Genome-wide function of MCM-BP in Trypanosoma brucei DNA replication and transcription.

In Trypanosoma brucei, genes are arranged in Polycistronic Transcription Units (PTUs), which are demarcated by transcription start and stop sites. Transcription start sites are also binding sites of Origin Recognition Complex 1 (ORC1). This spatial coincidence implies that transcription and replication in trypanosomes must occur in a highly ordered and cooperative manner. Interestingly, a previously published genetic screen identified the T. brucei MCM-BP, which interacts with subunits of MCM helicase, as a protein whose downregulation results in the loss of transcriptional silencing at subtelomeric loci. Here, I show that TbMCM-BP is required for DNA replication and transcription. TbMCM-BP depletion causes a significant reduction of replicating cells in S phase and genome-wide impairments of replication origin activation. Moreover, levels of sense and antisense transcripts increase at boundaries of PTUs in the absence of TbMCM-BP. TbMCM-BP is also important for transcriptional repression of the specialized subtelomeric PTUs, the Bloodstream-form Expression-Sites (BESs), which house the major antigenic determinant (the Variant Surface Glycoprotein, VSG gene) as well as TbORC1 binding sites. Overall, this study reveals that TbMCM-BP, a replication initiation protein, also guides the initiation, termination and directionality of transcription.

Chromosomal copy number variation analysis by next generation sequencing confirms ploidy stability in Trypanosoma brucei subspecies.

Although aneuploidy usually results in severe abnormalities in multicellular eukaryotes, recent data suggest that it could be beneficial for unicellular eukaryotes, such as yeast and trypanosomatid parasites, providing increased survival under stressful conditions. Among characterized trypanosomatids, Trypanosoma cruzi, Trypanosoma brucei and species from the genus Leishmania stand out due to their importance in public health, infecting around 20 million people worldwide. The presence of aneuploidies in T. cruzi and Leishmania was recently confirmed by analysis based on next generation sequencing (NGS) and fluorescence in situ hybridization, where they have been associated with adaptation during transmission between their insect vectors and mammalian hosts and in promoting drug resistance. Although chromosomal copy number variations (CCNVs) are present in the aforementioned species, PFGE and fluorescence cytophotometry analyses suggest that aneuploidies are absent from T. brucei. A re-evaluation of CCNV in T. b gambiense based on NGS reads confirmed the absence of aneuploidies in this subspecies. However, the presence of aneuploidies in the other two T. brucei subspecies, T. b. brucei and T. b. rhodesiense, has not been evaluated using NGS approaches. In the present work, we tested for aneuploidies in 26 T. brucei isolates, including samples from the three T. brucei subspecies, by both allele frequency and read depth coverage analyses. These analyses showed that none of the T. brucei subspecies presents aneuploidies, which could be related to differences in the mechanisms of DNA replication and recombination in these parasites when compared with Leishmania.

Resistance to Plasmodium falciparum in sickle cell trait erythrocytes is driven by oxygen-dependent growth inhibition.

Sickle cell trait (AS) confers partial protection against lethal Plasmodium falciparum malaria. Multiple mechanisms for this have been proposed, with a recent focus on aberrant cytoadherence of parasite-infected red blood cells (RBCs). Here we investigate the mechanistic basis of AS protection through detailed temporal mapping. We find that parasites in AS RBCs maintained at low oxygen concentrations stall at a specific stage in the middle of intracellular growth before DNA replication. We demonstrate that polymerization of sickle hemoglobin (HbS) is responsible for this growth arrest of intraerythrocytic P. falciparum parasites, with normal hemoglobin digestion and growth restored in the presence of carbon monoxide, a gaseous antisickling agent. Modeling of growth inhibition and sequestration revealed that HbS polymerization-induced growth inhibition following cytoadherence is the critical driver of the reduced parasite densities observed in malaria infections of individuals with AS. We conclude that the protective effect of AS derives largely from effective sequestration of infected RBCs into the hypoxic microcirculation.

Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum.

