Biology subjects

Francia, M. E.

Publications and source records attributed to Francia, M. E..

6 recordsLinked to original sources

bioRxiv2026

Histone H2A.X contributes to Gene Expression Regulation and Cell-Cycle Progression in Toxoplasma gondii Tachyzoites

Histone H2A.X is a conserved variant classically associated with the DNA damage response (DDR), where its phosphorylation ({gamma}H2A.X) marks double-strand breaks and recruits repair factors. H2A.X is absent in many parasitic protozoans such as Plasmodium, Trypanosoma and Leishmania. In Toxoplasma gondii, the obligate intracellular parasite, causative agent of toxoplasmosis, however, its presence raises the question of whether it contributes to genome stability. To tackle this, we generated a T. gondii knockout of H2A.X (RH{Delta}H2A.X) and evaluated its phenotype. Although H2A.X is phosphorylated in response to DNA damage in T. gondii as in other systems, RH{Delta}H2A.X parasites showed no increased sensitivity to genotoxic agents, indicating that H2A.X is dispensable for canonical DDR. Instead, loss of H2A.X caused reduced gliding motility, increased host cell invasion and clustering of extracellular parasites after egress. Despite no defects in replication rate, RH{Delta}H2A.X showed delayed mitotic progression with abnormal vacuole morphology. Transcriptomic profiling revealed a moderate yet coordinated reprogramming of protein-coding genes. Specifically, H2A.X loss altered the expression of key apical secretory and virulence factors, especially ROP18, while inducing the transcriptional activation of mitotic and DNA replication machinery. Differential enrichment analysis further revealed that upregulated genes were preferentially associated with S phase, whereas downregulated genes were enriched during mitosis and cytokinesis. This is consistent with the delayed mitotic progression and spindle abnormalities observed by microscopy and flow cytometry and suggests a cell cycle phase-specific mechanism of H2A.X-dependent transcriptional control. Rather than a driver of DDR, these findings suggest that T. gondii H2A.X operates as a chromatin organizer that coordinates cell division fidelity and virulence gene expression.

cell biology↗

bioRxiv2026

Toxoplasma gondii Fatty Acid Elongases are Important for Virulence and Persistence

Toxoplasma gondii is an apicomplexan parasite which can infect a diverse range of warm-blooded host species. This promiscuous lifestyle requires metabolic flexibility yet our understanding of the metabolic pathways in this parasite remains incomplete. This gap includes the endoplasmic reticulum localised fatty acid elongation (FAE) pathway, which is responsible for synthesis of long (LCFA) and very long chain fatty acids (VLCFA), thus contributing to critical functions such as lipid storage and membrane biogenesis. Analyses of FAE in the commonly studied type I T. gondii strain revealed its contribution to the production of LCFA and VLCFA in vitro. However, the cystogenic type II T. gondii, which is responsible for most toxoplasmosis cases worldwide, was not studied. These type II parasites can readily differentiate between virulent and immune-evasive stages whereas type I cannot. This ability to readily transition between life stages, which are metabolically fundamentally different, prompts the question of whether the FAE pathway may play different roles in their distinct phenotype. Here we address this question, first confirming the role of the FAE enzymes, TgELO-A & B, in the production of LCFA and VLCFA in a type II T. gondii strain. Next, we demonstrated that their deletion leads to defects in replication and invasion when grown under lipid-restricted conditions. The mutants were further shown to be attenuated in mice as demonstrated by extended survival. Importantly, FAE enzyme deletion caused reduced brain cyst burdens. These findings highlight the importance of FAE for parasite survival under metabolic stress and in vivo. ImportanceToxoplasma gondii infects a wide range of hosts and persists for life in multiple tissues, making metabolic flexibility essential for its success. Fatty acid elongation (FAE), mediated by elongase (ELO) enzymes, enables the parasite to modify both de novo synthesised and host-scavenged fatty acids to support membrane biogenesis, lipid storage, and adaptation to changing nutrient environments. Previous studies in the type I strain established the importance of FAE for parasite metabolism, but its role in the clinically relevant, cystogenic type II lineage--which causes most cases of human toxoplasmosis and forms long-lived, immune-evasive bradyzoite cysts-- remained unknown. Here, we address this gap by characterising type II ELO mutants and show that disruption of FAE remodels the parasite lipidome, impairs fitness under fatty acid-limiting conditions, and reduces virulence and chronic persistence in mice. These findings identify FAE as a key metabolic pathway underpinning adaptation and persistence during chronic infection.

microbiology↗

bioRxiv2026

Characterization of Ovine Abortion in Uruguay Reveals Extensive Non-Clonal Diversity and Multiple Evolutionary Origins of Toxoplasma gondii

Toxoplasma gondii is a globally prevalent zoonotic parasite with multiple life stages and transmission routes, including ingestion and transplacental transmission. It is a major cause of abortion in sheep, goats and pigs, among other production animals, worldwide. While Type II strains are common in livestock in North America and Europe, non-archetypal, non-clonal genotypes are highly prevalent in South America. This study aimed to determine the molecular epidemiology of T. gondii strains causing sheep abortion in Uruguay. Phylogenomic analyses confirmed significant divergence among typed strains and revealed similarities with genotypes previously detected in the human population. Two novel strains, were isolated and characterized, uncovering the connection between their genetic makeup and phenotypes. Differences in virulence could be correlated to differences in gene copy number of the pseudo kinase ROP5 - further highlighting this virulence factor as relevant in wild strains. Whole-genome sequencing further confirmed the divergence among Uruguayan isolates, uncovering at least three distinct evolutionary origins. Overall, our findings highlight the circulation of virulent non-clonal lineages with links to human infections and underscore the importance of furthering genomic surveillance in South America to better understand Toxoplasmas transmission dynamics, pathogenic potential, and zoonotic risk.

