scholarly journals Requirement of Toxoplasma gondii metacaspases for IMC1 maturation, endodyogeny and virulence in mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Muzi Li ◽  
Jing Liu ◽  
Yayun Wu ◽  
Yihan Wu ◽  
Xiaodong Sun ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Normal Growth ◽  
Double Knockout ◽  
Protein Aggregate ◽  
Knockout Strain ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Single Knockout

Abstract Background Metacaspases are multifunctional proteins found in plants, fungi and protozoa, and are involved in processes such as insoluble protein aggregate clearance and cell proliferation. Our previous study demonstrated that metacaspase-1 (MCA1) contributes to parasite apoptosis in Toxoplasma gondii. Deletion of MCA1 from T. gondii has no effect on the growth and virulence of the parasites. Three metacaspases were identified in the ToxoDB Toxoplasma Informatics Resource, and the function of metacaspase-2 (MCA2) and metacaspase-3 (MCA3) has not been demonstrated. Methods In this study, we constructed MCA1, MCA2 and MCA1/MCA2 transgenic strains from RHΔku80 (Δku80), including overexpressing strains and knockout strains, to clarify the function of MCA1 and MCA2 of T. gondii. Results MCA1 and MCA2 were distributed in the cytoplasm with punctuated aggregation, and part of the punctuated aggregation of MCA1 and MCA2 was localized on the inner membrane complex of T. gondii. The proliferation of the MCA1/MCA2 double-knockout strain was significantly reduced; however, the two single knockout strains (MCA1 knockout strain and MCA2 knockout strain) exhibited normal growth rates as compared to the parental strain, Δku80. In addition, endodyogeny was impaired in the tachyzoites whose MCA1 and MCA2 were both deleted due to multiple nuclei and abnormal expression of IMC1. We further found that IMC1 of the double-knockout strain was detergent-soluble, indicating that MCA1 and MCA2 are associated with IMC1 maturation. Compared to the parental Δku80 strain, the double-knockout strain was more readily induced from tachyzoites to bradyzoites in vitro. Furthermore, the double-knockout strain was less pathogenic in mice and was able to develop bradyzoites in the brain, which formed cysts and established chronic infection. Conclusion MCA1 and MCA2 are important factors which participate in IMC1 maturation and endodyogeny of T. gondii. The double-knockout strain has slower proliferation and was able to develop bradyzoites both in vitro and in vivo. Graphic abstract

scholarly journals The apical annuli of Toxoplasma gondii are composed of coiled-coil and signaling proteins embedded in the IMC sutures

2019 ◽  
Author(s):  
Klemens Engelberg ◽  
Chun-Ti Chen ◽  
Tyler Bechtel ◽  
Victoria Sánchez Guzmán ◽  
Allison A. Drozda ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Coiled Coil ◽  
Super Resolution ◽  
Building Blocks ◽  
Signaling Proteins ◽  
Waste Products ◽  
Membrane Complex ◽  
Inner Membrane Complex

AbstractThe apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2 and a methyltransferase (AAMT). Moreover, Inner Membrane Complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy (SR-SIM) identified a concentric organization comprising four rings with diameters ranging from 200-400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggest that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed the AAPs are conserved narrowly in Coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signaling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled-coil and signaling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.

scholarly journals A De novo Peptide from a High Throughput Peptide Library Blocks Myosin A -MTIP Complex Formation in Plasmodium falciparum

2020 ◽  
Vol 21 (17) ◽  
pp. 6158
Author(s):  
Zill e Anam ◽  
Nishant Joshi ◽  
Sakshi Gupta ◽  
Preeti Yadav ◽  
Ayushi Chaurasiya ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Peptide Library ◽  
Tail Domain ◽  
Interacting Protein ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
De Novo Peptide

