scholarly journals Identification of Fis1 Interactors in Toxoplasma gondii Reveals a Novel Protein Required for Peripheral Distribution of the Mitochondrion

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kylie Jacobs ◽  
Robert Charvat ◽  
Gustavo Arrizabalaga
Keyword(s):  
Life Cycle ◽  
Toxoplasma Gondii ◽  
Morphological Changes ◽  
Dominant Negative Effect ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Content Type ◽  
Membrane Contact Sites ◽  
Intracellular Parasites ◽  
Single Mitochondrion

ABSTRACT Toxoplasma gondii’s single mitochondrion is very dynamic and undergoes morphological changes throughout the parasite’s life cycle. During parasite division, the mitochondrion elongates, enters the daughter cells just prior to cytokinesis, and undergoes fission. Extensive morphological changes also occur as the parasite transitions from the intracellular environment to the extracellular environment. We show that treatment with the ionophore monensin causes reversible constriction of the mitochondrial outer membrane and that this effect depends on the function of the fission-related protein Fis1. We also observed that mislocalization of the endogenous Fis1 causes a dominant-negative effect that affects the morphology of the mitochondrion. As this suggests that Fis1 interacts with proteins critical for maintenance of mitochondrial structure, we performed various protein interaction trap screens. In this manner, we identified a novel outer mitochondrial membrane protein, LMF1, which is essential for positioning of the mitochondrion in intracellular parasites. Normally, while inside a host cell, the parasite mitochondrion is maintained in a lasso shape that stretches around the parasite periphery where it has regions of coupling with the parasite pellicle, suggesting the presence of membrane contact sites. In intracellular parasites lacking LMF1, the mitochondrion is retracted away from the pellicle and instead is collapsed, as normally seen only in extracellular parasites. We show that this phenotype is associated with defects in parasite fitness and mitochondrial segregation. Thus, LMF1 is necessary for mitochondrial association with the parasite pellicle during intracellular growth, and proper mitochondrial morphology is a prerequisite for mitochondrial division. IMPORTANCE Toxoplasma gondii is an opportunistic pathogen that can cause devastating tissue damage in the immunocompromised and congenitally infected. Current therapies are not effective against all life stages of the parasite, and many cause toxic effects. The single mitochondrion of this parasite is a validated drug target, and it changes its shape throughout its life cycle. When the parasite is inside a cell, the mitochondrion adopts a lasso shape that lies in close proximity to the pellicle. The functional significance of this morphology is not understood and the proteins involved are currently not known. We have identified a protein that is required for proper mitochondrial positioning at the periphery and that likely plays a role in tethering this organelle. Loss of this protein results in dramatic changes to the mitochondrial morphology and significant parasite division and propagation defects. Our results give important insight into the molecular mechanisms regulating mitochondrial morphology.

scholarly journals Identification of Fis1 interactors in Toxoplasma gondii reveals a novel protein required for peripheral distribution of the mitochondrion

2019 ◽  
Author(s):  
Kylie Jacobs ◽  
Robert Charvat ◽  
Gustavo Arrizabalaga
Keyword(s):  
Life Cycle ◽  
Toxoplasma Gondii ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Membrane Contact Sites ◽  
Intracellular Parasites

