scholarly journals Three Mitochondrial DNA Polymerases Are Essential for Kinetoplast DNA Replication and Survival of Bloodstream Form Trypanosoma brucei

2011 ◽  
Vol 10 (6) ◽  
pp. 734-743 ◽  
Author(s):  
David F. Bruhn ◽  
Mark P. Sammartino ◽  
Michele M. Klingbeil
Keyword(s):  
Mitochondrial Dna ◽  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Dna Polymerases ◽  
Bloodstream Form ◽  
Complex Life Cycle ◽  
Replication Proteins ◽  
Kinetoplast Dna ◽  
Content Type ◽  
Parasite Viability

ABSTRACT Trypanosoma brucei , the causative agent of human African trypanosomiasis, has a complex life cycle that includes multiple life cycle stages and metabolic changes as the parasite switches between insect vector and mammalian host. The parasite's single mitochondrion contains a unique catenated mitochondrial DNA network called kinetoplast DNA (kDNA) that is composed of minicircles and maxicircles. Long-standing uncertainty about the requirement of kDNA in bloodstream form (BF) T. brucei has recently eroded, with reports of posttranscriptional editing and subsequent translation of kDNA-encoded transcripts as essential processes for BF parasites. These studies suggest that kDNA and its faithful replication are indispensable for this life cycle stage. Here we demonstrate that three kDNA replication proteins (mitochondrial DNA polymerases IB, IC, and ID) are required for BF parasite viability. Silencing of each polymerase was lethal, resulting in kDNA loss, persistence of prereplication DNA monomers, and collapse of the mitochondrial membrane potential. These data demonstrate that kDNA replication is indeed crucial for BF T. brucei . The contributions of mitochondrial DNA polymerases IB, IC, and ID to BF parasite viability suggest that these and other kDNA replication proteins warrant further investigation as a new class of targets for the development of antitrypanosomal drugs.

scholarly journals Functional Analyses of Cytokinesis Regulators in Bloodstream Stage Trypanosoma brucei Parasites Identify Functions and Regulations Specific to the Life Cycle Stage

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Xuan Zhang ◽  
Tai An ◽  
Kieu T. M. Pham ◽  
Zhao-Rong Lun ◽  
Ziyin Li
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Bloodstream Form ◽  
Signaling Cascade ◽  
Insect Vector ◽  
Content Type ◽  
Procyclic Form ◽  
Life Cycle Stages ◽  
Evolutionarily Conserved

ABSTRACT The early divergent protozoan parasite Trypanosoma brucei alternates between the insect vector and the mammalian hosts during its life cycle and proliferates through binary cell fission. The cell cycle control system in T. brucei differs substantially from that in its mammalian hosts and possesses distinct mitosis-cytokinesis checkpoint controls between two life cycle stages, the procyclic form and the bloodstream form. T. brucei undergoes an unusual mode of cytokinesis, which is controlled by a novel signaling cascade consisting of evolutionarily conserved protein kinases and trypanosome-specific regulatory proteins in the procyclic form. However, given the distinct mitosis-cytokinesis checkpoints between the two forms, it is unclear whether the cytokinesis regulatory pathway discovered in the procyclic form also operates in a similar manner in the bloodstream form. Here, we showed that the three regulators of cytokinesis initiation, cytokinesis initiation factor 1 (CIF1), CIF2, and CIF3, are interdependent for subcellular localization but not for protein stability as in the procyclic form. Further, we demonstrated that KLIF, a regulator of cytokinesis completion in the procyclic form, plays limited roles in cytokinesis in the bloodstream form. Finally, we showed that the cleavage furrow-localizing protein FRW1 is required for cytokinesis initiation in the bloodstream form but is nonessential for cytokinesis in the procyclic form. Together, these results identify conserved and life cycle-specific functions of cytokinesis regulators, highlighting the distinction in the regulation of cytokinesis between different life cycle stages of T. brucei. IMPORTANCE The early divergent protozoan parasite Trypanosoma brucei is the causative agent of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. This parasite has a complex life cycle by alternating between the insect vector and the mammalian hosts and proliferates by binary cell fission. The control of cell division in trypanosomes appears to be distinct from that in its human host and differs substantially between two life cycle stages, the procyclic (insect) form and the bloodstream form. Cytokinesis, the final step of binary cell fission, is regulated by a novel signaling cascade consisting of two evolutionarily conserved protein kinases and a cohort of trypanosome-specific regulators in the procyclic form, but whether this signaling pathway operates in a similar manner in the bloodstream form is unclear. In this report, we performed a functional analysis of multiple cytokinesis regulators and discovered their distinct functions and regulations in the bloodstream form.