Apicomplexan parasites, through their motor machinery, produce the required propulsive force critical for host cell-entry. The conserved components of this so-called glideosome machinery are myosin A and myosin A Tail Interacting Protein (MTIP). MTIP tethers myosin A to the inner membrane complex of the parasite through 20 amino acid-long C-terminal end of myosin A that makes direct contacts with MTIP, allowing the invasion of Plasmodium falciparum in erythrocytes. Here, we discovered through screening a peptide library, a de-novo peptide ZA1 that binds the myosin A tail domain. We demonstrated that ZA1 bound strongly to myosin A tail and was able to disrupt the native myosin A tail MTIP complex both in vitro and in vivo. We then showed that a shortened peptide derived from ZA1, named ZA1S, was able to bind myosin A and block parasite invasion. Overall, our study identified a novel anti-malarial peptide that could be used in combination with other antimalarials for blocking the invasion of Plasmodium falciparum.

scholarly journals Growth-Regulated Hsp70 Phosphorylation Regulates Stress Responses and Prion Maintenance

2020 ◽  
Vol 40 (12) ◽  
Author(s):  
Chung-Hsuan Kao ◽  
Seung W. Ryu ◽  
Min J. Kim ◽  
Xuemei Wen ◽  
Oshadi Wimalarathne ◽  
...  
Keyword(s):  
Stress Responses ◽  
Normal Growth ◽  
Serine Phosphorylation ◽  
Metal Exposure ◽  
Negative Effects ◽  
Protein Aggregate ◽  
Binding Domains ◽  
Heat Shock Responses

ABSTRACT Maintenance of protein homeostasis in eukaryotes under normal growth and stress conditions requires the functions of Hsp70 chaperones and associated cochaperones. Here, we investigate an evolutionarily conserved serine phosphorylation that occurs at the site of communication between the nucleotide-binding and substrate-binding domains of Hsp70. Ser151 phosphorylation in yeast Hsp70 (Ssa1) is promoted by cyclin-dependent kinase (Cdk1) during normal growth. Phosphomimetic substitutions at this site (S151D) dramatically downregulate heat shock responses, a result conserved with HSC70 S153 in human cells. Phosphomimetic forms of Ssa1 also fail to relocalize in response to starvation conditions, do not associate in vivo with Hsp40 cochaperones Ydj1 and Sis1, and do not catalyze refolding of denatured proteins in vitro in cooperation with Ydj1 and Hsp104. Despite these negative effects on HSC70/HSP70 function, the S151D phosphomimetic allele promotes survival of heavy metal exposure and suppresses the Sup35-dependent [PSI+] prion phenotype, consistent with proposed roles for Ssa1 and Hsp104 in generating self-nucleating seeds of misfolded proteins. Taken together, these results suggest that Cdk1 can downregulate Hsp70 function through phosphorylation of this site, with potential costs to overall chaperone efficiency but also advantages with respect to reduction of metal-induced and prion-dependent protein aggregate production.

scholarly journals Novel Approaches Reveal that Toxoplasma gondii Bradyzoites within Tissue Cysts Are Dynamic and Replicating Entities In Vivo

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Elizabeth Watts ◽  
Yihua Zhao ◽  
Animesh Dhara ◽  
Becca Eller ◽  
Abhijit Patwardhan ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Packing Density ◽  
Critical Role ◽  
Chronic Phase ◽  
Life Cycle Stage ◽  
Content Type ◽  
Symptomatic Disease ◽  
Membrane Complex ◽  
Inner Membrane Complex

