Unraveling the replication process of Toxoplasma gondii through the MOB1 protein

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e Biomédicas, área científica - Sanidade AnimalABSTRACT - MOB1 is a conserved protein that regulates cellular proliferation versus apoptosis, centrosome duplication and cellular differentiation in multicellular eukaryotes and also cytokinesis and division axis orientation in unicellular and multicellular eukaryotes. Toxoplasma gondii, an obligate intracellular parasite of veterinary and medical importance, presents one MOB1 protein. T. gondii interconverts between several cellular stages during its life cycle, namely between fast replicating tachyzoite and slow replicating bradyzoite stages during its asexual cycle, a key ability for its success as a parasite. Bradyzoites produce tissue cysts, establishing a chronic infection that enables recrudescence. Conversion is dependent on cell cycle regulation and involves cell differentiation and regulation of replication. This led us to select MOB1 as a strong candidate to be involved in the Toxoplasma replication process. We employed reverse genetics to assess the Mob1 function in T. gondii. In opposition to what was observed in other unicellular eukaryotes, as Tetrahymena and Trypanosoma, Mob1 knockout in T. gondii showed no cytokinesis impairment in its asexual cycle. Instead, we observed an increase in replication, a decrease in parasitophorous vacuole regularity and a significant loss in tachyzoite to bradyzoite conversion. Additionally, recombinant MOB1 accumulates in a midline between the daughter nuclei at the end of mitosis, suggesting MOB1 may be involved in this process. To elucidate how MOB1 acts in T. gondii, we employed a proximity biotinylation method and identified the MOB1 interactome. This analysis detected proteins related to several functional categories, indicating a multivalent role for MOB1 regulated by the ubiquitin proteasome system. We also verified that the Mob1 locus is transcribed from both genomic strands and gives rise to alternatively spliced variants. Our results indicate that MOB1 is tightly regulated along the cell cycle and along the life cycle of T. gondii, contributing to the control of replication and tachyzoite-bradyzoite differentiation.RESUMO - Desvendar o processo de replicação de Toxoplasma gondii através da proteína MOB1 - As proteínas da família monospindle one binder (MOB) são cinases adaptadoras de sinal altamente conservadas em eucariotas que são frequentemente essenciais para a sobrevivência das células e dos organismos. Historicamente, as proteínas MOB foram descritas como ativadoras de cinases que participam nas vias de sinalização mitotic exit network/ septation initiation network (MEN/SIN) que têm papéis centrais na regulação da citocinese, polaridade celular, proliferação celular e destino celular para controlar o crescimento e a regeneração dos órgãos. Nos metazoários, as proteínas MOB atuam como adaptadores centrais de sinal do módulo nuclear de cinases mammalian sterile 20 (MST) 1/2, large tumor suppressor (LATS) 1/2 e nuclear dbf2-related (NDR) 1/2 que fosforilam os co-ativadores transcricionais Yes associated protein (YAP)/ WW domain containing transcription regulator 1 (TAZ), efetores da via de sinalização Hippo. Mais recentemente, as proteínas MOB mostraram também ter parceiros não-cinase e estar envolvidos na biologia dos cílios, indicando que a sua atividade e regulação é mais diversificada do que inicialmente foi admitido. Em particular, as proteínas MOB1 regulam o equilíbrio entre proliferação celular versus apoptose, a duplicação de centrossomas e a diferenciação celular em eucariotas multicelulares mas também a citocinese e a orientação do eixo de divisão em eucariotas unicelulares e multicelulares. Toxoplasma gondii é um parasita intracelular obrigatório de grande importância veterinária e médica, afetando uma grande variedade de espécies animais e estando presente por todo o mundo. A sua distribuição e o facto de poder parasitar potencialmente todos os animais de sangue quente contribuíram para ser considerado o parasita de maior sucesso. Apesar de os seus hospedeiros geralmente não desenvolverem formas clínicas, T. gondii pode causar doença grave em hospedeiros imunocomprometidos e levar a aborto e malformações congénitas se a infeção é adquirida durante a gestação. T. gondii apresenta um ciclo de vida complexo, com uma fase assexuada e uma fase sexuada. Ao longo do seu ciclo de vida, é capaz de interconverter entre vários estádios celulares, nomeadamente entre os estádios de replicação rápida (taquizoítos) e os estádios de replicação lenta (bradizoítos) durante a fase assexuada, uma capacidade fundamental para o seu sucesso como parasita. ...N/