scholarly journals Identification by Random Mutagenesis of Functional Domains in KREPB5 That Differentially Affect RNA Editing between Life Cycle Stages of Trypanosoma brucei

2015 ◽  
Vol 35 (23) ◽  
pp. 3945-3961 ◽  
Author(s):  
Suzanne M. McDermott ◽  
Jason Carnes ◽  
Kenneth Stuart
Keyword(s):  
Life Cycle ◽  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Bloodstream Form ◽  
Single Amino Acid ◽  
Wild Type ◽  
Rnase Iii ◽  
Content Type ◽  
Life Cycle Stages

KREPB5 is an essential component of ∼20S editosomes inTrypanosoma bruceiwhich contains a degenerate, noncatalytic RNase III domain. To explore the function of this protein, we used a novel approach to make and screen numerous conditional nullT. bruceibloodstream form cell lines that express randomly mutagenized KREPB5 alleles. We identified nine single amino acid substitutions that could not complement the conditional loss of wild-type KREPB5. Seven of these were within the RNase III domain, and two were in the C-terminal region that has no homology to known motifs. Exclusive expression of these mutated KREPB5 alleles in the absence of wild-type allele expression resulted in growth inhibition, the loss of ∼20S editosomes, and inhibition of RNA editing in BF cells. Eight of these mutations were lethal in bloodstream form parasites but not in procyclic-form parasites, showing that multiple domains function in a life cycle-dependent manner. Amino acid changes at a substantial number of positions, including up to 7 per allele, allowed complementation and thus did not block KREPB5 function. Hence, the degenerate RNase III domain and a newly identified domain are critical for KREPB5 function and have differential effects between the life cycle stages ofT. bruceithat differentially edit mRNAs.

scholarly journals Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

2012 ◽  
Vol 11 (7) ◽  
pp. 844-855 ◽  
Author(s):  
Jeniffer Concepción-Acevedo ◽  
Juemin Luo ◽  
Michele M. Klingbeil
Keyword(s):  
Mitochondrial Dna ◽  
Trypanosoma Brucei ◽  
Replication Proteins ◽  
Dynamic Localization ◽  
Content Type ◽  
Specific Subset ◽  
Dnase Treatment ◽  
Essential Components ◽  
Detergent Extraction ◽  
Mitochondrial Dna Polymerase

ABSTRACT Trypanosomes contain a unique form of mitochondrial DNA called kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Several proteins are essential for network replication, and most of these localize to the antipodal sites or the kinetoflagellar zone. Essential components for kDNA synthesis include three mitochondrial DNA polymerases TbPOLIB, TbPOLIC, and TbPOLID). In contrast to other kDNA replication proteins, TbPOLID was previously reported to localize throughout the mitochondrial matrix. This spatial distribution suggests that TbPOLID requires redistribution to engage in kDNA replication. Here, we characterize the subcellular distribution of TbPOLID with respect to the Trypanosoma brucei cell cycle using immunofluorescence microscopy. Our analyses demonstrate that in addition to the previously reported matrix localization, TbPOLID was detected as discrete foci near the kDNA. TbPOLID foci colocalized with replicating minicircles at antipodal sites in a specific subset of the cells during stages II and III of kDNA replication. Additionally, the TbPOLID foci were stable following the inhibition of protein synthesis, detergent extraction, and DNase treatment. Taken together, these data demonstrate that TbPOLID has a dynamic localization that allows it to be spatially and temporally available to perform its role in kDNA replication.

scholarly journals Distinct Roles of a Mitogen-Activated Protein Kinase in Cytokinesis between Different Life Cycle Forms of Trypanosoma brucei