molecular biology↗

bioRxiv2024

The essential kinase TgGSK regulates centrosome division and endodyogeny in Toxoplasma gondii

Intracellular replication is crucial for the success of apicomplexan parasites, including Toxoplasma gondii. Therefore, essential players in parasite replication present potential targets for drug development. In this study, we have characterized TgGSK, a glycogen synthase kinase homolog that plays an important role in Toxoplasma endodyogeny. We have shown that TgGSK has a dynamic localization that is concurrent with the cell cycle. In non-dividing parasites, this kinase is highly concentrated in the nucleus. However, during division, TgGSK displays a cytosolic localization, with concentration foci at the centrosomes, a key organelle involved in parasite division, and the basal end. Conditional knockdown of TgGSK determined that it is essential for the completion of the lytic cycle and proper parasite division. Parasites lacking endogenous protein levels of TgGSK exhibited defects in division synchronicity and the segregation of the nucleus and apicoplast into forming daughter cells. These phenotypes are associated with defects in centrosome duplication and fission. Global phosphoproteomic analysis determined TgGSK-dependent phosphorylation of RNA-processing, basal end, and centrosome proteins. Consistent with the putative regulation of RNA-processing proteins, global transcriptomic analysis suggests that TgGSK is needed for proper splicing. Finally, we show that TgGSK interacts with GCN5b, a well-characterized acetyltransferase with roles in transcriptional control. Conversely, GCN5b chemical inhibition results in specific degradation of TgGSK. Thus, these studies reveal the involvement of TgGSK in various crucial processes, including endodyogeny and splicing, and identify acetylation as a possible mechanism by which this essential kinase is regulated. AUTHOR SUMMARYToxoplasma gondii infects nearly a third of the worlds human population. While infection is largely asymptomatic in healthy adults, in immunocompromised or immunosuppressed individuals it can lead to brain lesions and even death. Similarly, toxoplasmosis can result in stillbirth, birth defects, and blindness of a developing fetus in the case of a congenital infection. With minimal treatments for Toxoplasmosis available, it is crucial to study parasite-specific processes that could be potential drug targets for the treatment of Toxoplasmosis. In this study, we investigated the protein TgGSK that is essential for parasite survival and proper division. We showed that TgGSK may perform its essential functions through interaction with the centrosome, an organelle that plays a major role in cell division in many organisms. We also show in this study a role for TgGSK in proper processing of messenger RNAs. Taken together, we have performed an in-depth study of the functional role of the essential protein TgGSK in Toxoplasma gondii. Importantly, TgGSK was shown to have more similarity to plant proteins than mammalian proteins which may allow for the possibility of targeting of this protein for therapeutic treatment of toxoplasmosis.

microbiology↗

bioRxiv2022

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. The Toxoplasma centrosome is constituted by two domains; an outer core, distal from the nucleus, and an inner core, proximal to the nucleus. This dual organization has been proposed to underlie T. gondiis cell division plasticity. Homeostasis of the outer core has been shown to be critical for the proper assembly of the daughter cells. However, the role of the inner core remains undeciphered. Here, we focus on understanding the function of the inner core by studying the dynamics and role of its only known molecular marker; TgCEP250L1. We show that upon conditional degradation of TgCEP250L1, parasites are unable to survive. Mutants exhibit nuclear segregation defects, whilst normally forming daughter cells. 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 high resolution microscopy. We show that TgCEP250L1s location is dynamic and encompasses the formation of the mitotic spindle. Moreover, we show that in the absence of TgCEP250L1, the microtubule binding protein TgEB1, fails to translocate from the nucleus to the mitotic spindle, while polyploid nuclei accumulate. Overall, our data supports 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.

cell biology↗

bioRxiv2020

Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences and chromosomal rearrangements

Neospora caninum primarily infects cattle causing abortions with an estimated impact of a billion dollars on worldwide economy, annually. However, the study of its biology has been unheeded by the established paradigm that it is virtually identical to its close relative, the widely studied human pathogen, Toxoplasma gondii. By revisiting the genome sequence, assembly and annotation using third generation sequencing technologies, here we show that the N. caninum genome was originally incorrectly assembled under the presumption of synteny with T. gondii. We show that major chromosomal rearrangements have occurred between these species. Importantly, we show that chromosomes originally annotated as ChrVIIb and VIII are indeed fused, reducing the karyotype of both N. caninum and T. gondii to 13 chromosomes. We reannotate the N. caninum genome, revealing over 500 new genes. We sequence and annotate the non-photosynthetic plastid and mitochondrial genomes, and show that while apicoplast genomes are virtually identical, high levels of gene fragmentation and reshuffling exists between species and strains. Our results correct assembly artifacts that are currently widely distributed in the genome database of N. caninum and T. gondii, but more importantly, highlight the mitochondria as a previously oversighted source of variability and pave the way for a change in the paradigm of synteny, encouraging rethinking the genome as basis of the comparative unique biology of these pathogens.

genomics↗