Trypanosome Prereplication Machinery: A Potential New Target for an Old Problem

Approximately ten million people suffer from Chagas disease worldwide, caused by Trypanosoma cruzi, with the disease burden predominately focused in Latin America. Sleeping sickness is another serious health problem, caused by Trypanosoma brucei, especially in sub-Saharan countries. Unfortunately, the drugs currently available to treat these diseases have toxic effects and are not effective against all disease phases or parasite strains. Therefore, there is a clear need for the development of novel drugs and drug targets to treat these diseases. We propose the trypanosome prereplication machinery component, Orc1/Cdc6, as a potential target for drug development. In trypanosomes, Orc1/Cdc6 is involved in nuclear DNA replication, and, despite its involvement in such a conserved process, Orc1/Cdc6 is distinct from mammalian Orc1 and Cdc6 proteins. Moreover, RNAi-mediated silencing of trypanosome Orc1/Cdc6 expression in T. brucei decreased cell survival, indicating that Orc1/Cdc6 is critical for trypanosome survival

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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Universidade Federal de São Paulo, Parasitol Lab, Inst Butantan, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Ultraestrutura Celular Hertha Meyer, BR-21941 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Parasitol Lab, Inst Butantan, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

O obračunu kamata i kredita

<div><p>Background</p><p>Infection with <i>Trypanosoma cruzi</i> causes Chagas disease, a major public health problem throughout Latin America. There is no vaccine and the only drugs have severe side effects. Efforts to generate new therapies are hampered by limitations in our understanding of parasite biology and disease pathogenesis. Studies are compromised by the complexity of the disease, the long-term nature of the infection, and the fact that parasites are barely detectable during the chronic stage. In addition, functional dissection of <i>T</i>. <i>cruzi</i> biology has been restricted by the limited flexibility of the genetic manipulation technology applicable to this parasite.</p><p>Methodology/Principal findings</p><p>Here, we describe two technical innovations, which will allow the role of the parasite in disease progression to be better assessed. First, we generated a <i>T</i>. <i>cruzi</i> reporter strain that expresses a fusion protein comprising red-shifted luciferase and green fluorescent protein domains. Bioluminescence allows the kinetics of infection to be followed within a single animal, and specific foci of infection to be pinpointed in excised tissues. Fluorescence can then be used to visualise individual parasites in tissue sections to study host-parasite interactions at a cellular level. Using this strategy, we have been routinely able to find individual parasites within chronically infected murine tissues for the first time. The second advance is the incorporation of a streamlined CRISPR/Cas9 functionality into this reporter strain that can facilitate genome editing using a PCR-based approach that does not require DNA cloning. This system allows the rapid generation of null mutants and fluorescently tagged parasites in a background where the <i>in vivo</i> phenotype can be rapidly assessed.</p><p>Conclusions/Significance</p><p>The techniques described here will have multiple applications for studying aspects of <i>T</i>. <i>cruzi</i> biology and Chagas disease pathogenesis previously inaccessible to conventional approaches. The reagents and cell lines have been generated as a community resource and are freely available on request.</p></div

Molecular dynamics at Trypanosoma cruzi nucleus.

Em Trypanosoma cruzi os sítios de replicação estão localizados na periferia nuclear. A fim de entender a dinâmica das moléculas envolvidas na replicação, Orc/Cdc6 foi usado como marcador da maquinaria de pré-replicação e PCNA como marcador da maquinaria de replicação durante o ciclo celular da forma epimastigota. Ambas as moléculas apresentaram dois padrões de localização nuclear: padrão disperso e periférico. Em ensaio de dupla marcação três combinações dos padrões de Orc/Cdc6 e TcPCNA foram encontrados durante G1/S: Orc/Cdc6 periférico e PCNA disperso, ambos dispersos e ambos periféricos. Por meio destes resultados pudemos concluir que durante G1 ambas as moléculas se encontram dispersas. Ao final desta fase, Orc/Cdc6 migra para periferia nuclear enquanto PCNA permanece disperso, migrando para a periferia nuclear quando a célula entra em S, quando a replicação do DNA irá ocorrer. Assim, a replicação na periferia nuclear não se deve à localização prévia das moléculas de replicação nesta região, mas sim à migração destas moléculas para os sítios apropriados.In Trypanosoma cruzi the replication sites are located at nuclear periphery. In order to analyse the dynamics of molecules involved in replication, Orc/Cdc6 was used as a marker of pre-replication machinery and PCNA as a marker of replication machinery during the cell cycle of epimastigote form. Both molecules presented two nuclear patterns: dispersed pattern and peripheral pattern. Double-labeling assay showed three different patterns of Orc/Cdc6 and PCNA in the nucleus: Orc/Cdc6 at nuclear periphery and PCNA dispersed, both dispersed and both at nuclear periphery. This data allowed us to conclude that during early G1 phase both molecules are dispersed in the nucleus and during late G1 Orc/Cdc6 goes to nuclear periphery while TcPCNA remains dispersed, moving to nuclear periphery, where DNA replication will take place, when S phase starts. Thus, the replication of DNA at nuclear periphery is not due to localization of replications factors at nuclear periphery; instead it depends on the movement of these factors to the appropriated sites

DNA replication alternatives: control pathways and fork dynamic in trypanosomas.