ABSTRACTToxoplasma gondii’s singular mitochondrion is very dynamic and undergoes morphological changes throughout the parasite’s life cycle. During parasite division, the mitochondrion elongates, enters the daughter cells just prior to cytokinesis and undergoes fission. Extensive morphological changes also occur as the parasite transitions from the intracellular to the extracellular environment. We show that treatment with the ionophore monensin causes reversible constriction of the mitochondrial outer membrane, and that this effect depends on the function of the fission related protein Fis1. We also observed that mislocalization of the endogenous Fis1 causes a dominant negative effect that affects the morphology of the mitochondrion. As this suggests Fis1 interacts with proteins critical for maintenance of mitochondrial structure, we performed various protein interaction trap screens. In this manner we identified a novel outer mitochondrial membrane protein, LMF1, which is essential for positioning of the mitochondrion in intracellular parasites. Normally, while inside a host cell, the parasite mitochondrion is maintained in a lasso shape that stretches around the parasite periphery where it has regions of coupling with the parasite pellicle, suggesting the presence of membrane contact sites. In intracellular parasites lacking LMF1 the mitochondrion is retracted away from the pellicle and instead is collapsed, as only normally seen in extracellular parasites. We show that this phenotype is associated with defects in parasite fitness and mitochondrial segregation. Thus, LMF1 is necessary for mitochondrial association with the parasite pellicle during intracellular growth and proper mitochondrial morphology is a prerequisite for mitochondrial division.IMPORTANCEToxoplasma gondii is an opportunistic pathogen that can cause devastating tissue damage in the immunocompromised and the congenitally infected. Current therapies are not effective against all life stages of the parasite and many cause toxic effects. The single mitochondrion of this parasite is a validated drug target and it changes its shape throughout its life cycle. When the parasite is inside of a cell, the mitochondrion adopts a lasso shape that lies in close proximity to the pellicle. The functional significance of this morphology is not understood nor are the proteins involved currently known. We have identified a protein that is required for proper mitochondrial positioning at the periphery and that likely plays a role in tethering this organelle. Loss of this protein results in dramatic changes to the mitochondrial morphology and significant parasite division and propagation defects. Our results give important insight into the molecular mechanisms regulating mitochondrial morphology.

scholarly journals Dispensable Role of Mitochondrial Fission Protein 1 (Fis1) in the Erythrocytic Development of Plasmodium falciparum

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Mulaka Maruthi ◽  
Liqin Ling ◽  
Jing Zhou ◽  
Hangjun Ke
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Outer Membrane ◽  
Malaria Parasite ◽  
Mitochondrial Fission ◽  
Adaptor Proteins ◽  
Mitochondrial Outer Membrane ◽  
Content Type ◽  
Single Mitochondrion

ABSTRACT Malaria remains a huge global health burden, and control of this disease has run into a severe bottleneck. To defeat malaria and reach the goal of eradication, a deep understanding of the parasite biology is urgently needed. The mitochondrion of the malaria parasite is essential throughout the parasite’s life cycle and has been validated as a clinical drug target. In the asexual development of Plasmodium spp., the single mitochondrion grows from a small tubular structure to a complex branched network. This branched mitochondrion is divided at the end of schizogony when 8 to 32 daughter cells are produced, distributing one mitochondrion to each forming merozoite. In mosquito and liver stages, the giant mitochondrial network is split into thousands of pieces and daughter mitochondria are segregated into individual progeny. Despite the significance of mitochondrial fission in Plasmodium, the underlying mechanism is largely unknown. Studies of mitochondrial fission in model eukaryotes have revealed that several mitochondrial fission adaptor proteins are involved in recruiting dynamin GTPases to physically split mitochondrial membranes. Apicomplexan parasites, however, share no identifiable homologs of mitochondrial fission adaptor proteins with yeast or humans, except for Fis1. Here, we investigated the localization and essentiality of the Fis1 homolog in Plasmodium falciparum, PfFis1 (PF3D7_1325600), during the asexual life cycle. We found that PfFis1 requires an intact C terminus for mitochondrial localization but is not essential for parasite development or mitochondrial fission. The dispensable role of PfFis1 indicates that Plasmodium contains additional fission adaptor proteins on the mitochondrial outer membrane that could be essential for mitochondrial fission. IMPORTANCE Malaria is responsible for over 230 million clinical cases and ∼half a million deaths each year. The single mitochondrion of the malaria parasite functions as a metabolic hub throughout the parasite’s developmental cycle (DC) and also as a source of ATP in certain stages. To pass on its essential functions, the parasite’s mitochondrion needs to be properly divided and segregated into all progeny during cell division via a process termed mitochondrial fission. Due to the divergent nature of Plasmodium spp., the molecular players involved in mitochondrial fission and their mechanisms of action remain largely unknown. Here, we found that the only identifiable mitochondrial fission adaptor protein that is evolutionarily conserved in the Apicomplexan phylum, Fis1, it not essential in P. falciparum asexual stages. Our data suggest that malaria parasites use redundant fission adaptor proteins on the mitochondrial outer membrane to mediate the fission process.

scholarly journals Biofilms Comprise a Component of the Annual Cycle ofVibrio choleraein the Bay of Bengal Estuary

