17th International Congress on Toxoplasmosis

Unraveling Morphological and Biomechanical Responses to Harsh Environmental Conditions in Coccidian Oocysts

Cell Biology

Coccidia are environmentally resistant parasites with significant implications for global health and economy due to severe tissue infections they can cause in humans and/or animals. Understanding the transmission and host infection by coccidia requires a close examination of the biomechanical responses of their oocyst form to environmental factors and disinfectant treatments. Our study delved into the effects of a chlorinated household disinfectant and thermal treatment on the oocyst wall, with a focus on Eimeria species as a model of coccidia. For this, we employed fluorescence microscopy to quantify the morphological characteristics and autofluorescence of oocysts. To improve automated data processing, a StarDist model was trained to segment and classify oocysts. Complementing our microscopy-based investigation, we used microindentation techniques with a flexible micropipette as a spring to evaluate the rupture force of the oocyst wall under various treatment conditions. Our microscopy findings show a significant reduction in autofluorescence and oocyst apparent area for both Eimeria acervulina and E. tenella, with the most dramatic effects found with combined disinfectant and thermal treatments, indicating impaired wall structure. Our innovative micromanipulation system quantified, for the first time to our knowledge, the rupture force of the oocyst wall, which revealed significant variations in E. acervulina in response to the same treatments, while E. tenella displayed less discernible differences. This work sheds light on the varied effects of chemical and physical factors on coccidian oocyst features, providing important information about potential changes in responses of such parasites to environmental conditions. Future investigations will be performed to study the correlation between their infectivity and biomechanics.

Poster Turbo Talk

Professor

Preliminary data of a time-controlled splitCas9 genome wide screen in Toxoplasma gondii

Genomics

CRISPR/Cas9 technology has revolutionized genome editing and gene knockout studies across various organisms. Its successful application in Toxoplasma gondii has provided crucial insights into this parasite&quot;s biology. Previously, we utilized splitCas9, an inducible Cas9 system relying on rapamycin-induced dimerization of FRB and FKBP domains fused to either the N- or C-terminal domain of Cas9, for targeted gene knockout in T. gondii. This approach facilitated a time-controlled induction of gene knockouts through continuous co-expression of suitable guide RNAs (gRNAs), leading to the identification of essential egress genes in a small-scale phenotypic screen. Here, we present a genome-wide screen employing the splitCas9 system, targeting all annotated genes of T. gondii. After integrating a genome-wide gRNA library into a strain constitutively expressing splitCas9, we induced dimerization of the N- and C-terminal Cas9 domains. We collected samples at various time points post-induction, allowing for the assignment of genes to their respective "dropout times" and providing first insights into their potential roles in the cell cycle. Specifically, we anticipated dropout of genes responsible for replication processes at 24 hours post-induction, followed by a reduction in essential egress, motility, and invasion genes at later time points such as 48 and 96 hours.

Exploring the Seroprevalence and Molecular Epidemiology of Toxoplasma gondii in Sheep Populations of Uruguay

Epidemiology, Public Health and Clinical Aspects of Toxoplasmosis

Toxoplasmosis poses a substantial risk as a globally prevalent foodborne zoonotic disease. This disease does not only impact public health, but also results in noteworthy economic losses for countries dependent on rural activities, particularly those associated to the livestock industry, particularly pronounced in sheep and goats, where reproductive failure is a prevailing consequence. With a history deeply rooted in sheep farming, Uruguay ranks as the fifth-largest global exporter of sheep meat and the third-largest exporter of combed wool. However, the reproductive performance of sheep flocks remains suboptimal in the country. Our previous studies unveiled a concerning proportion of abortions attributable to Toxoplasma gondii 27% among aborted fetuses/placentas subjected to laboratory diagnostic investigations to assess for abortion causality. However, the overall prevalence of the parasite and the genotypes infecting Uruguayan sheep flocks have not been investigated. In this study, we aimed to assess T. gondii seroprevalence in sheep using an in-house ELISA assay, elucidate the prevailing genotypes in sheep employing molecular and serological typing, and characterize the impact of genetic diversity on parasite phenotype. Our findings indicate that around 17% of sheep in Uruguay are seropositive for T. gondii, and there is notable genetic variability in T. gondii detected in aborted ovine fetuses. In addition, we detected a predominance of type III strains, along with atypical and previously unreported genetic types. Furthermore, isolation and characterization of two Uruguayan strains obtained from aborted sheep, named TgUru1 and TgUru2, revealed diversity in terms of virulence and the ability to cause chronic/persistent infections. Thorough genetic and phenotypic analysis of these strains, encompassing in vivo and in vitro assessments revealed that both exhibit markedly distinct phenotypes, with TgUru1 displaying high virulence and rapid growth, and TgUru2 exhibiting slow growth and high rates of spontaneous cystogenesis. These differences extend to in vivo studies and gene-specific developmental stage expression. We are currently delving into the genetic and transcriptomic underpinnings of these variations, aiming to comprehend their potential impact on disease outcome. Additionally, our research extends to exploring the correlation between our animal findings and the human population in Uruguay concerning the prevalent strain's genetic background.