Cryptosporidium parvum is a zoonotic protozoan that can cause a life-threatening gastrointestinal syndrome in children and in immunocompromised adults. Currently, the only approved drug for treatment of Cryptosporidium infections in humans is nitazoxanide, but it is not effective in immunocompromised individuals or in children with malnutrition. This is compounded by the lack of genetic methods for studying and validating potential drug targets in the parasite. Therefore, in this study, we endeavoured to adapt the use of a phosphorodiamidate morpholino oligomer (morpholino) antisense approach to develop a targeted gene knockdown assay for use in C. parvum. We show that morpholinos, at non-toxic concentrations, are rapidly internalised by both C. parvum and host cells (HCT-8), and distribute diffusely throughout the cytosol. Using morpholinos to separately target C. parvum lactate dehydrogenase and putative arginine n-methyltransferase genes, within 36h of in vitro culture, we achieved over 10-fold down-regulation of the respective encoded proteins in C. parvum. Pursuant to this, we observed that knockdown of C. parvum lactate dehydrogenase produced a dramatic reduction in intracellular growth and development of C. parvum by 56h of culture. On the other hand, C. parvum putative arginine n-methyltransferase knockdown did not appear to have any effect on parasite growth, but nevertheless provided the proof-of-principle that the morpholino knockdown assay in C. parvum was consistent. Together, our findings present a gene regulation approach for interrogating gene function in C. parvum in vitro, and further provide genetic evidence for the essential role of C. parvum lactate dehydrogenase in fueling the growth and development of intracellular C. parvum.

Plasmodium falciparum RuvB2 translocates in 5'-3' direction, relocalizes during schizont stage and its enzymatic activities are up regulated by RuvB3 of the same complex.

Two similar proteins RuvB like1 (Rvb1/Pontin) and RuvB like2 (Rvb2/Reptin) of AAA+ family of enzymes are present in yeast to human and are well known to be involved in diverse cellular activities. The human malaria parasite Plasmodium falciparum contains three different RuvB like proteins. Thus it has been of interest to explore why P. falciparum requires three RuvB like proteins and how these enzymes are biochemically regulated. In this study, we present the detailed biochemical characterization of PfRuvB2. The complex of PfRuvB3 was immunopurified and the presence of PfRuvB2 was confirmed. The in vitro interaction study shows that PfRuvB2 interacts only with PfRuvB3 but not with PfRuvB1. The recombinant as well as endogenous PfRuvB2 contains ATPase as well as weak DNA helicase activities. The presence of PfRuvB3 in the helicase reaction of PfRuvB2 increases the helicase activity significantly. Interestingly PfRuvB2/PfRuvB3 complex preferentially translocates and unwinds DNA in the 5'-3' direction. In vivo studies showed that PfRuvB2 is expressed in all the asexual intraerythrocytic developmental stages and localizes mainly in the nucleus during merozoite, ring and trophozoite stages while during schizont stage it relocalizes partially in the nucleus and partially towards cytoplasm. As PfRuvB3 is specific to intraerythrocytic mitosis so we interpret that PfPuvB3 interacts with PfRuvB2 during schizont/intraerythrocytic mitosis and acts as its modulator mainly for the appreciable helicase activity.

Asexual populations of the human malaria parasite, Plasmodium falciparum, use a two-step genomic strategy to acquire accurate, beneficial DNA amplifications.

Malaria drug resistance contributes to up to a million annual deaths. Judicious deployment of new antimalarials and vaccines could benefit from an understanding of early molecular events that promote the evolution of parasites. Continuous in vitro challenge of Plasmodium falciparum parasites with a novel dihydroorotate dehydrogenase (DHODH) inhibitor reproducibly selected for resistant parasites. Genome-wide analysis of independently-derived resistant clones revealed a two-step strategy to evolutionary success. Some haploid blood-stage parasites first survive antimalarial pressure through fortuitous DNA duplications that always included the DHODH gene. Independently-selected parasites had different sized amplification units but they were always flanked by distant A/T tracks. Higher level amplification and resistance was attained using a second, more efficient and more accurate, mechanism for head-to-tail expansion of the founder unit. This second homology-based process could faithfully tune DNA copy numbers in either direction, always retaining the unique DNA amplification sequence from the original A/T-mediated duplication for that parasite line. Pseudo-polyploidy at relevant genomic loci sets the stage for gaining additional mutations at the locus of interest. Overall, we reveal a population-based genomic strategy for mutagenesis that operates in human stages of P. falciparum to efficiently yield resistance-causing genetic changes at the correct locus in a successful parasite. Importantly, these founding events arise with precision; no other new amplifications are seen in the resistant haploid blood stage parasite. This minimizes the need for meiotic genetic cleansing that can only occur in sexual stage development of the parasite in mosquitoes.

Target specific-trisubstituted pyrrole inhibits Babesia bovis erythrocytic growth.

Babesiosis, a significant veterinary disease and an emerging zoonotic human infection, is caused by certain species of the protozoan parasite, Babesia. Here we report that a trisubstituted pyrrole is a potent inhibitor of Babesia bovis, a bovine parasite. Furthermore, B. bovis expresses the known target of the compound, the cGMP dependent protein kinase. Target conservation and the in vitro efficacy support further investigation of this compound and validation of Babesia cGMP dependent protein kinase as its in vivo target.

