNA) is the mitochondrial genome of trypanosomatids. It consists of a few dozen maxicircles and several thousand minicircles, all catenated topologically to form a two-dimensional DNA network. Minicircles are heterogeneous in size and sequence among species. They present one or several conserved regions that contain three highly conserved sequence blocks. CSB-1 (10?bp sequence) and CSB-2 (8?bp sequence) present lower interspecies homology, while CSB-3 (12?bp sequence) or the Universal Minicircle Sequence is conserved within most trypanosomatids. The Universal Minicircle Sequence is located at the replication origin of the minicircles, and is the binding site for the UMS binding protein, a protein involved in trypanosomatid survival and virulence. Here, we describe the structure and organisation of the kDNA of Trypanosoma copemani, a parasite that has been shown to infect mammalian cells and has been associated with the drastic decline of the endangered Australian marsupial, the woylie (Bettongia penicillata). Deep genomic sequencing showed that T. copemani presents two classes of minicircles that share sequence identity and organisation in the conserved sequence blocks with those of Trypanosoma cruzi and Trypanosoma lewisi. A 19,257?bp partial region of the maxicircle of T. copemani that contained the entire coding region was obtained. Comparative analysis of the T. copemani entire maxicircle coding region with the coding regions of T. cruzi and T. lewisi showed they share 71.05% and 71.28% identity, respectively. The shared features in the maxicircle/minicircle organisation and sequence between T. copemani and T. cruzi/T. lewisi suggest similarities in their process of kDNA replication, and are of significance in understanding the evolution of Australian trypanosomes.     

Evidence for horizontal gene transfer from bacteroidetes bacteria to dinoflagellate minicircles

Dinoflagellate protists harbor a characteristic peridinin-containing plastid that evolved from a red or haptophyte alga. In contrast to typical plastids that have ～100-200 kb circular genomes, the dinoflagellate plastid genome is composed of minicircles that each encode 0-5 genes. It is commonly assumed that dinoflagellate minicircles are derived from a standard plastid genome through drastic reduction and fragmentation. However, we demonstrate that the ycf16 and ycf24 genes (encoded on the Ceratium AF490364 minicircle), as well as rpl28 and rpl33 (encoded on the Pyrocystis AF490367 minicircle), are related to sequences from Algoriphagus and/or Cytophaga bacteria belonging to the Bacteroidetes clade. Moreover, we identified a new open reading frame on the Pyrocystis minicircle encoding a SRP54 N domain, which is typical of FtsY proteins. Because neither of these minicircles share sequence similarity with any other dinoflagellate minicircles, and their genes resemble bacterial operons, we propose that these Ceratium and Pyrocystis minicircles resulted from a horizontal gene transfer (HGT) from a Bacteroidetes donor. Our findings are the first indication of HGT to dinoflagellate minicircles, highlighting yet another peculiar aspect of this plastid genome.     

Plastid-derived single gene minicircles of the dinoflagellate Ceratium horridum are localized in the nucleus

Recent reports show that numerous chloroplast-specific proteins of peridinin-containing dinoflagellates are encoded on minicircles-small plasmidlike molecules containing one or two polypeptide genes each. The genes for these polypeptides are chloroplast specific because their homologs from other photosynthetic eukaryotes are exclusively encoded in the chloroplast genome. Here, we report the isolation, sequencing, and subcellular localization of minicircles from the peridinin-containing dinoflagellate Ceratium horridum. The C. horridum minicircles are organized in the same manner as in other peridinin-containing dinoflagellates and encode the same kinds of plastid-specific proteins, as previous studies reported. However, intact plastids isolated from C. horridum do not contain minicircles, nor do they contain DNA that hybridizes to minicircle-specific probes. Rather, C. horridum minicircles are localized in the nucleus as shown by cell fractionation, Southern hybridization, and in situ hybridization with minicircle-specific probes. A high-molecular-weight DNA was detected in purified C. horridum plastids, but it is apparently not minicircular in organization, as hybridization with a cloned probe from the plastid-localized DNA suggests. The distinction between C. horridum and other peridinin-containing dinoflagellates at the level of their minicircle localization is paralleled by C. horridum thylakoid organization, which also differs from that of other peridinin-containing dinoflagellates, indicating that a hitherto underestimated diversity of minicircle DNA localization and thylakoid organization exists across various dinoflagellate groups.     