Professor Dr.

Establishment of an E. coli model for studying the interaction and flexibility of apicoplast resident ferredoxin and bacterial flavodoxin redox systems towards IspH of the isoprenoid pathway

Metabolism, Biochemistry & Drug Development

The apicoplast ferredoxin (Fd) redox system of Apicomplexa and flavodoxin (fldA) redox system of bacteria serves as electron donors to aid the catalytic activity of enzymes of various metabolic pathways. One of the enzymes that benefit from the redox activity of both proteins in the respective organisms is the 4Fe-4S containing IspH, involved in the last step of the MEP pathway for isoprenoid precursor synthesis, an essential pathway for survival in both, Escherichia coli and apicomplexan parasites harboring an apicoplast. We are interested in knowing whether these two structurally very different redox proteins can replace each other and if it would allow us to use E. coli as a surrogate for the apicoplast for drug screening purposes. We report the complementation of EcDIspH by Plasmodium falciparum and Toxoplasma gondii IspH (PfIspH and TgIspH). Using homologous recombination, we deleted the essential EcfldA and introduced the mevalonate by-pass pathway to make the E. coli double mutant strain entirely dependent on mevalonate. Removing mevalonate in a simple growth assay allowed us to further use this strain as a model to show that whereas Cyanobacteria (Nostoc) flavodoxin complements EcMP2, Chromera velia flavodoxin does not, raising interesting evolutionary questions. Strain EcMP2 is entirely dependent on the simultaneous expression of plasmid-encoded PfFd, PfFNR, PfIspH, which shows that the three parasite proteins are active and required to replace the respective endogenous E. coli proteins. We have also used the model to test for likely residues at the interaction interface between PfFd and PfIspH based on AlphaFold2 predictions and verified their importance for growth. Metabolomic studies are on the way to unravel the impact of the various complementation results on the MEP pathway. Altogether, we describe an engineered E. coli strain useful for functional and drug screening purposes for inhibitors of the essential MEP pathway. It will be very useful as a first screening platform for functional mutations or drug effects before more laborious and time-consuming assays in the parasite are performed.

Structural insight into the apicomplexan drug target cytochrome bc1

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Complex III, also known as the cytochrome bc1 complex, is the target of clinically used drugs such as atovaquone. The apicomplexan mETC is highly divergent from the mammalian system. Our previous proteomic work uncovered the composition of the Toxoplasma mETC complexes and F1Fo-ATP synthase identifying 70 proteins, including 20 newly discovered protein subunits, highlighting their divergence from mammals. To understand how these divergent complexes work and elucidate the mechanism of action of drug binding we used Cryo-EM to determine the structure of complex III and IV. Using native purification approaches we solved the structure of the respiratory supercomplex of complex III-IV from Toxoplasma, identifying new subunits and parasite-specific domains, as well as unique supercomplex architecture. Using a combination of native and immunoprecipitation approaches we were able to determine high resolution structures of the drug target Toxoplasma complex III in Toxoplasma with the inhibitors atovaquone or ELQ-300 bound. This gave us a detailed understanding of the mechanism of inhibitor binding and species specificity of drug action. This includes insight into why atovaquone displays much higher potency against apicomplexan complex III, compared to host, as well as showing that ELQ-300 has a different binding mode in apicomplexans compared to mammals. Insights from structural work opens the way for future drug design in both Toxoplasma and Plasmodium.

Turbo Talks for Poster Session II

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