Trypanosoma brucei Orc1 is essential for nuclear DNA replication and affects both VSG silencing and VSG switching.

Binding of the Origin Recognition Complex (ORC) to replication origins is essential for initiation of DNA replication, but ORC has non-essential functions outside of DNA replication, including in heterochromatic gene silencing and telomere maintenance. Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis, uses antigenic variation as a major virulence mechanism to evade the host's immune attack by expressing its major surface antigen, the Variant Surface Glycoprotein (VSG), in a monoallelic manner. An Orc1/Cdc6 homologue has been identified in T. brucei, but its role in DNA replication has not been directly confirmed and its potential involvement in VSG repression or switching has not been thoroughly investigated. In this study, we show that TbOrc1 is essential for nuclear DNA replication in mammalian-infectious bloodstream and tsetse procyclic forms (BF and PF). Depletion of TbOrc1 resulted in derepression of telomere-linked silent VSGs in both BF and PF, and increased VSG switching particularly through the in situ transcriptional switching mechanism. TbOrc1 associates with telomere repeats but appears to do so independently of two known T. brucei telomere proteins, TbRAP1 and TbTRF. We conclude that TbOrc1 has conserved functions in DNA replication and is also required to control telomere-linked VSG expression and VSG switching.

DNA content alterations in Tetrahymena pyriformis macronucleus after exposure to food preservatives sodium nitrate and sodium benzoate.

The toxicity, in terms of changes in the DNA content, of two food preservatives, sodium nitrate and sodium benzoate was studied on the protozoan Tetrahymena pyriformis using DNA image analysis technology. For this purpose, selected doses of both food additives were administered for 2 h to protozoa cultures and DNA image analysis of T. pyriformis nuclei was performed. The analysis was based on the measurement of the Mean Optical Density which represents the cellular DNA content. The results have shown that after exposure of the protozoan cultures to doses equivalent to ADI, a statistically significant increase in the macronuclear DNA content compared to the unexposed control samples was observed. The observed increase in the macronuclear DNA content is indicative of the stimulation of the mitotic process and the observed increase in MOD, accompanied by a stimulation of the protozoan proliferation activity is in consistence with this assumption. Since alterations at the DNA level such as DNA content and uncontrolled mitogenic stimulation have been linked with chemical carcinogenesis, the results of the present study add information on the toxicogenomic profile of the selected chemicals and may potentially lead to reconsideration of the excessive use of nitrates aiming to protect public health.

Regulation of the cell division cycle in Trypanosoma brucei.

The cell division cycle is tightly regulated by the activation and inactivation of a series of proteins that control the replication and segregation of organelles to the daughter cells. During the past decade, we have witnessed significant advances in our understanding of the cell cycle in Trypanosoma brucei and how the cycle is regulated by various regulatory proteins. However, many other regulators, especially those unique to trypanosomes, remain to be identified, and we are just beginning to delineate the signaling pathways that drive the transitions through different cell cycle stages, such as the G(1)/S transition, G(2)/M transition, and mitosis-cytokinesis transition. Trypanosomes appear to employ both evolutionarily conserved and trypanosome-specific molecules to regulate the various stages of its cell cycle, including DNA replication initiation, spindle assembly, chromosome segregation, and cytokinesis initiation and completion. Strikingly, trypanosomes lack some crucial regulators that are well conserved across evolution, such as Cdc6 and Cdt1, which are involved in DNA replication licensing, the spindle motor kinesin-5, which is required for spindle assembly, the central spindlin complex, which has been implicated in cytokinesis initiation, and the actomyosin contractile ring, which is located at the cleavage furrow. Conversely, trypanosomes possess certain regulators, such as cyclins, cyclin-dependent kinases, and mitotic centromere-associated kinesins, that are greatly expanded and likely play diverse cellular functions. Overall, trypanosomes apparently have integrated unique regulators into the evolutionarily conserved pathways to compensate for the absence of those conserved molecules and, additionally, have evolved certain cell cycle regulatory pathways that are either different from its human host or distinct between its own life cycle forms.

The RNA accordion model for template positioning by telomerase RNA during telomeric DNA synthesis.

Telomerase is a ribonucleoprotein (RNP) enzyme that maintains the ends of linear eukaryotic chromosomes and whose activation is a hallmark of 90% of all cancers. This RNP minimally contains a reverse transcriptase protein subunit (TERT) that catalyzes telomeric DNA synthesis and an RNA subunit (TER) that has templating, architectural and protein-scaffolding roles. Telomerase is unique among polymerases in that it synthesizes multiple copies of the template on the 3' end of a primer following a single binding event, a process known as repeat addition processivity (RAP). Using biochemical assays and single-molecule Förster resonance energy transfer (smFRET) experiments on Tetrahymena thermophila telomerase, we now directly demonstrate that TER contributes to template positioning within the active site and to the template translocation required for RAP. We propose that the single-stranded RNA elements flanking the template act as a molecular accordion, undergoing reciprocal extension and compaction during telomerase translocation.

