17th International Congress on Toxoplasmosis

Professor

Exploring the ultrastructure of cell division coordination in Toxoplasma gondii through expansion microscopy

Intracellular Replication & Survival

The ability of protozoan parasites to rapidly proliferate within their host is at the core of their mechanisms of pathogenesis. Toxoplasma gondii, resorts to flexible cell division modes, resulting in variable outputs. It can proliferate by means of endodyogeny, endopolygeny and schizogony. These modes of division share mechanistic features such as lack of chromatin condensation, nuclear fission by semi-closed mitosis and de novo daughter cell assembly. However, the underlying mechanisms of this flexibility are only partially understood. The centrosome has long been staged at the center of regulation. However, its biogenesis and homeostasis are poorly understood. Here, we have dissected the contribution of different centrosomal components in T. gondii, using ultrastructure expansion microscopy. We analyze the phenotypes displayed by conditional mutants of centrosomal proteins TgSAS6 and TgCEP250L1 during asexual cell division and delve into pre-sexual division leading up to sexual differentiation. This work uncovers unexpected features of microtubule nucleation and centriole biogenesis. Overall, our work proposes a model for the modular organization of compartmentalized functional domains which may ultimately underly cell division flexibility allowing these parasites to adapt to different niches and proliferate accordingly.

Oral presentation

FIT plays a role in iron acquisition in Toxoplasma gondii

The obligate intracellular apicomplexan parasite Toxoplasma gondii requires iron as a cofactor for essential metabolic proteins. Due to its importance to almost all life, iron uptake and regulation have been well studied across model eukaryotes. Iron acquisition has been studied in some detail in parasitic kinetoplastids like Leishmania. However, despite its importance, little is known about how Toxoplasma gondii acquires iron. Many organisms make use of specific transporters, named Zinc and Iron Permeases (ZIPs) to take up iron from their environments. T. gondii encodes four ZIP-domain containing proteins, one of which, named here as FIT, is predicted to localize to the plasma membrane and is essential for parasite growth. We endogenously tagged FIT with 3HA tags and show that its localization is dynamic, localizing initially peripherally, before moving basal during parasite replication. Overexpression of FIT provides a protective effect when iron is depleted, but cells are hypersensitive to excess iron, when compared to wildtype parasites. Interestingly, ICP-MS experiments show that FIT overexpression leads to Zn accumulation in the parasite, however no change was seen in parasite associated iron. To determine function, we conditionally depleted of FIT, which led to a severe growth defect in vitro, suggesting it is essential for parasite survival. X-ray fluorescence microscopy (XFM) data show that FIT knockdown leads to reduced parasite iron. Moreover, ICP-MS data show that knockdown of FIT results in a decrease in Zn in the parasites. Finally, we show that knocking down FIT leads to an increased expression of bradyzoite markers including BAG1 and DBL, suggesting that knockdown of FIT may lead to the conversion/differentiation of tachyzoite parasites to the cyst-forming bradyzoite form of the parasite. Interestingly, depletion of iron by chelation also promotes differentiation, suggesting a similar mechanism and a role for iron in triggering differentiation. Together, we show that FIT has a role in iron uptake, and possibly zinc uptake in Toxoplasma gondii, and that this is important for parasite survival in vitro.

Functional dissection of NudCL1 protein in Toxoplasma gondii cell division.

Nuclear distribution protein C (NudC) is a highly conserved eukaryotic protein initially identified in Aspergillus nidulans as a dynein-mediated nuclear migration factor. However, vertebrates typically contain four paralogs sharing a common p23 domain, which engages the cochaperone HSP90 involved in protein folding and interactions. Additionally, a conserved NudC-N-terminal domain is present in two vertebrate NudC proteins. The annotated genome of Toxoplasma gondii encodes four NudC proteins based on the conservation of the p23 domain. So far, NudC proteins have not been characterized in apicomplexan parasites. We studied all four TgNudC family genes, which are all essential. Here, we focus on NudC-like 1 protein (NudC-L1), which possesses both conserved NudC and p23 domains. Surprisingly, despite its predicted fitness score of -0.47, we demonstrated that NudC-L1 is essential for the parasite lytic cycle. Depleting NudC-L1 inhibits parasite replication and leads to the development of multinucleated, unindividualized parasites, associated to elongated intercellular bridges between daughter cells. Pan Expansion Microscopy was used to assess parasite ultrastructure, revealing that NudC-L1 knockdown induces a Russian doll-like, &quot;bud-in-bud&quot; parasite development, with no observed defects in nuclear division. In humans, NudC protein regulates cytokinesis. In fact, Aurora B, a kinase essential for cell abscission, phosphorylates NudC at a conserved Threonine 40 residue. Remarkably, we found that TgNudC-L1 possesses this conserved Threonine residue. Moreover, previous work has demonstrated a similar Russian doll phenotype upon depletion of T. gondii Aurora kinase 1 (Ark1). This suggests that TgArk1 directly phosphorylates NudC-L1, which implies a putative role for the spindle midbody in promoting cytokinesis. This study represents the first comprehensive characterization of NudC proteins in T. gondii and has uncovered a putative connection between Ark1 and NudC-L1 in cytokinesis.

