New Insights into the Role of the Trypanosoma cruzi Aldo-Keto Reductase TcAKR.

Chagas disease is a zoonotic infectious disease caused by the protozoan parasite Trypanosoma cruzi. It is distributed worldwide, affecting around 7 million people; there is no effective treatment, and it constitutes a leading cause of disability and premature death in the Americas. Only two drugs are currently approved for the treatment, Benznidazole and Nifurtimox, and both have to be activated by reducing the nitro-group. The T. cruzi aldo-keto reductase (TcAKR) has been related to the metabolism of benznidazole. TcAKR has been extensively studied, being most efforts focused on characterizing its implication in trypanocidal drug metabolism; however, little is known regarding its biological role. Here, we found that TcAKR is confined, throughout the entire life cycle, into the parasite mitochondria providing new insights into its biological function. In particular, in epimastigotes, TcAKR is associated with the kinetoplast, which suggests additional roles of the protein. The upregulation of TcAKR, which does not affect TcOYE expression, was correlated with an increase in PGF2α, suggesting that this enzyme is related to PGF2α synthesis in T. cruzi. Structural analysis showed that TcAKR contains a catalytic tetrad conserved in the AKR superfamily. Finally, we found that TcAKR is also involved in Nfx metabolization.

Insights into the Cell Division of Neospora caninum.

Neospora caninum is an apicomplexan protozoan parasite responsible for causing neosporosis in a range of animal species. It results in substantial economic losses in the livestock industry and poses significant health risks to companion and wild animals. Central to its survival and pathogenicity is the process of cell division, which remains poorly understood in this parasite. In this study, we explored the cell division of Neospora caninum using a combination of modern and classic imaging tools, emphasizing its pivotal role in perpetuating the parasite's life cycle and contributing to its ability to persist within host organisms. We described the intricacies of endodyogeny in Neospora caninum, detailing the dynamics of the cell assembly and the nuclear division by ultrastructure expansion microscopy and regular confocal microscopy. Furthermore, we explored the centrosome dynamics, the centrioles and the apicoplast through the advancement of the cell cycle. Our analysis described with unprecedented detail, the endodyogeny in this parasite. By advancing our understanding of these molecular mechanisms, we aimed to inspire innovative strategies for disease management and control, with the ultimate goal of mitigating the devastating impact of neosporosis on animal health and welfare.

Separate To Operate: the Centriole-Free Inner Core of the Centrosome Regulates the Assembly of the Intranuclear Spindle in Toxoplasma gondii.

Centrosomes are the main microtubule-organizing center of the cell. They are normally formed by two centrioles, embedded in a cloud of proteins known as pericentriolar material (PCM). The PCM ascribes centrioles with their microtubule nucleation capacity. Toxoplasma gondii, the causative agent of toxoplasmosis, divides by endodyogeny. Successful cell division is critical for pathogenesis. The centrosome, one of the microtubule organizing centers of the cell, plays central roles in orchestrating the temporal and physical coordination of major organelle segregation and daughter cell formation during endodyogeny. The Toxoplasma centrosome is constituted by multiple domains: an outer core, distal from the nucleus; a middle core; and an inner core, proximal to the nucleus. This modular organization has been proposed to underlie T. gondii's cell division plasticity. However, the role of the inner core remains undeciphered. Here, we focus on understanding the function of the inner core by finely studying the localization and role of its only known molecular marker; TgCep250L1. We show that upon conditional degradation of TgCep250L1 parasites are unable to survive. Mutants exhibit severe nuclear segregation defects. In addition, the rest of the centrosome, defined by the position of the centrioles, disconnects from the nucleus. We explore the structural defects underlying these phenotypes by ultrastructure expansion microscopy. We show that TgCep250L1's location changes with respect to other markers, and these changes encompass the formation of the mitotic spindle. Moreover, we show that in the absence of TgCep250L1, the microtubule binding protein TgEB1, fails to localize at the mitotic spindle, while unsegregated nuclei accumulate at the residual body. Overall, our data support a model in which the inner core of the T. gondii centrosome critically participates in cell division by directly impacting the formation or stability of the mitotic spindle. IMPORTANCE Toxoplasma gondii parasites cause toxoplasmosis, arguably the most widespread and prevalent parasitosis of humans and animals. During the clinically relevant stage of its life cycle, the parasites divide by endodyogeny. In this mode of division, the nucleus, containing loosely packed chromatin and a virtually intact nuclear envelope, parcels into two daughter cells generated within a common mother cell cytoplasm. The centrosome is a microtubule-organizing center critical for orchestrating the multiple simultaneously occurring events of endodyogeny. It is organized in two distinct domains: the outer and inner cores. We demonstrate here that the inner core protein TgCEP250L1 is required for replication of T. gondii. Lack of TgCEP250L1 renders parasites able to form daughter cells, while unable to segregate their nuclei. We determine that, in the absence of TgCEP250L1, the mitotic spindle, which is responsible for karyokinesis, does not assemble. Our results support a role for the inner core in nucleation or stabilization of the mitotic spindle in T. gondii.