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e00483-18 ◽  
Author(s):  
Marzia Sultana ◽  
Suraia Nusrin ◽  
Nur A. Hasan ◽  
Abdus Sadique ◽  
Kabir U. Ahmed ◽  
...  
Keyword(s):  
Life Cycle ◽  
Vibrio Cholerae ◽  
Causative Agent ◽  
Aquatic Environment ◽  
Morphological Changes ◽  
Living Cells ◽  
Estuarine Environment ◽  
Annual Life Cycle ◽  
Free Living ◽  
Content Type

ABSTRACTVibrio cholerae, an estuarine bacterium, is the causative agent of cholera, a severe diarrheal disease that demonstrates seasonal incidence in Bangladesh. In an extensive study ofV. choleraeoccurrence in a natural aquatic environment, water and plankton samples were collected biweekly between December 2005 and November 2006 from Mathbaria, an estuarine village of Bangladesh near the mangrove forests of the Sundarbans. ToxigenicV. choleraeexhibited two seasonal growth peaks, one in spring (March to May) and another in autumn (September to November), corresponding to the two annual seasonal outbreaks of cholera in this region. The total numbers of bacteria determined by heterotrophic plate count (HPC), representing culturable bacteria, accounted for 1% to 2.7% of the total numbers obtained using acridine orange direct counting (AODC). The highest bacterial culture counts, including toxigenicV. cholerae, were recorded in the spring. The direct fluorescent antibody (DFA) assay was used to detectV. choleraeO1 cells throughout the year, as free-living cells, within clusters, or in association with plankton.V. choleraeO1 varied significantly in morphology, appearing as distinctly rod-shaped cells in the spring months, while small coccoid cells within thick clusters of biofilm were observed during interepidemic periods of the year, notably during the winter months. ToxigenicV. choleraeO1 was culturable in natural water during the spring when the temperature rose sharply. The results of this study confirmed biofilms to be a means of persistence for bacteria and an integral component of the annual life cycle of toxigenicV. choleraein the estuarine environment of Bangladesh.IMPORTANCEVibrio cholerae, the causative agent of cholera, is autochthonous in the estuarine aquatic environment. This study describes morphological changes in naturally occurringV. choleraeO1 in the estuarine environment of Mathbaria, where the bacterium is culturable when the water temperature rises and is observable predominantly as distinct rods and dividing cells. In the spring and fall, these morphological changes coincide with the two seasonal peaks of endemic cholera in Bangladesh.V. choleraeO1 cells are predominantly coccoid within biofilms but are rod shaped as free-living cells and when attached to plankton or to particulate matter in interepidemic periods of the year. It is concluded that biofilms represent a stage of the annual life cycle ofV. choleraeO1, the causative agent of cholera in Bangladesh.

scholarly journals Stage-Specific and Selective Delivery of Caged Azidosugars into the Intracellular Parasite Toxoplasma gondii by Using an Esterase-Ester Pair Technique

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Tadakimi Tomita ◽  
Hua Wang ◽  
Peng Wu ◽  
Louis M. Weiss
Keyword(s):  
Toxoplasma Gondii ◽  
Small Molecules ◽  
Click Chemistry ◽  
Cell Biology ◽  
Host Cells ◽  
Intracellular Parasite ◽  
Content Type ◽  
Intracellular Parasites ◽  
Specific Manner ◽  
Selective Delivery

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that chronically infects up to a third of the human population. The parasites persist in the form of cysts in the central nervous system and serve as a reservoir for the reactivation of toxoplasmic encephalitis. The cyst wall is known to have abundant O-linked N-acetylgalactosamine glycans, but the existing metabolic labeling methods do not allow selective labeling of intracellular parasite glycoproteins without labeling of host glycans. In this study, we have integrated Cu(I)-catalyzed bioorthogonal click chemistry with a specific esterase-ester pair system in order to selectively deliver azidosugars to the intracellular parasites. We demonstrated that α-cyclopropyl modified GalNAz was cleaved by porcine liver esterase produced in the parasites but not in the host cells. Our proof-of-concept study demonstrates the feasibility and potential of this esterase-ester click chemistry approach for the selective delivery of small molecules in a stage-specific manner. IMPORTANCE Selective delivery of small molecules into intracellular parasites is particularly problematic due to the presence of multiple membranes and surrounding host cells. We have devised a method that can deliver caged molecules into an intracellular parasite, Toxoplasma gondii, that express an uncaging enzyme in a stage-specific manner without affecting host cell biology. This system provides a valuable tool for studying many intracellular parasites.