The apicomplexan parasite Toxoplasma gondii

PTDC/CVT-CVT/31840/2017EXPL/CVT-EPI/1945/2013Toxoplasma gondii is a ubiquitous zoonotic parasite with an obligatory intracellular lifestyle. It relies on a specialized set of cytoskeletal and secretory organelles for host cell invasion. When infecting its felid definitive host, T. gondii undergoes sexual reproduction in the intestinal epithelium, producing oocysts that are excreted with the feces and sporulate in the environment. In other hosts and/or tissues, T. gondii multiplies by asexual reproduction. Rapidly dividing tachyzoites expand through multiple tissues, particularly nervous and muscular tissues, and eventually convert to slowly dividing bradyzoites which produce tissue cysts, structures that evade the immune system and remain infective within the host. Infection normally occurs through ingestion of sporulated oocysts or tissue cysts. While T. gondii is able to infect virtually all warm-blooded animals, most infections in humans are asymptomatic, with clinical disease occurring most often in immunocompromised hosts or fetuses carried by seronegative mothers that are infected during pregnancy.info:eu-repo/semantics/publishedVersio

Characterization of a MOB1 homolog in the Apicomplexan parasite toxoplasma gondii

FCT_UIDB/00100/2020. FCT_UIDP/00100/2020.Monopolar spindle One Binder1 (MOB1) proteins are conserved components of the tumor-suppressing Hippo pathway, regulating cellular processes such as cytokinesis. Apicomplexan parasites present a life cycle that relies on the parasites’ ability to differentiate between stages and regulate their proliferation; thus, Hippo signaling pathways could play an important role in the regulation of the apicomplexan life cycle. Here, we report the identification of one MOB1 protein in the apicomplexan Toxoplasma gondii. To characterize the function of MOB1, we generated gain-of-function transgenic lines with a ligand-controlled destabilization domain, and loss-of-function clonal lines obtained through CRISPR/Cas9 technology. Contrary to what has been characterized in other eukaryotes, MOB1 is not essential for cytokinesis in T. gondii. However, this picture is complex since we found MOB1 localized between the newly individualized daughter nuclei at the end of mitosis. Moreover, we detected a significant delay in the replication of overexpressing tachyzoites, contrasting with increased replication rates in knockout tachyzoites. Finally, using the proximity-biotinylation method, BioID, we identified novel members of the MOB1 interactome, a probable consequence of the observed lack of conservation of some key amino acid residues. Altogether, the results point to a complex evolutionary history of MOB1 roles in apicomplexans, sharing properties with other eukaryotes but also with divergent features, possibly associated with their complex life cycle.EXPL/CVT-EPI/1945/2013info:eu-repo/semantics/publishedVersio

Origin and Epidemiological History of HIV-1 CRF14_BG

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Users must also make clear the license terms under which the work was published. CC BY Licence: http://creativecommons.org/licenses/by/4.0/Background: CRF14_BG isolates, originally found in Spain, are characterized by CXCR4 tropism and rapid disease progression. This study aimed to identify the origin of CRF14_BG and reconstruct its epidemiological history based on new isolates from Portugal.Methodology/Principal Findings: C2V3C3 env gene sequences were obtained from 62 samples collected in 1993–1998 from Portuguese HIV-1 patients. Full-length genomic sequences were obtained from three patients. Viral subtypes, diversity, divergence rate and positive selection were investigated by phylogenetic analysis. The molecular structure of the genomes was determined by bootscanning. A relaxed molecular clock model was used to date the origin of CRF14_BG. Geno2pheno was used to predict viral tropism. Subtype B was the most prevalent subtype (45 sequences; 73%) followed by CRF14_BG (8; 13%), G (4; 6%), F1 (2; 3%), C (2; 3%) and CRF02_AG (1; 2%). Three CRF14_BG sequences were derived from 1993 samples. Near full-length genomic sequences were strongly related to the CRF14_BG isolates from Spain. Genetic diversity of the Portuguese isolates was significantly higher than the Spanish isolates (0.044 vs 0.014, P,0.0001). The mean date of origin of the CRF14_BG cluster was estimated to be 1992 (range, 1989 and 1996) based on the subtype G genomic region and 1989 (range, 1984–1993) based on the subtype B genomic region. Most CRF14_BG strains (78.9%) were predicted to be CXCR4. Finally, up to five amino acids were under selective pressure in subtype B V3 loop whereas only one was found in the CRF14_BG cluster.Conclusions: CRF14_BG emerged in Portugal in the early 1990 s soon after the beginning of the HIV-1 epidemics, spread to Spain in late 1990 s as a consequence of IVDUs migration and then to the rest of Europe. CXCR4 tropism is a general characteristic of this CRF that may have been selected for by escape from neutralizing antibody response