ABSTRACTDespite their critical role in chronic toxoplasmosis, the biology ofToxoplasma gondiibradyzoites is poorly understood. In an attempt to address this gap, we optimized approaches to purify tissue cysts and analyzed the replicative potential of bradyzoites within these cysts. In order to quantify individual bradyzoites within tissue cysts, we have developed imaging software, BradyCount 1.0, that allows the rapid establishment of bradyzoite burdens within imaged optical sections of purified tissue cysts. While in general larger tissue cysts contain more bradyzoites, their relative “occupancy” was typically lower than that of smaller cysts, resulting in a lower packing density. The packing density permits a direct measure of how bradyzoites develop within cysts, allowing for comparisons across progression of the chronic phase. In order to capture bradyzoite endodyogeny, we exploited the differential intensity of TgIMC3, an inner membrane complex protein that intensely labels newly formed/forming daughters within bradyzoites and decays over time in the absence of further division. To our surprise, we were able to capture not only sporadic and asynchronous division but also synchronous replication of all bradyzoites within mature tissue cysts. Furthermore, the time-dependent decay of TgIMC3 intensity was exploited to gain insights into the temporal patterns of bradyzoite replicationin vivo. Despite the fact that bradyzoites are considered replicatively dormant, we find evidence for cyclical, episodic bradyzoite growth within tissue cystsin vivo. These findings directly challenge the prevailing notion of bradyzoites as dormant nonreplicative entities in chronic toxoplasmosis and have implications on our understanding of this enigmatic and clinically important life cycle stage.IMPORTANCEThe protozoanToxoplasma gondiiestablishes a lifelong chronic infection mediated by the bradyzoite form of the parasite within tissue cysts. Technical challenges have limited even the most basic studies on bradyzoites and the tissue cystsin vivo. Bradyzoites, which are viewed as dormant, poorly replicating or nonreplicating entities, were found to be surprisingly active, exhibiting not only the capacity for growth but also previously unrecognized patterns of replication that point to their being considerably more dynamic than previously imagined. These newly revealed properties force us to reexamine the most basic questions regarding bradyzoite biology and the progression of the chronic phase of toxoplasmosis. By developing new tools and approaches to study the chronic phase at the level of bradyzoites, we expose new avenues to tackle both drug development and a better understanding of events that may lead to reactivated symptomatic disease.

scholarly journals Raft microdomain localized in the luminal leaflet of inner membrane complex of living Toxoplasma gondii

2021 ◽  
Vol 100 (2) ◽  
pp. 151149
Author(s):  
Rikako Konishi ◽  
Yuna Kurokawa ◽  
Kanna Tomioku ◽  
Tatsunori Masatani ◽  
Xuenan Xuan ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Inner Membrane ◽  
Membrane Complex ◽  
Inner Membrane Complex

Anti-parasitic activity of Annona muricata L. leaf ethanolic extract and its fractions against Toxoplasma gondii in vitro and in vivo

2021 ◽  
pp. 114019
Author(s):  
Natália Carnevalli Miranda ◽  
Ester Cristina Borges Araujo ◽  
Allisson Benatti Justino ◽  
Yusmaris Cariaco ◽  
Caroline Martins Mota ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Ethanolic Extract ◽  
Annona Muricata ◽  
Parasitic Activity

scholarly journals DIPG-43. CAN WE REPROGRAM DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)? EXPLORING THE ROLE OF DISTALLESS/DLX HOMEOBOX GENE REGULATION OF OLIGODENDROGLIAL PROGENITOR CELLS (OPC) IN THE DEVELOPING VERTEBRATE NERVOUS SYSTEM

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii295-iii295
Author(s):  
Mikaela Nevin ◽  
Janine Gallego ◽  
Xiaohua Song ◽  
Qiang Jiang ◽  
Alan Underhill ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Homeobox Gene ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Cell Of Origin ◽  
Double Knockout ◽  
Promoter Dna ◽  
And Migration ◽  
Developmental Reprogramming