A replicação do DNA tem início nas origens de replicação que são licenciadas na transição das fases M/G1, pelo complexo de pré-replicação (CPR), e ativadas apenas na fase S. Existem diversas origens de replicação no genoma, mas apenas parte destas origens é disparada em diferentes momentos de S, havendo assim origens early (disparadas no início de S) e late (disparadas mais tardiamente). Em trypanosomas as origens de replicação são reconhecidas por um CPR formado por Orc1/Cdc6 e pelo complexo MCM2-7. Em T. cruzi observamos que existem dois mecanismos diferentes para controlar a replicação do DNA. Durante o ciclo celular da forma epimastigota, as proteínas do CPR são sempre expressas e ligadas ao DNA, mas durante o ciclo de vida Orc1/Cdc6 se liga ao DNA apenas nas formas que replicam, e Mcm7 não é expressa nas que não replicam. Também foi analisado o perfil das forquilhas de replicação em T. brucei utilizando a técnica de SMARD onde vimos que a velocidade da forquilha é semelhante a dos demais eucariontes, além de encontrarmos a primeira origem de replicação late.The DNA replication starts at the origins of replication, which are licensed at M/G1 transition, by the pre replication complex (PRC), and are activated just at S phase. There are many origins of replication along genome, but some of them are fired at different moments of S phase. So there are early and late origins fired at the beginning or later in S phase, respectively. The PRC of trypanosomes is composed of Orc1/Cdc6 and Mcm2-7. We could observe that in T. cruzi there are two distinct ways to control DNA replication. Whereas in epimastigote cell cycle the PRC are expressed and bound to DNA in all phases, during T. cruzi life cycle Orc1/Cdc6 is bound to DNA only in replicative forms and Mcm7 is absent in the non-replicative forms. We also analyzed the fork profile in T. brucei through SMARD technique. We found that the speed of replication fork is similar from other eukaryotes and that different replication origins are fired every cell cycle. Finally, we found a new origin of replication that is the first late origin described in this organism

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Approximately ten million people suffer from Chagas disease worldwide, caused by Trypanosoma cruzi, with the disease burden predominately focused in Latin America. Sleeping sickness is another serious health problem, caused by Trypanosoma brucei, especially in sub-Saharan countries. Unfortunately, the drugs currently available to treat these diseases have toxic effects and are not effective against all disease phases or parasite strains. Therefore, there is a clear need for the development of novel drugs and drug targets to treat these diseases. We propose the trypanosome prereplication machinery component, Orc1/Cdc6, as a potential target for drug development. In trypanosomes, Orc1/Cdc6 is involved in nuclear DNA replication, and, despite its involvement in such a conserved process, Orc1/Cdc6 is distinct from mammalian Orc1 and Cdc6 proteins. Moreover, RNAi-mediated silencing of trypanosome Orc1/Cdc6 expression in T. brucei decreased cell survival, indicating that Orc1/Cdc6 is critical for trypanosome survival

A single amastigotes can be identified in a whole tissue section during chronic stage infections.

<p><b>A.</b><i>Ex vivo</i> bioluminescence image of organs from a chronically infected mouse (day 117), showing parasite presence in stomach, colon and lungs (organs placed as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006388#pntd.0006388.g002" target="_blank">Fig 2D</a>). The red box identifies the tissue sample from which the section shown in B-F was taken. <b>B.</b> mNeonGreen fluorescence. <b>C.</b> Higher magnification of boxed area in B, showing a single amastigote in close conjunction with the nucleus of the host cell. The kinetoplast DNA (K) is indicated by an arrow. <b>D.</b> DNA staining of section shown in B. <b>E.</b> Phase image illustrating architecture of tissue section. <b>F.</b> Merged image of B, D and E, showing phase, DNA and mNeonGreen. The single amastigote is indicated by an arrow and appears yellow. For panel C, bar represents 5 μm; for all other panels, bar indicates 100 μm. Images B, D, E and F are taken with a 40x objective at 0.7 scan zoom, image C is taken with 100x objective, scan zoom 2.7. <b>G.</b> Schematic indicating individual layers of the section. MC, mucosa; MM, muscularis mucosae; SM, submucosa; MEC, muscularis externae (circular); MEL, muscularis externae (longitudinal). The approximate location of the infected cell is indicated within this schematic (red oval and green circle).</p

Location of parasites in the stomach and cecum during chronic stage infections.

<p><b>A.</b> Section of stomach tissue from a chronically infected mouse (day 117)—a single slice from a Z-stack through the infected cell (63x scan zoom 0.7). The left-hand panel is a magnified view of the boxed section (63x scan zoom 2.8). The bars represent 5 μm (left hand panel) and 50 μm (right hand panel). <b>B.</b> Section of cecum tissue from a chronically infected mouse (day 117), with a cluster of amastigotes surrounding the nucleus of an infected cell (left hand image, 63x scan zoom 2.5; right hand image, 63x scan zoom 0.7).</p