Genome-Wide Association Study and Functional Characterization Identifies Candidate Genes for Insulin-Stimulated Glucose Uptake

Distinct tissue-specific mechanisms mediate insulin action in fasting and postprandial states. Previous genetic studies have largely focused on insulin resistance in the fasting state, where hepatic insulin action dominates. Here we studied genetic variants influencing insulin levels measured 2 h after a glucose challenge in \u3e55,000 participants from three ancestry groups. We identified ten new loci (P \u3c 5 × 10-8) not previously associated with postchallenge insulin resistance, eight of which were shown to share their genetic architecture with type 2 diabetes in colocalization analyses. We investigated candidate genes at a subset of associated loci in cultured cells and identified nine candidate genes newly implicated in the expression or trafficking of GLUT4, the key glucose transporter in postprandial glucose uptake in muscle and fat. By focusing on postprandial insulin resistance, we highlighted the mechanisms of action at type 2 diabetes loci that are not adequately captured by studies of fasting glycemic traits

The genetics of blood pressure regulation and its target organs from association studies in 342,415 individuals

To dissect the genetic architecture of blood pressure and assess effects on target-organ damage, we analyzed 128,272 SNPs from targeted and genome-wide arrays in 201,529 individuals of European ancestry and genotypes from an additional 140,886 individuals were used for validation. We identified 66 blood pressure loci, of which 17 were novel and 15 harbored multiple distinct association signals. The 66 index SNPs were enriched for cis-regulatory elements, particularly in vascular endothelial cells, consistent with a primary role in blood pressure control through modulation of vascular tone across multiple tissues. The 66 index SNPs combined in a risk score showed comparable effects in 64,421 individuals of non-European descent. The 66-SNP blood pressure risk score was significantly associated with target-organ damage in multiple tissues, with minor effects in the kidney. Our findings expand current knowledge of blood pressure pathways and highlight tissues beyond the classic renal system in blood pressure regulation

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

The trans-ancestral genomic architecture of glycemic traits

Glycemic traits are used to diagnose and monitor type 2 diabetes and cardiometabolic health. To date, most genetic studies of glycemic traits have focused on individuals of European ancestry. Here we aggregated genome-wide association studies comprising up to 281,416 individuals without diabetes (30% non-European ancestry) for whom fasting glucose, 2-h glucose after an oral glucose challenge, glycated hemoglobin and fasting insulin data were available. Trans-ancestry and single-ancestry meta-analyses identified 242 loci (99 novel; P < 5 x 10(-8)), 80% of which had no significant evidence of between-ancestry heterogeneity. Analyses restricted to individuals of European ancestry with equivalent sample size would have led to 24 fewer new loci. Compared with single-ancestry analyses, equivalent-sized trans-ancestry fine-mapping reduced the number of estimated variants in 99% credible sets by a median of 37.5%. Genomic-feature, gene-expression and gene-set analyses revealed distinct biological signatures for each trait, highlighting different underlying biological pathways. Our results increase our understanding of diabetes pathophysiology by using trans-ancestry studies for improved power and resolution. A trans-ancestry meta-analysis of GWAS of glycemic traits in up to 281,416 individuals identifies 99 novel loci, of which one quarter was found due to the multi-ancestry approach, which also improves fine-mapping of credible variant sets.Peer reviewe

A global experiment on motivating social distancing during the COVID-19 pandemic

Finding communication strategies that effectively motivate social distancing continues to be a global public health priority during the COVID-19 pandemic. This cross-country, preregistered experiment (n = 25,718 from 89 countries) tested hypotheses concerning generalizable positive and negative outcomes of social distancing messages that promoted personal agency and reflective choices (i.e., an autonomy-supportive message) or were restrictive and shaming (i.e., a controlling message) compared with no message at all. Results partially supported experimental hypotheses in that the controlling message increased controlled motivation (a poorly internalized form of motivation relying on shame, guilt, and fear of social consequences) relative to no message. On the other hand, the autonomy-supportive message lowered feelings of defiance compared with the controlling message, but the controlling message did not differ from receiving no message at all. Unexpectedly, messages did not influence autonomous motivation (a highly internalized form of motivation relying on one’s core values) or behavioral intentions. Results supported hypothesized associations between people’s existing autonomous and controlled motivations and self-reported behavioral intentions to engage in social distancing. Controlled motivation was associated with more defiance and less long-term behavioral intention to engage in social distancing, whereas autonomous motivation was associated with less defiance and more short- and long-term intentions to social distance. Overall, this work highlights the potential harm of using shaming and pressuring language in public health communication, with implications for the current and future global health challenges