Trypanosoma cruzi DNA replication includes the sequential recruitment of pre-replication and replication machineries close to nuclear periphery.

In eukaryotes, many nuclear processes are spatially compartmentalized. Previously, we have shown that in Trypanosoma cruzi, an early-divergent eukaryote, DNA replication occurs at the nuclear periphery where chromosomes remain constrained during the S phase of the cell cycle. We followed Orc1/Cdc6, a pre-replication machinery component and the proliferating cell nuclear antigen (PCNA), a component of replication machinery, during the cell cycle of this protozoon. We found that, at the G(1) stage, TcOrc1/Cdc6 and TcPCNA are dispersed throughout the nuclear space. During the G(1)/S transition, TcOrc1/Cdc6 migrates to a region close to nuclear periphery. At the onset of S phase, TcPCNA is loaded onto the DNA and remains constrained close to nuclear periphery. Finally, in G(2), mitosis and cytokinesis, TcOrc1/Cdc6 and TcPCNA are dispersed throughout the nuclear space. Based on these findings, we propose that DNA replication in T. cruzi is accomplished by the organization of functional machineries in a spatial-temporal manner.

The Cdc45·Mcm2-7·GINS protein complex in trypanosomes regulates DNA replication and interacts with two Orc1-like proteins in the origin recognition complex.

Accurate DNA replication requires a complex interplay of many regulatory proteins at replication origins. The CMG (Cdc45·Mcm2-7·GINS) complex, which is composed of Cdc45, Mcm2-7, and the GINS (Go-Ichi-Ni-San) complex consisting of Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the heterohexameric origin recognition complex (ORC) and functions as a replicative helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein. However, unlike archaea, trypanosomes possess components of the eukaryote-like CMG complex, but whether they form an active helicase complex, associate with the ORC, and regulate DNA replication remains unknown. Here, we demonstrated that the CMG complex is formed in vivo in trypanosomes and that Mcm2-7 helicase activity is activated by the association with Cdc45 and the GINS complex in vitro. Mcm2-7 and GINS proteins are confined to the nucleus throughout the cell cycle, whereas Cdc45 is exported out of the nucleus after DNA replication, indicating that nuclear exclusion of Cdc45 constitutes one mechanism for preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6, and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an additional component of the ORC and showed that both Orc1b and Orc1/Cdc6 associate with Mcm2-7 via interactions with Mcm3. All together, we identified the Cdc45·Mcm2-7·GINS complex as the replicative helicase that interacts with two Orc1-like proteins in the unusual origin recognition complex in trypanosomes.

Entamoeba invadens: dynamics of DNA synthesis during differentiation from trophozoite to cyst.

The DNA dynamics which mediate conversion of uni-nucleate trophozoite into quadrinucleate cyst in Entamoeba histolytica is not well understood. Here, we have addressed this question in Entamoeba invadens (a model system for encystation) through a detailed time course study of the differentiation process. We combined flow cytometric analysis with the change in rate of thymidine incorporation and the number of nuclei per cell. Our data shows that during encystment the cell population passes through three phases: (1) Early phase (0-8h); of rapid DNA synthesis which may correspond to completion of ongoing DNA replication. Bi-nucleated cells increase with concomitant drop in uni-nucleated cells. (2) Commitment phase (8-24h); in which DNA synthesis rate slows down. Possibly new rounds of replication are initiated which proceed slowly, followed by mitosis at 20 h. After this the number of bi- and uni-nucleated cells gradually decline and the tri- and tetra-nucleated cells begin to increase. (3) Consolidation phase (24-72 h); in which the rate of DNA synthesis shows a small increase till 32 h and then begins to decline. The G2/M peak reappears at 48 h, showing that more rounds of DNA replication may be getting completed, followed by nuclear division. By 72 h the encystment is virtually complete. The bi-nucleated stage could be an intermediate both in the conversion of trophozoite to cyst and back. Our study provides a comprehensive view of DNA dynamics during encystation and excystation of E. invadens.

The trypanocidal activity of the alkaloid oliverine involves inhibition of DNA synthesis.