2013 ◽  
Vol 13 (1) ◽  
pp. 110-118 ◽  
Author(s):  
Ying Wei ◽  
Ziyin Li
Keyword(s):  
Life Cycle ◽  
Protein Kinase ◽  
Map Kinase ◽  
Trypanosoma Brucei ◽  
Bloodstream Form ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Procyclic Form ◽  
Moderate Growth ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein kinase (MAPK) modules are evolutionarily conserved signaling cascades that function in response to the environment and play crucial roles in intracellular signal transduction in eukaryotes. The involvement of a MAP kinase in regulating cytokinesis in yeast, animals, and plants has been reported, but the requirement for a MAP kinase for cytokinesis in the early-branching protozoa is not documented. Here, we show that a MAP kinase homolog (TbMAPK6) from Trypanosoma brucei plays distinct roles in cytokinesis in two life cycle forms of T. brucei . TbMAPK6 is distributed throughout the cytosol in the procyclic form but is localized in both the cytosol and the nucleus in the bloodstream form. RNA interference (RNAi) of TbMAPK6 results in moderate growth inhibition in the procyclic form but severe growth defects and rapid cell death in the bloodstream form. Moreover, TbMAPK6 appears to be implicated in furrow ingression and cytokinesis completion in the procyclic form but is essential for cytokinesis initiation in the bloodstream form. Despite the distinct defects in cytokinesis in the two forms, RNAi of TbMAPK6 also caused defective basal body duplication/segregation in a small cell population in both life cycle forms. Altogether, our results demonstrate the involvement of the TbMAPK6-mediated pathway in regulating cytokinesis in trypanosomes and suggest distinct roles of TbMAPK6 in cytokinesis between different life cycle stages of T. brucei .

scholarly journals A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Marta Tibúrcio ◽  
Annie S. P. Yang ◽  
Kazuhide Yahata ◽  
Pablo Suárez-Cortés ◽  
Hugo Belda ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Genetic Modification ◽  
Gene Deletion ◽  
Genetically Modify ◽  
Complex Life Cycle ◽  
Mosquito Vector ◽  
Parasite Line ◽  
Content Type ◽  
First Time

ABSTRACT Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1. IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

scholarly journals A Second Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast Minicircle DNA Replication in Trypanosoma brucei

2011 ◽  
Vol 10 (3) ◽  
pp. 445-454 ◽  
Author(s):  
Jane C. Hines ◽  
Dan S. Ray
Keyword(s):  
Mitochondrial Dna ◽  
Trypanosoma Brucei ◽  
Rna Recognition Motif ◽  
Dependent Manner ◽  
Dna Viruses ◽  
Content Type ◽  
Amino Terminal ◽  
Circular Dnas ◽  
Nucleocytoplasmic Large Dna Viruses ◽  
Minicircle Dna

ABSTRACT The mitochondrial DNA of trypanosomes contains two types of circular DNAs, minicircles and maxicircles. Both minicircles and maxicircles replicate from specific replication origins by unidirectional theta-type intermediates. Initiation of the minicircle leading strand and also that of at least the first Okazaki fragment involve RNA priming. The Trypanosoma brucei genome encodes two mitochondrial DNA primases, PRI1 and PRI2, related to the primases of eukaryotic nucleocytoplasmic large DNA viruses. These primases are members of the archeoeukaryotic primase superfamily, and each of them contain an RNA recognition motif and a PriCT-2 motif. In Leishmania species, PRI2 proteins are approximately 61 to 66 kDa in size, whereas in Trypanosoma species, PRI2 proteins have additional long amino-terminal extensions. RNA interference (RNAi) of T. brucei PRI2 resulted in the loss of kinetoplast DNA and accumulation of covalently closed free minicircles. Recombinant PRI2 lacking this extension (PRI2ΔNT) primes poly(dA) synthesis on a poly(dT) template in an ATP-dependent manner. Mutation of two conserved aspartate residues (PRI2ΔNTCS) resulted in loss of enzymatic activity but not loss of DNA binding. We propose that PRI2 is directly involved in initiating kinetoplast minicircle replication.