Structural and Functional Insights into the Microtubule Organizing Centers of Toxoplasma gondii and Plasmodium spp.

Microtubule organizing centers (MTOCs) perform critical cellular tasks by nucleating, stabilizing, and anchoring microtubule's minus ends. These capacities impact tremendously a wide array of cellular functions ranging from ascribing cell shape to orchestrating cell division and generating motile structures, among others. The phylum Apicomplexa comprises over 6000 single-celled obligate intracellular parasitic species. Many of the apicomplexan are well known pathogens such as Toxoplasma gondii and the Plasmodium species, causative agents of toxoplasmosis and malaria, respectively. Microtubule organization in these parasites is critical for organizing the cortical cytoskeleton, enabling host cell penetration and the positioning of large organelles, driving cell division and directing the formation of flagella in sexual life stages. Apicomplexans are a prime example of MTOC diversity displaying multiple functional and structural MTOCs combinations within a single species. This diversity can only be fully understood in light of each organism's specific MT nucleation requirements and their evolutionary history. Insight into apicomplexan MTOCs had traditionally been limited to classical ultrastructural work by transmission electron microscopy. However, in the past few years, a large body of molecular insight has emerged. In this work we describe the latest insights into nuclear MTOC biology in two major human and animal disease causing Apicomplexans: Toxoplasma gondii and Plasmodium spp.

The Structural and Molecular Underpinnings of Gametogenesis in Toxoplasma gondii.

Toxoplasma gondii is a widely prevalent protozoan parasite member of the phylum Apicomplexa. It causes disease in humans with clinical outcomes ranging from an asymptomatic manifestation to eye disease to reproductive failure and neurological symptoms. In farm animals, and particularly in sheep, toxoplasmosis costs the industry millions by profoundly affecting their reproductive potential. As do all the parasites in the phylum, T. gondii parasites go through sexual and asexual replication in the context of an heteroxenic life cycle involving members of the Felidae family and any warm-blooded vertebrate as definitive and intermediate hosts, respectively. During sexual replication, merozoites differentiate into female and male gametes; their combination gives rise to a zygotes which evolve into sporozoites that encyst and are shed in cat's feces as environmentally resistant oocysts. During zygote formation T. gondii parasites are diploid providing the parasite with a window of opportunity for genetic admixture making this a key step in the generation of genetic diversity. In addition, oocyst formation and shedding are central to dissemination and environmental contamination with infectious parasite forms. In this minireview we summarize the current state of the art on the process of gametogenesis. We discuss the unique structures of macro and microgametes, an insight acquired through classical techniques, as well as the more recently attained molecular understanding of the routes leading up to these life forms by in vitro and in vivo systems. We pose a number of unanswered questions and discuss these in the context of the latest findings on molecular cues mediating stage switching, and the implication for the field of newly available in vitro tools.