scholarly journals Effect of 3-Bromopyruvate and Atovaquone on Infection duringIn VitroInteraction of Toxoplasma gondii and LLC-MK2 Cells

2015 ◽  
Vol 59 (9) ◽  
pp. 5239-5249 ◽  
Author(s):  
Loyze Paola O. de Lima ◽  
Sergio H. Seabra ◽  
Henrique Carneiro ◽  
Helene S. Barbosa
Keyword(s):  
Toxoplasma Gondii ◽  
Metabolic Pathways ◽  
Morphological Characteristics ◽  
Cellular Mechanisms ◽  
Dolichos Biflorus ◽  
Content Type ◽  
Intracellular Parasites ◽  
Current Therapies ◽  
Therapeutic Alternatives

ABSTRACTToxoplasma gondiiinfection can be severe during pregnancy and in immunocompromised patients. Current therapies for toxoplasmosis are restricted to tachyzoites and have little or no effect on bradyzoites, which are maintained in tissue cysts. Consequently, new therapeutic alternatives have been proposed as the use of atovaquone has demonstrated partial efficacy against tachyzoites and bradyzoites. This work studies the effect of 3-bromopyruvate (3-BrPA), a compound that is being tested against cancer cells, on the infection of LLC-MK2 cells withT. gondiitachyzoites, RH strain. No effect of 3-BrPA on host cell proliferation or viability was observed, but it inhibited the proliferation ofT. gondii. The incubation of cultures with lectinDolichos biflorusagglutinin (DBA) showed the development of cystogenesis, and an ultrastructural analysis of parasite intracellular development confirmed morphological characteristics commonly found in tissue cysts. Moreover, the presence of degraded parasites and the influence of 3-BrPA on endodyogeny were observed. Infected cultures were alternatively treated with a combination of this compound plus atovaquone. This resulted in a 73% reduction in intracellular parasites after 24 h of treatment and a 71% reduction after 48 h; cyst wall formation did not occur in these cultures. Therefore, we conclude that the use of 3-BrPA may serve as an important tool for the study of (i)in vitrocystogenesis; (ii) parasite metabolism, requiring a deeper understanding of the target of action of this compound onT. gondii; (iii) the alternative parasite metabolic pathways; and (iv) the molecular/cellular mechanisms that trigger parasite death.

scholarly journals Direct and Indirect Inhibition Effects of Resveratrol against Toxoplasma gondii Tachyzoites In Vitro

2018 ◽  
Vol 63 (3) ◽  
Author(s):  
Qi-Wei Chen ◽  
Kai Dong ◽  
Han-Xiao Qin ◽  
Yi-Kai Yang ◽  
Jin-Lei He ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Toxoplasma Gondii ◽  
Redox Homeostasis ◽  
Inhibitory Effects ◽  
Natural Plant ◽  
Content Type ◽  
Multiple Functions ◽  
Therapeutic Drugs ◽  
Intracellular Parasites

ABSTRACT Toxoplasma gondii is one of the most widespread obligatory parasitic protozoa and infects nearly all warm-blooded animals, leading to toxoplasmosis. The therapeutic drugs currently administered, like the combination of pyrimethamine and sulfadiazine, show high rates of toxic side effects, and drug resistance is encountered in some cases. Resveratrol is a natural plant extract with multiple functions, such as antibacterial, anticancer, and antiparasite activities. In this study, we evaluated the inhibitory effects of resveratrol on tachyzoites of the Toxoplasma gondii RH strain extracellularly and intracellularly. We demonstrate that resveratrol possesses direct antitoxoplasma activity by reducing the population of extracellularly grown tachyzoites, probably by disturbing the redox homeostasis of the parasites. Moreover, resveratrol was also able to release the burden of cellular stress, promote apoptosis, and maintain the autophagic status of macrophages, which turned out to be regulated by intracellular parasites, thereby functioning indirectly in eliminating T. gondii. In conclusion, resveratrol has both direct and indirect antitoxoplasma effects against RH tachyzoites and may possess the potential to be further evaluated and employed for toxoplasmosis treatment.

scholarly journals A uvs-5 Strain Is Deficient for a Mitofusin Gene Homologue, fzo1 , Involved in Maintenance of Long Life Span in Neurospora crassa