Development and Validation of Non-Integrative,Self-Limited,and Replicating Minicircles for Safe Reporter Gene Imaging of Cell-Based Therapies

Reporter gene (RG) imaging of cell-based therapies provides a direct readout of therapeutic efficacy by assessing the fate of implanted cells. To permit long-term cellular imaging, RGs are traditionally required to be integrated into the cellular genome. This poses a potential safety risk and regulatory bottleneck for clinical translation as integration can lead to cellular transformation. To address this issue, we have developed non-integrative, replicating minicircles (MCs) as an alternative platform for safer monitoring of cells in living subjects. We developed both plasmids and minicircles containing the scaffold/matrix attachment regions (S/MAR) of the human interferon-beta gene, driven by the CMV promoter, and expressing the bioluminescence RG firefly luciferase. Constructs were transfected into breast cancer cells, and expanded S/MAR minicircle clones showed luciferase signal for greater than 3 months in culture and minicircles remained as episomes. Importantly, luciferase activity in clonal populations was slowly lost over time and this corresponded to a loss of episome, providing a way to reversibly label cells. To monitor cell proliferation in vivo, 1.5×10⁶ cells carrying the S/MAR minicircle were implanted subcutaneously into mice (n = 5) and as tumors developed significantly more bioluminescence signal was noted at day 35 and 43 compared to day 7 post-implant (p<0.05). To our knowledge, this is the first work examining the use of episomal, self-limited, replicating minicircles to track the proliferation of cells using non-invasive imaging in living subjects. Continued development of S/MAR minicircles will provide a broadly applicable vector platform amenable with any of the numerous RG technologies available to allow therapeutic cell fate to be assessed in individual patients, and to achieve this without the need to manipulate the cell''s genome so that safety concerns are minimized. This will lead to safe tools to assess treatment response at earlier time points and improve the precision of cell-based therapies.     

TbPIF5 Is a Trypanosoma brucei Mitochondrial DNA Helicase Involved in Processing of Minicircle Okazaki Fragments

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.     

Formation and transposition of the covalently closed IS30 circle: the relation between tandem dimers and monomeric circles

In the present study, we demonstrate that a circular IS30 element acts as an intermediate for simple insertion. Covalently closed IS and Tn circles constructed in vitro are suitable for integration into the host genome. Minicircle integration displays all the characteristics of transpositional fusion mediated by the (IS30 )2 dimer regarding target selection and target duplication. Evidence is provided for in vivo circularization of the element located either on plasmids or on the genome. It is shown that circle formation can occur through alternative pathways. One of them is excision of IS30 from a hot spot via joining the IRs. This reaction resembles the site-specific dimerization that leads to (IS30 )2 establishment. The other process is the dissolution of (IS30 )2 dimer, when the element is excised from an IR-IR joint. These pathways differ basically in the fate of the donor replicon: only dimer dissolution gives rise to resealed donor backbone. Analysis of minicircles and the rearranged donor replicons led us to propose a molecular model that can account for differences between the circle-generating processes. Our focus was to the dissolution of IR-IR joints located on the host genome, because these events promoted extensive genomic rearrangements and accompanied minicircle formation. The results present the possibility of host genome reorganization by IS30-like transposition.     

Replication of kinetoplast DNA in isolated kinetoplasts from Crithidia fasciculata. Identification of minicircle DNA replication intermediates

The kinetoplast DNA (kDNA) of trypanosomes is comprised of thousands of DNA minicircles and 20-50 maxicircles catenated into a single network. We show that kinetoplasts isolated from the trypanosomatid species Crithidia fasciculata incorporate labeled nucleotides and support minicircle DNA replication in a manner which mimics two characteristics of minicircle replication in vivo: 1) the minicircles are replicated as free molecules and subsequently reattached to the kDNA network, and 2) a replication intermediate having a structure consistent with a highly gapped minicircle species is generated. In addition, a class of minicircle DNA replication intermediates is observed containing discontinuities at specific sites within each of the newly synthesized DNA strands. By using a strain of C. fasciculata possessing nearly homogenous minicircles, we were able to map the discontinuities to two small regions situated 180 degrees apart on the minicircle. Each region has two sites at which a discontinuity can occur, one on each strand and separated by approximately 100 base pairs. These sites may represent origins of minicircle DNA replication.     

The kinetoplast duplication cycle in Trypanosoma brucei is orchestrated by cytoskeleton-mediated cell morphogenesis

The mitochondrial DNA of Trypanosoma brucei is organized in a complex structure called the kinetoplast. In this study, we define the complete kinetoplast duplication cycle in T. brucei based on three-dimensional reconstructions from serial-section electron micrographs. This structural model was enhanced by analyses of the replication process of DNA maxi- and minicircles. Novel insights were obtained about the earliest and latest stages of kinetoplast duplication. We show that kinetoplast S phase occurs concurrently with the repositioning of the new basal body from the anterior to the posterior side of the old flagellum. This emphasizes the role of basal body segregation in kinetoplast division and suggests a possible mechanism for driving the rotational movement of the kinetoplast during minicircle replication. Fluorescence in situ hybridization with minicircle- and maxicircle-specific probes showed that maxicircle DNA is stretched out between segregated minicircle networks, indicating that maxicircle segregation is a late event in the kinetoplast duplication cycle. This new view of the complexities of kinetoplast duplication emphasizes the dependencies between the dynamic remodelling of the cytoskeleton and the inheritance of the mitochondrial genome.