scholarly journals Mitochondrial DNA Ligases of Trypanosoma brucei

2005 ◽  
Vol 4 (4) ◽  
pp. 765-774 ◽  
Author(s):  
Nick Downey ◽  
Jane C. Hines ◽  
Krishna M. Sinha ◽  
Dan S. Ray
Keyword(s):  
Mitochondrial Dna ◽  
Trypanosoma Brucei ◽  
Topoisomerase Ii ◽  
Leishmania Major ◽  
Nuclear Dna ◽  
Protein Complexes ◽  
Dna Ligase ◽  
Kinetoplast Dna ◽  
Rapid Loss ◽  
Dna Ligases

ABSTRACT The mitochondrial DNA of Trypanosoma brucei, termed kinetoplast DNA or kDNA, consists of thousands of minicircles and a small number of maxicircles catenated into a single network organized as a nucleoprotein disk at the base of the flagellum. Minicircles are replicated free of the network but still contain nicks and gaps after rejoining to the network. Covalent closure of remaining discontinuities in newly replicated minicircles after their rejoining to the network is delayed until all minicircles have been replicated. The DNA ligase involved in this terminal step in minicircle replication has not been identified. A search of kinetoplastid genome databases has identified two putative DNA ligase genes in tandem. These genes (LIG kα and LIG kβ) are highly diverged from mitochondrial and nuclear DNA ligase genes of higher eukaryotes. Expression of epitope-tagged versions of these genes shows that both LIG kα and LIG kβ are mitochondrial DNA ligases. Epitope-tagged LIG kα localizes throughout the kDNA, whereas LIG kβ shows an antipodal localization close to, but not overlapping, that of topoisomerase II, suggesting that these proteins may be contained in distinct structures or protein complexes. Knockdown of the LIG kα mRNA by RNA interference led to a cessation of the release of minicircles from the network and resulted in a reduction in size of the kDNA networks and rapid loss of the kDNA from the cell. Closely related pairs of mitochondrial DNA ligase genes were also identified in Leishmania major and Crithidia fasciculata.

scholarly journals Protein Prenylation and Hsp40 in Thermotolerance of Plasmodium falciparum Malaria Parasites

mBio ◽  
2021 ◽  
Author(s):  
Emily S. Mathews ◽  
Andrew J. Jezewski ◽  
Audrey R. Odom John
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Malaria Parasite ◽  
Plasmodium Falciparum Malaria ◽  
Complex Life Cycle ◽  
Large Temperature ◽  
Protein Prenylation ◽  
Content Type

During its complex life cycle, the malaria parasite survives dramatic environmental stresses, including large temperature shifts. Protein prenylation is required during asexual replication of Plasmodium falciparum , and the canonical heat shock protein 40 protein (HSP40; PF3D7_1437900) is posttranslationally modified with a 15-carbon farnesyl isoprenyl group.

scholarly journals Unexpected plasticity in the life cycle of Trypanosoma brucei

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sarah Schuster ◽  
Jaime Lisack ◽  
Ines Subota ◽  
Henriette Zimmermann ◽  
Christian Reuter ◽  
...  
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Parasite Load ◽  
Tsetse Fly ◽  
Productive Infection ◽  
Complex Life Cycle ◽  
Chronic Infections ◽  
Population Densities ◽  
African Trypanosomes ◽  
Stage Transition

African trypanosomes cause sleeping sickness in humans and nagana in cattle. These unicellular parasites are transmitted by the bloodsucking tsetse fly. In the mammalian host's circulation, proliferating slender stage cells differentiate into cell cycle-arrested stumpy stage cells when they reach high population densities. This stage transition is thought to fulfil two main functions: first, it auto-regulates the parasite load in the host; second, the stumpy stage is regarded as the only stage capable of successful vector transmission. Here, we show that proliferating slender stage trypanosomes express the mRNA and protein of a known stumpy stage marker, complete the complex life cycle in the fly as successfully as the stumpy stage, and require only a single parasite for productive infection. These findings suggest a reassessment of the traditional view of the trypanosome life cycle. They may also provide a solution to a long-lasting paradox, namely the successful transmission of parasites in chronic infections, despite low parasitemia.

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