Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts

ABSTRACTMany of the world’s warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including up to an estimated one third of the global human population. The cellular processes that permit long-term parasite persistence within the cyst are largely unknown, not only for T. gondii but also for related coccidian parasites that impact human and animal health. A previous study revealed an accumulation of autophagic material in the lysosome-like Vacuolar Compartment (VAC) of chronic stage bradyzoites lacking functional cathepsin L protease (TgCPL) activity. Furthermore, it was shown that TgCPL knockout bradyzoites have compromised viability, indicating the turnover of autophagic material could be necessary for bradyzoite survival. However, the extent to which autophagy itself contributes to bradyzoite development and fitness remained unknown. Herein we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed structural abnormalities occurring in Δatg9 bradyzoites, including disorganization of the inner membrane complex and plasma membrane, the occurrence of multiple nuclei within a single bradyzoite cell, as well as various anomalies associated with the VAC. TgATG9-deficient bradyzoites accumulated significantly less undigested material in the VAC upon inhibition of TgCPL activity, suggesting that autophagy contributes material to the VAC for degradation. Intriguingly, abnormal mitochondria networks were observed in TgATG9-deficient bradyzoites. They were thin and elongated and often adopted a horseshoe conformation. Some abnormal mitochondrial structures were found to contain numerous different cytoplasmic components and organelles. Bradyzoite fitness was found to be drastically compromised, both in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.

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Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts

eLife ◽

10.7554/elife.59384 ◽

2021 ◽

Vol 10 ◽

Author(s):

David Smith ◽

Geetha Kannan ◽

Isabelle Coppens ◽

Fengrong Wang ◽

Hoa Mai Nguyen ◽

...

Keyword(s):

Animal Health ◽

Genetic Ablation ◽

Cellular Processes ◽

Transmission Electron ◽

Structural Abnormalities ◽

Coccidian Parasites ◽

Brain Cysts ◽

Mitochondrial Networks

Many of the world's warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including an estimated one third of the global human population. The cellular processes that permit long-term persistence within the cyst are largely unknown for T. gondii and related coccidian parasites that impact human and animal health. Herein we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed numerous structural abnormalities occurring in ∆atg9 bradyzoites. Intriguingly, abnormal mitochondrial networks were observed in TgATG9-deficient bradyzoites, some of which contained numerous different cytoplasmic components and organelles. ∆atg9 bradyzoite fitness was drastically compromised in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.

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Hedgehog pathway permissive conditions allow generation of immortal cell lines from granule cells derived from cancerous and non-cancerous cerebellum

Open Biology ◽

10.1098/rsob.180145 ◽

2019 ◽

Vol 9 (1) ◽

pp. 180145 ◽

Cited By ~ 2

Author(s):

Constantin Heil

Keyword(s):

Cell Lines ◽

Hedgehog Pathway ◽

Simple Method ◽

Genetic Ablation ◽

Neural Lineage ◽

Shh Pathway ◽

Pathway Activation

Cerebellar granule cell progenitors (GCPs) undergo proliferation in the post-natal cerebellum that is dependent on sonic hedgehog (SHH) signalling. Deregulated SHH signalling leads to type 2 medulloblastoma (MB). In this work, a novel cell culture protocol is described, which is suitable for the establishment and long-term maintenance of GCP-derived cells. This method is first applied to SHH pathway active MB cells from Atoh1 -cre; Ptch1 FL/FL tumours, which leads to the generation of neurosphere-like cell lines expressing GCP markers and an active SHH signalling pathway. These cells also show high sensitivity to the Smoothened inhibitor vismodegib, therefore recapitulating the SHH pathway requirement for survival shown by type 2 MB. Analysis of culture supplements reveals that bFGF and fetal bovine serum act as inhibitors of the SHH pathway and therefore preclude generation of cell lines that are relevant to the study of the SHH pathway. Consequently, these insights are transferred from the context of MB to non-transformed, post-natal day 7 cerebellum-derived cellular explants. In contrast to other, previously used methods, these GCP cultures proliferate indefinitely and depend on SHH pathway activation, either by means of the small molecule SAG or through genetic ablation of Ptch1 . This culture method therefore leads to the generation of immortal neurosphere-like cell lines, that are named murine SAG-dependent spheres (mSS). Despite long-term culture, mSS cells remain dependent on continuous stimulation of the SHH pathway. Further, mSS cells maintain their lineage after extensive periods in vitro, as demonstrated by their differentiation towards the neural lineage. Herein a simple method for the generation of immortal cell lines from murine cerebella is defined. These lines can be maintained indefinitely through hedgehog pathway activation and maintain the GCP lineage.

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Neuroblastoma therapy: what is in the pipeline?

