17th International Congress on Toxoplasmosis

Professor

Is Toxoplasma gondii an under diagnosed cause of morbidity and mortality in humans? - My personal experience.

Epidemiology, Public Health and Clinical Aspects of Toxoplasmosis

Toxoplasma is known to infect ~20% of the human population in the UK but is not considered a major cause of disease. However, there is an old saying &quot;you will not find something unless you look for it&quot;. This presentation reflects my experience in a teaching hospital in Oxford, UK where pathologists knew of my interest in Toxoplasma (unrelated to my role in providing an ultrastructural diagnostic service). I had access to anti-Toxoplasma stage specific antibodies (tackyzoite-SAG1 and bradyzoite-Bag1). In the UK, where clinicians know of Toxoplasma in theory, it is rarely considered in differential diagnosis, except in cases of pregnancy. In other difficult cases, I was asked to examine pathology sections using immuno-cytochemistry to identify the presence or absence of Toxoplasma. In a period of 10 years, 10 cases were diagnosed at biopsy or autopsy plus two cases of congenital infection. These ranged from a middle-age woman (with unexplained headaches) to transplant patient (with negative serology). All the cases, except for the congenital cases, appeared to be associated with recrudescence rather than primary infection. In all cases, host tissue destruction is due to the uncontrolled proliferation of the tachyzoite stage. The devastating effect of uncontrolled proliferation will be illustrated. On review, most of these cases were subsequently shown to be associated with patients with a possibly impaired immune system. It is possible that earlier diagnosis and treatment could have prevented death but still cause some morbidity. In the congenital cases (diagnosed at 20 and 32 weeks scans due to foetal abnormalities), when the blood samples, taken at 12 weeks, was examined they were positive for Toxoplasma antibodies consistent with a recent primary infection. However, Toxoplasmosis is not routinely checked for in pregnant women in the UK. Could treatment at 12 weeks have protected the foetus? In adult cases, without the autopsy examination these deaths would have attributed to unknown infectious agent. Two changes could reduce the mortality: 1. Test the blood sample taken from pregnant women at 12 weeks for evidence of recent infection (IgM positive) and treat. 2. Check the immune status of patients with infections of unknown cause and, if immuno-compromised, consider Toxoplasma infection. From this local experience it could be suggested that Toxoplasma is an under diagnosed cause of mortality in the UK and probably many other countries.

Oral presentation

Uncovering the minimalist adhesive strategy of the Toxoplasma parasite for high-speed motility

Cell Biology

Toxoplasma gondii is a intracellular protozoan and the etiological agent of toxoplasmosis, a set of chronic diseases. The T. gondii tachyzoite morphotype serves as model to study cell motility in simple living non swimming systems, since with a permanent apicobasal polarity, it bypasses the cell symmetry breaking needed for motility. Its moves by gliding front first between cells or into and out from host cells with a repetitive helical trajectory. Gliding is thought to proceed through a series of sub-membranous myosin motors which direct retrograde translocation of actin filaments along the parasite length, and power continuous sub-membranous forces. In this scenario, the tachyzoite secretes adhesins that, once exposed at the cell apex, engage with ECMs ligands, and flow backward with the newly formed actin filaments. We recently uncover a critical specific anchorage site for the generation and transmission of a periodic traction force during gliding (Pavlou et al, 2020). In this new study, we have interrogated the minimal spatial and molecular requirements for productive adhesion and force transmission beneath efficient gliding. To this end, we have combined submicron resolution micropatterning with live, quantitative reflection interference contrast microscopy and expansion microscopy. Using 4D image modeling, we bring first nanoscale evidence that the tachyzoite needs to build only one apical anchoring contact with the substrate, which spatially defines a minimal force transmission platform over which it slides. We clarify the relationship between surface flow and force generation, by monitoring microbead flow at 200 Hz and uncover that the apicobasal driven surface flow is set up independently from adhesin release and adhesion, prior to motile activity. Finally, to screen for the essential adhesion requirements for helical gliding at the single molecular species level in absence of any absorption of molecular components from the environment, we developed biochemical and biophysical quantitative assays using tunable surface chemistry and quartz crystal microbalance with dissipation monitoring. These approaches uncover the sufficiency of glycosaminoglycan (GAG)-parasite interaction to promote a productive contact for helical gliding and offer a new versatile platform to dissect the structure and density of the molecules functionally involved in the T. gondii gliding force. Pavlou et al., ACS Nano 2020. doi: 10.1021/acsnano.0c01893