2012 ◽  
Vol 12 (2) ◽  
pp. 233-243 ◽  
Author(s):  
Kiminori Kurashima ◽  
Michael Chae ◽  
Hirokazu Inoue ◽  
Shin Hatakeyama ◽  
Shuuitsu Tanaka
Keyword(s):  
Life Span ◽  
Neurospora Crassa ◽  
Mitochondrial Fission ◽  
Single Amino Acid ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Wild Type ◽  
Content Type ◽  
Long Life ◽  
Mutant Phenotypes

ABSTRACT Mitochondria are highly dynamic organelles that continuously fuse and divide. To maintain mitochondria, cells establish an equilibrium of fusion and fission events, which are mediated by dynamin-like GTPases. We previously showed that an mus-10 strain, a mutagen-sensitive strain of the filamentous fungus Neurospora crassa , is defective in an F-box protein that is essential for the maintenance of mitochondrial DNA (mtDNA), long life span, and mitochondrial morphology. Similarly, a uvs-5 mutant accumulates deletions within its mtDNA, has a shortened life span, and harbors fragmented mitochondria, the latter of which is indicative of an imbalance between mitochondrial fission and fusion. Since the uvs-5 mutation maps very close to the locus of fzo1 , encoding a mitofusin homologue thought to mediate mitochondrial outer membrane fusion, we determined the sequence of the fzo1 gene in the uvs-5 mutant. A single amino acid substitution (Q368R) was found in the GTPase domain of the FZO1 protein. Expression of wild-type FZO1 in the uvs-5 strain rescued the mutant phenotypes, while expression of a mutant FZO1 protein did not. Moreover, when knock-in of the Q368R mutation was performed on a wild-type strain, the resulting mutant displayed phenotypes identical to those of the uvs-5 mutant. Therefore, we concluded that the previously unidentified uvs-5 gene is fzo1 . Furthermore, we used immunoprecipitation analysis to show that the FZO1 protein interacts with MUS-10, which suggests that these two proteins may function together to maintain mitochondrial morphology.

scholarly journals The The TOB/SAM complex composition in mitochondria of Dictyostelium discoideum during progression from unicellularity to multicellularity

2019 ◽  
Author(s):  
Monika Mazur ◽  
Małgorzata Wojtkowska ◽  
Marcin Skalski ◽  
Małgorzata Słocińska ◽  
Hanna Kmita
Keyword(s):  
Life Cycle ◽  
Outer Membrane ◽  
Dictyostelium Discoideum ◽  
Protein Import ◽  
Single Cells ◽  
Slime Mold ◽  
Complex Life Cycle ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
The Impact

Despite its complex life cycle including unicellular and multicellular stages, the slime mold Dictyostelium discoideum, a well-known model in biomedical research, has not been used as a model organism in studies on mitochondrial import, including its significance in cellular processes. Moreover, data concerning mitochondrial protein import machinery in D. discoideum mitochondria is limited and nothing is known about the impact of that machinery on slime mold life cycle. Here, we focused on the TOB/SAM (topogenesis of the mitochondrial outer membrane β-barrel proteins/sorting and assembly machinery) complex. This complex is localized in the mitochondrial outer membrane and is indispensable for the formation of metabolite exchange and protein import pathways in the membrane, and substantially contributes to the regulation of mitochondrial morphology and distribution. Furthermore, the available data suggests that the TOB/SAM complex variants differ between mitochondria of multicellular and unicellular eukaryotes. Therefore, we decided to determine these variants of the TOB/SAM in mitochondria of D. discoideum progressing from single cells to early multicellular stages, when the cells stream together to form a multicellular organism. The results revealed two complex variants of the TOB/SAM complex of about 160 and 600 kDa molecular weight, present in mitochondria of D. discoideum cells at the studied stages. The discussed complex variants resemble the ones that have been already detected for the yeast Saccharomyces cerevisiae, fungus Neurospora crassa and human cells, and one of investigated variants differentiates unicellular and initial multicellular stages of the D. discoideum life cycle.

scholarly journals Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 476
Author(s):  
Joachim Kloehn ◽  
Matteo Lunghi ◽  
Emmanuel Varesio ◽  
David Dubois ◽  
Dominique Soldati-Favre
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Rna Degradation ◽  
Major Facilitator Superfamily ◽  
Untargeted Metabolomics ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Major Facilitator ◽  
Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

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