The Trypanosoma cruzi parasite is an etiologic agent of the American trypanosomiasis called Chagas disease. This pathology affects more than 24 million persons and represents one of the most important public health problems in Latin America. Taking into account this, it is necessary the search of new antitrypanosomal agents that show a major level of efficacy and minor indexes of toxicity in affected patients. Vast source of them are the natural products from plants with enormous structural diversity. A particular type of these compounds is represented by aporphinoid alkaloids. In our experiments, anonaine (2), oliverine (3) and guatterine (5) displayed antitrypanosomal activity. The compound 3 showed the most important activity with an IC50 = 12.00 ± 0.36 μM. Its mechanism of action may include inhibition of DNA synthesis.

The FACT subunit TbSpt16 is involved in cell cycle specific control of VSG expression sites in Trypanosoma brucei.

The African trypanosome Trypanosoma brucei monoallelically expresses one of more than 1000 Variant Surface Glycoprotein (VSG) genes. The active VSG is transcribed from one of about 15 telomeric VSG expression sites (ESs). It is unclear how monoallelic expression of VSG is controlled, and how inactive VSG ESs are silenced. Here, we show that blocking synthesis of the T. brucei FACT subunit TbSpt16 triggers a G2/early M phase cell cycle arrest in both bloodstream and insect form T. brucei. Segregation of T. brucei minichromosomes in these stalled cells is impaired, implicating FACT in maintenance of centromeres. Strikingly, knock-down of TbSpt16 results in 20- to 23-fold derepression of silent VSG ES promoters in bloodstream form T. brucei, with derepression specific to the G2/M cell cycle stage. In insect form T. brucei TbSpt16 knock-down results in 16- to 25-fold VSG ES derepression. Using chromatin immunoprecipitation (ChIP), TbSpt16 was found to be particularly enriched at the promoter region of silent but not active VSG ESs in bloodstream form T. brucei. The chromatin remodeler FACT is therefore implicated in maintenance of repressed chromatin present at silent VSG ES promoters, but is also essential for chromosome segregation presumably through maintenance of functional centromeres.

Evaluation of drug effects on Toxoplasma gondii nuclear and plastid DNA replication using real-time PCR.

Toxoplasma gondii Nicolle and Manceaux, 1908 is a unicellular protozoan that can infect a broad spectrum of organisms including humans. In addition to a nuclear genome, it also carries a circular DNA within a plastid-like organelle (apicoplast) and a linear genome within its mitochondria. The plastid organelle has been shown to be the target of various anti-parasitic drugs or antibiotics. To evaluate the effects of agents on the DNA replication of T. gondii, we tested six drugs (ciprofloxacin, acetylspiramycin, clindamycin, azithromycin, artemether, and sulfadiazine) on the parasite cultured in Hela cells. After drug treatment for 48 h, the parasite growth and DNA replication were evaluated and quantitated using TaqMan real-time quantitative PCR with oligonucleotide primers synthesized based on a gene from the apicoplast genome (ycf24, Genbank accession no. U87145) and a gene from the nuclear genome (uprt, Genbank accession no. U10246). Our results showed that ciprofloxacin was the most effective in inhibiting the replication of the plastid DNA after 48 h drug treatment, with a reduction of 22% in the copy number of the plastid DNA. Artemether was the most effective drug in suppressing the proliferation of tachyzoites. This study also demonstrates that real-time quantitative PCR is a simple and useful technique for monitoring parasite growth and DNA replication.

Giardia intestinalis: aphidicolin influence on the trophozoite cell cycle.

This study is a thorough examination of the effects of the DNA polymerase inhibitor aphidicolin on the nuclear cycle and cell cycle progression characteristics, as well as their reversibility, in Giardia intestinalis. Giardia trophozoites are arrested in the G1/S-junction after aphidicolin treatment according to their DNA content. However, cell growth continues and trophozoites arrested with aphidicolin resemble cells in the G2 phase and trophozoites in ageing cultures. Extensive treatment with aphidicolin causes side effects and we detected positive signals for phosphorylated histone H2A, which, in mammalian cells, is involved in a signalling pathway triggered as a reaction to double stranded DNA breaks. These results suggest that aphidicolin causes dissociation of the nuclear and cytoplasmic cycles, a phenomenon that has also been described for other inhibitors in mammalian cell lines. Thus, if aphidicolin is used for synchronization of Giardia trophozoites, this fact must be accounted for, and treatment with aphidicolin must be minimal.

Unraveling the secrets of regulating mitochondrial DNA replication.

In this issue, Liu et al. (2009) report that maxicircle DNA copy number in trypanosomes is regulated by proteolysis of a helicase; the complex kinetoplast DNA system yields a clear view of how mitochondrial DNA replication can be regulated.