Endocrine Related Cancer ◽

10.1530/erc-11-0251 ◽

2011 ◽

Vol 18 (6) ◽

pp. R213-R231 ◽

Cited By ~ 17

Author(s):

Carla S Verissimo ◽

Jan J Molenaar ◽

Carlos P Fitzsimons ◽

Erno Vreugdenhil

Keyword(s):

Cathepsin L ◽

Molecular Targets ◽

Kinase Gene ◽

Microtubule Associated Proteins ◽

Micro Rnas ◽

Associated Proteins ◽

Selection Of

Despite the expansion of knowledge about neuroblastoma (NB) in recent years, the therapeutic outcome for children with a high-risk NB has not significantly improved. Therefore, more effective therapies are needed. This might be achieved by aiming future efforts at recently proposed but not yet developed targets for NB therapy. In this review, we discuss the recently proposed molecular targets that are in clinical trials and, in particular, those that are not yet explored in the clinic. We focus on the selection of these molecular targets for which promisingin vitroandin vivoresults have been obtained by silencing/inhibiting them. In addition, these selected targets are involved at least in one of the NB tumorigenic processes: proliferation, anti-apoptosis, angiogenesis and/or metastasis. In particular, we will review a recently proposed target, the microtubule-associated proteins (MAPs) encoded by doublecortin-like kinase gene (DCLK1).DCLK1-derived MAPs are crucial for proliferation and survival of neuroblasts and are highly expressed not only in NB but also in other tumours such as gliomas. Additionally, we will discuss neuropeptide Y, its Y2 receptor and cathepsin L as examples of targets to decrease angiogenesis and metastasis of NB. Furthermore, we will review the micro-RNAs that have been proposed as therapeutic targets for NB. Detailed investigation of these not yet developed targets as well as exploration of multi-target approaches might be the key to a more effective NB therapy, i.e. increasing specificity, reducing toxicity and avoiding long-term side effects.

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Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases

Nucleic Acids Research ◽

10.1093/nar/gkaa1228 ◽

2021 ◽

Author(s):

Karin Johanna Ferrari ◽

Simona Amato ◽

Roberta Noberini ◽

Cecilia Toscani ◽

Daniel Fernández-Pérez ◽

...

Keyword(s):

Transcriptional Control ◽

Cathepsin L ◽

Histone H3 ◽

Regulatory Function ◽

Post Translational Modifications ◽

Cellular Processes ◽

Differentiated Cells ◽

Proteolytic Activities

Abstract The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.

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In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center

10.1101/2021.08.19.457048 ◽

2021 ◽

Author(s):

Pratick Khara ◽

Liqiang Song ◽

Peter J. Christie ◽

Bo Hu

Keyword(s):

Electron Microscopy ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Membrane Complex ◽

Type Iv ◽

Inner Membrane Complex ◽

Cytoplasmic Complex

ABSTRACTBacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as ‘minimized’ or ‘expanded’ based on whether they are composed of a core set of signature subunits or additional system-specific components. Prototypical ‘minimized’ systems mediating Agrobacterium tumefaciens T-DNA transfer and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1-VirB11 and VirD4. We visualized the pKM101-encoded T4SS in the native cellular context by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388 analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three ‘expanded’ T4SSs previously visualized by in situ CryoET, but differs strikingly from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SSpKM101 function supplied evidence that TraBB4 as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers-of-dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially-dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer.SIGNIFICANCEBacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryoelectron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely-related T4SS analyzed in vitro by single particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines, and support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate early-stage machine biogenesis and substrate entry passage through the T4SS. The VirB4 ATPases are therefore excellent targets for development of intervention strategies aimed at suppressing the action of T4SS nanomachines.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21176158 ◽

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.

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

10.1101/711432 ◽

2019 ◽

Cited By ~ 1

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.

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

Parasites & Vectors ◽

10.1186/s13071-021-04878-0 ◽

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

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Ultrastructural investigations of MDL 19,660-induced effects on the canine platelet and megakaryocyte

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100159412 ◽

1990 ◽

Vol 48 (3) ◽

pp. 374-375

Author(s):

D.E. Loudy ◽

J. Sprinkle-Cavallo ◽

J.T. Yarrington ◽

F.Y. Thompson ◽

J.P. Gibson

Keyword(s):

Antidepressant Drug ◽

Beagle Dogs ◽

Long Term Effects ◽

Short Term ◽

Platelet Counts ◽

Toxicological Studies ◽

Induced Aggregation ◽

Internal Architecture

Previous short term toxicological studies of one to two weeks duration have demonstrated that MDL 19,660 (5-(4-chlorophenyl)-2,4-dihydro-2,4-dimethyl-3Hl, 2,4-triazole-3-thione), an antidepressant drug, causes a dose-related thrombocytopenia in dogs. Platelet counts started to decline after two days of dosing with 30 mg/kg/day and continued to decrease to their lowest levels by 5-7 days. The loss in platelets was primarily of the small discoid subpopulation. In vitro studies have also indicated that MDL 19,660: does not spontaneously aggregate canine platelets and has moderate antiaggregating properties by inhibiting ADP-induced aggregation. The objectives of the present investigation of MDL 19,660 were to evaluate ultrastructurally long term effects on platelet internal architecture and changes in subpopulations of platelets and megakaryocytes.Nine male and nine female beagle dogs were divided equally into three groups and were administered orally 0, 15, or 30 mg/kg/day of MDL 19,660 for three months. Compared to a control platelet range of 353,000- 452,000/μl, a doserelated thrombocytopenia reached a maximum severity of an average of 135,000/μl for the 15 mg/kg/day dogs after two weeks and 81,000/μl for the 30 mg/kg/day dogs after one week.

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