Abstract BACKGROUND The identification of H3.3/H3.1K27M in most DIPG has changed our understanding of this disease. H3K27M mutations usually demonstrate global loss of H3K27 trimethylation (me3) with gain of H3K27 acetylation (ac). Single cell RNAseq has identified the putative cell of origin as oligodendroglial progenitor cells (OPC). The distalless gene family is necessary for the differentiation and tangential migration of committed neural progenitors to become GABAergic interneurons. Dlx1/Dlx2 double knockout (DKO) cells from the ganglionic eminences (GE) transplanted into a wild-type environment become oligodendrocytes. RESULTS We identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo and confirmed direct DLX2 protein-promoter DNA binding in vitro. Co-expression of Dlx2 with target sequences reduced reporter gene expression in vitro. There was increased expression of OLIG2, NKX2.2 and PLP-1 expression in vivo, consistent with de-repression in the absence of Dlx1/Dlx2 function. Transient over-expression of a Dlx2-GFP construct into murine DIPG cells from a GEMM that develops DIPG resulted in significant increases in expression of Gad isoforms with concomitant decreases in Olig2 and Nkx2.2. Dlx2-transfected mDIPG cells also demonstrated reduced migration, invasion and colony formation in vitro. Of significance, there was global restoration of H3K27me3 with corresponding loss of H3K27ac expression in transfected cells compared to controls. CONCLUSIONS DLX2 promotes GABAergic differentiation and migration while concomitantly repressing OPC differentiation in vivo. Developmental reprogramming of mDIPG cells by DLX2 demonstrates the potential role for directed differentiation strategies towards improving patient outcomes for this devastating pediatric cancer.

Leishmania infantum and Toxoplasma gondii: Mixed infection of macrophages in vitro and in vivo

2011 ◽  
Vol 128 (3) ◽  
pp. 279-284 ◽  
Author(s):  
Vasiliki Christodoulou ◽  
Ippokratis Messaritakis ◽  
Eleni Svirinaki ◽  
Christos Tsatsanis ◽  
Maria Antoniou
Keyword(s):  
Toxoplasma Gondii ◽  
Mixed Infection ◽  
Leishmania Infantum

scholarly journals Toxoplasma gondii myosins B/C

2001 ◽  
Vol 155 (4) ◽  
pp. 613-624 ◽  
Author(s):  
Frédéric Delbac ◽  
Astrid Sänger ◽  
Eva M. Neuhaus ◽  
Rolf Stratmann ◽  
James W. Ajioka ◽  
...  
Keyword(s):  
Cell Division ◽  
Toxoplasma Gondii ◽  
Daughter Cell ◽  
Gliding Motility ◽  
Apicomplexan Parasites ◽  
Daughter Cells ◽  
Membrane Complex ◽  
Alternatively Spliced ◽  
Butanedione Monoxime ◽  
Inner Membrane Complex

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

scholarly journals Omics profiling identifies MAPK/ERK pathway as a gatekeeper of nephron progenitor metabolism

2021 ◽  
Author(s):  
Hyuk Nam Kwon ◽  
Kristen Kurtzeborn ◽  
Xing Jin ◽  
Bruno Reversade ◽  
Sunghyouk Park ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Mitogen Activated Protein Kinase ◽  
Metabolic Effects ◽  
Erk Pathway ◽  
Double Knockout ◽  
Nephron Progenitor ◽  
Progenitor Maintenance

Nephron endowment is defined by fetal kidney growth and it critically dictates renal health in adults. Despite the advances in understanding the molecular regulation of nephron progenitor maintenance, propagation, and differentiation, the causes for low congenital nephron count and contribution of basic metabolism to nephron progenitor regulation remain poorly studied. Here we have analyzed the metabolic effects that depend on and are triggered by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which is an essential intracellular cascade required for nephron progenitor maintenance. Our combined approach utilizing LC/MS-based metabolomics and transcriptional profiling of MAPK/ERK-deficient cells identified 18 out of total 46 metabolites (38 untargeted and 8 targeted) that were down-regulated. These represent glycolysis, gluconeogenesis, pentose phosphate, glycine, and proline pathways among others. We focused our functional characterization of identified metabolites on pyruvate and proline. Use of in vitro kidney cultures revealed dosage-specific functions for pyruvate in not only controlling ureteric bud branching but also determining progenitor and differentiated (tip-trunk) cell identities. Our in vivo characterization of Pycr1/2 double knockout kidneys revealed functional requirement for proline metabolism in nephron progenitor maintenance. In summary, our results demonstrate that MAPK/ERK cascade regulates energy and amino acid metabolism in developing kidney where these metabolic pathways specifically regulate progenitor preservation.

Sign in / Sign up

Export Citation Format

Share Document