The Toxoplasma gondii mitoribosome reveals novel features of ribosome evolution

Mitochondrial ribosomes (mitoribosomes) are fundamental, and their function of synthesising mitochondrial proteins is universal, including in parasites. The apicomplexan mitochondrion is essential for parasite survival, virulence, and dissemination, and the same is thus expected for its mitoribosome. In agreement with this prediction, evidence of the essentiality of conserved mitoribosomal proteins in apicomplexan, accumulate, including from our own work. In addition to being essential, indirect observations further suggest that this mitoribosome is highly divergent from its human parallel. Divergence is expected from the prediction of rRNA fragmentation, based on different apicomplexan mitochondrial genome sequences, as well as on the sensitivity profile of some apicomplexan parasites to mitoribosome inhibitors. Despite its essentiality and divergence, the biology of the apicomplexan mitoribosome is poorly studied. Here, using Toxoplasma gondii as a model organism, we employed a combination of complementary approaches to expand our understanding of the apicomplexan mitoribosome function and assembly. We discovered an rRNA fragmentation that is much more extensive than predicted according to the parasite mitochondrial genome, and revealed several novel features that enable this highly divergent ribosome to still perform its critical function. One example of an apicomplexan, and likely myzozoan, mitoribosome signature feature we discovered is the repurposing of several transcription factors as new mitoribosomal proteins, which we believe compensate for rRNA remodelling and we postulate that they effectively replace conserved and critical ribosomal domains. On top of addressing the fundamental question of how divergent ribosomes function, our work has further potential to inform apicomplexan drug discovery, which we demonstrate by revisiting a previously proposed resistance mechanism of apicomplexan to a known mitoribosome inhibitor.

Professor Dr.

Evolutionary Insights into the Composition and Assembly of Mitoribosomes in Alveolates

The Myzozoa, a grouping of phyla within the Alveolates, emerged through endosymbiosis with a red alga, resulting in the acquisition of a plastid. This cluster includes Apicomplexa, Chromerida, Perkinsozoa, and Dinoflagellates, all sharing a notably reduced mitochondrial genome that encodes only three proteins for the mitochondrial electron transport chain, cytochrome c oxidase subunits 1 and 3 and cytochrome b. Intriguingly, the mitoribosomal rRNAs in these phyla exhibit high segmentation, prompting questions about the process of mitoribosome assembly. Furthermore, these phyla possess four conserved proteins with domains resembling the plant Apetala2/ethylene response factor (AP2/ERF) integrase DNA binding domain. In Toxoplasma gondii, these four AP2 proteins localize within the mitochondrion and play a crucial role in oxidative phosphorylation and parasite viability. Surprisingly, instead of acting as transcription factors, these mtAP2s form tight interactions with the small and large mitoribosomal subunits. These genes are highly fitness-conferring and prove critical for mitoribosome assembly and integrity. Applying mass spectrometry and small RNA sequencing to the purified T. gondii mitoribosome revealed its distinctive protein-rich composition, abundance of fragmented rRNAs, and assembly factors. Besides mtAP2s, other contributors, such as the RAP (RNA binding domain abundant in apicomplexans ) proteins, and HPR (heptatricopeptide repeat) proteins, contribute to molecular diversity and evolutionary adaptations. Together, our results provide insight into the composition, biogenesis and evolution of the ribosomal machinery in Alveolates.

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