AP2XII-9 is a crucial factor regulating the cell cycle of Toxoplasma gondii tachyzoites.

A rapid and efficient division is key for the ability of apicomplexan parasites to proliferate and therefore is crucial for their pathogenesis. During the Toxoplasma gondii tachyzoite cell cycle, division and segregation of organelles is a highly coordinated process that ensures that each daughter parasite is properly complemented with all organelles. This highly regulated mechanism implies a tight regulation of gene expression that is crucial for the production of daughter cells in this parasite. However, which proteins are key actors in this regulation remain to be discovered. We discovered that TgAP2IX-5 is a cell-cycle-dependent ApiAP2 transcription factor (TF) bearing a crucial regulatory role during the tachyzoite cell cycle.TgAP2IX-5 also acts as a master regulator that controls the expression of other TFs that may be essential for the completion of the cell cycle. TgAP2IX-5 directly controls the expression of TgAP2XII-9 (Gissot M, Nat Comm. 2021). Nevertheless, the putative TFs' role in the biology of T. gondii has not been studied. To investigate the role of TgAP2XII-9, we produced conditional knock-down strain and characterized its phenotypes. TgAP2XII-9 is essential for the growth of parasites indicating that it may exert essential roles during the cell cycle. We have shown that TgAP2XII-9 localizes to the nucleus and is expressed during the S/M phase in line with its transcript expression profile. Depletion of TgAP2XII-9 causes significant defects in the formation of the daughter cells inner membrane complex therefore producing a significant number of multinucleated parasites. Our CUT&amp;Tag and RNA-seq data unveiled AP2XII-9's direct control over genes including other AP2 TFs, IMC coding genes, and notably, microneme and rhoptry coding genes. There is a strong correlation in these results with the phenotypes observed by investigating cell-cycle markers by IFA with clear IMC and daughter cell formation defects. Surprisingly, most of the genes that are found in both the RNA-seq and CUT&amp;Tag datasets are upregulated suggesting that AP2XII-9 is a repressor of gene expression that controls the cell cycle of T. gondii. To our knowledge, this is the first description of a transcriptional repressor with a direct role in the tachyzoite cell cycle.

Defining the Role of the Kinase TgGSK in Toxoplasma gondii

TgGSK is a serine/threonine kinase in T. gondii that is most closely related to the glycogen synthase kinase BIN2 in plants. In plants, BIN2 is a negative regulator of brassinosteroid signaling, which plays a role in plant growth and development and stress responses. Previously, we have shown that another T. gondii brassinosteroid signaling homolog, PPKL, is essential for the regulation of proper parasite division. Therefore, we wanted to investigate if TgGSK plays a similar role. We have established a conditional knockdown strain and determined that TgGSK is essential for parasite propagation. Using immunofluorescence, we have determined that TgGSK has dynamic localization, which is concurrent with the parasite&quot;s division cycle. Moreover, based on expansion microscopy we observe TgGSK in the centrosomes, corroborating a possible role in parasite division. Partial knockdown of TgGSK causes abnormal division phenotypes such as asynchronous division, uneven segregation of nuclear material, and abnormal amounts of centrosomes in each parasite as shown by both immunofluorescence and expansion microscopy. Immunoprecipitation has shown that TgGSK interacts with the GCN5b lysine acetyltransferase complex. This complex acetylates histones to open up chromatin for gene transcription and has been shown to interact with ~40% of T. gondii genes. While global transcriptomics did not show a clear pattern of differential gene transcription after TgGSK knockdown, we are currently performing global phosphoproteome analysis to determine differences in phosphorylation. These experiments will begin to elucidate a unique signaling pathway that is essential for T. gondii survival and replication.

A pair of large centrosomal proteins are crucial for daughter cell formation in Toxoplasma gondii.

Cell Biology

Toxoplasma gondii fast replication is key to its pathogenesis in intermediate hosts such as humans. The centrosome is central to the organization and coordination of the cell cycle and division of these parasites. One of the main roles of the centrosome is to ensure proper positioning and production of the daughter cell scaffold. Daughter cell assembly is a complex process that is regulated at distance by the centrosome through the nucleation of a fiber that is visible by electron microscopy. However, the centrosome components involved are poorly described. To gain novel insights into the biology and the composition of the T. gondii centrosome, we identified and characterized TgCep404 and TgCep359, two proteins associated with the centrosome, which dynamically elongate towards the apical pole of the forming daughter cells. TgCep404 and TgCep359 interact with each other, playing an essential role in the survival of the parasite. Combined depletion of these proteins leads to the disorganization of the daughter cell inner membrane complex and disruption of the normal cell cycle. It also affects the division and segregation of the centrosome. We propose that TgCep404 and TgCep359 could form a structure that positions the apical plaque of the IMC in the budding daughter cells, hence explaining its essential role in the construction of the daughter cell IMC.

VIII
Session III: Intracelluar Replication & Survival

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Vorsitzende

Programm

VIIISession III: Intracelluar Replication & Survival

Termin

Vorsitzende

Programm

VIII
Session III: Intracelluar Replication & Survival