17th International Congress on Toxoplasmosis

Does T cell Exhaustion occur in pregnant women with chronic Toxoplasma gondii infection?

Immunology of Acute & Chronic Infection

We have recently reported finding significantly suppressed levels of inflammatory cytokines, IFN- γ , IL-6, IL-17, and IL-12 across pregnancy in women with T.gondii (TG) chronic infection (n=158) (Prescott S, et al. (2023). A mechanism that may be related to the reduced cytokines in latent infection is T cell exhaustion due to lifelong exposure of the immune system to parasite antigens. Latent, persistent, infections may continuously provide antigen exposure to T cell receptors (TCRs), leading to exhaustion of TCRs on antigen specific T cells. Pregnancy related fetal antigen exposure at the placenta also can produce T cell exhaustion. T cell exhaustion is marked by increased expression of inhibitory receptors on CD8 cells, loss of the ability to secrete cytokines, and development of different transcriptional programs. Exhaustion progresses over the time of continuous antigen exposure and is marked by cell signature immune checkpoint molecules. The cells most affected are the CD8 effector cells, which, in the case of TG, produce the proinflammatory milieu suppressing bradyzoite cysts. A loss of these CD8 effectors allows for the loss of memory responses to the antigens. Our data also suggested TG associated significant suppressed activation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which is activated by inflammatory cytokines, and acts to reduce tryptophan availability to cysts. Both the immune and tryptophan/kynurenine effects could potentially cause low level parasitemia. Consistently lower levels of peripheral circulating inflammatory cytokines in the latently infected pregnant women suggest a global immunosuppression related to T.gondii and pregnancy induced T cell exhaustion. We are planning immunophenotyping and flow cytometry studies to determine if pregnancy associated TG latent infection produces T cell exhaustion. Prescott, S., Mutka, T., Baumgartel, K., Yoo, J. Y., Morgan, H., Postolache, T. T., Seyfang, A., Gostner, J. M., Fuchs, D., Kim, K., &amp; Groer, M. E. (2023). Tryptophan metabolism and immune alterations in pregnant Hispanic women with chronic Toxoplasma gondii infection. American journal of reproductive immunology 90(3), e13768.

Poster Turbo Talk

Professor

Uncovering the mechanisms employed by latent Toxoplasma gondii within neurons to evade immune surveillance

IFN-γ-induced anti-microbial mechanisms are instrumental for parasite control and host protection during T. gondii (Tg) infection. Upon brain invasion by Tg, neurons upregulate surface MHC class I molecules, allowing them to present tachyzoite antigens to CD8+ T cells, which is key to avoid excessive parasite burden (Salvioni, Cell Rep 2019 PMID: 31189109). Neurons also restrict parasite growth in response to IFN-γ, although neurons may be less able to do so than other cells. Tg eventually persists in the brain throughout the latent phase of infection as bradyzoites within intra-neuronal cysts. The mechanisms by which Tg bradyzoite-infected neurons evade CD8+ T cell recognition, remain unclear. Here we investigate to which extent Tg effectors that hijack IFN-γ responses in various cell types, also modulate the responses of infected neurons to IFN-γ. Using primary cultures of hippocampal neurons stimulated or not with IFN-γ, we assessed the nuclear translocation of IRF1 transcription factor and MHC I surface expression in neurons containing mostly tachyzoites (24 hpi) or bradyzoites (72 hpi). IRF1 was translocated in the host cell nucleus and surface expression of MHC I was upregulated after IFN-γ stimulation. Neurons stimulated by IFN-γ and infected by WT tachyzoites or bradyzoites failed to accumulate IRF1 in their nuclei. Conversely, neurons infected with TgIST-deficient parasites showed higher nuclear IRF1 intensity. Strikingly, neurons infected by tachyzoites upregulated surface MHC I similarly as uninfected ones, but neurons infected by bradyzoites showed a clear TgIST-dependent inhibition of surface MHC I upregulation. These results suggest that the impairment of neuronal MHC I is TgIST-dependent but is delayed compared to the subversion of IRF1, suggesting that MHC I subversion may occur after or upon bradyzoite differentiation. Experiments are underway to explore the impact of bradyzoite-derived TgIST and other parasite effectors on the neuronal responses to IFN-γ, and ultimately, on the ability of CD8+ T cells to detect infected neurons and control the parasite in vivo.

In silico, transcriptomic and proteomic comparative analysis of Dense Granule Proteins orthologues in Toxoplasma gondii and Neospora caninum

Innate Immunity and Immune Evasion

Apicomplexa parasites require a parasitophorous vacuole (PV) for replication and infection maintenance, which is formed through protein secretion from organelles such as dense granules. Thus, Dense granule proteins (GRAs) are important for the intracellular survival of these parasites, as they influence host-parasite interactions, the acquisition of nutrients and the modulation of the host's immune responses. Research has focused on GRAs of Toxoplasma gondii, but little is known about related Neospora caninum. Thus, the aim of this study is to fill this gap by carrying out a bioinformatic, transcriptomic and proteomic analysis, providing information on the conservation and divergence of GRAs in these parasites. For that purpose, we generated a list of predicted GRAs obtained at the Toxoplasma Informatics Resources Database (ToxoDB) and the literature, and checked for N. caninum orthologues, in order to initially analyze the genes for synteny, identity, conserved or unique motifs and to compare their predicted conformational structure. We found a high level of synteny and a reasonable ammino acid similarity. Differences in transmembrane domains and signal peptides indicate possible disparities in subcellular localization and functionality between GRAs, whereas little divergence was found in the presence of other conserved domains and predicted 3D structures. On the other hand, gene expression of the GRAs in T. gondii and N. caninum presented a notable distinction in profile for part of the genes, indicating possible adaptations in the host-parasite interactions. In order to confirm the presence of those proteins in the host-N. caninum interface, we used proximity biotinylation to identify the predicted targets in the PV. We have also generated knockout parasites of selected genes and submitted those to in vitro and in vivo assays to assess their role in host resistance to the infections. In conclusion, we believe our work contributes to a deeper understanding of the molecular aspects, functional diversity and evolutionary history of TgGRAs and NcGRAs, and may have far-reaching implications for the development of interventions and therapies for toxoplasmosis and neosporosis.

Human toxoplasmosis in the Czech Republic 1923-2023: From the first case through a massive outbreak to a long-term decline

Epidemiology, Public Health and Clinical Aspects of Toxoplasmosis

When the Prague ophthalmologist Josef Janků in March 1922 noted and a year later described strange symptoms in a deceased newborn, he did not know that this would be the first documented case of toxoplasmosis in humans, moreover with congenital transmission and eye damage. Only the improvement of serological diagnostics revealed that toxoplasmosis was one of the most common serious parasitoses in the Czech Republic. The annual number of reported cases of clinical toxoplasmosis increased from one hundred in the early 1970s to 513-860 between 1983 and 1993. In 1994, the number of cases of clinical toxoplasmosis rose transiently to a total of 2056 cases as a result of an unusually widespread epidemic. Since then, the numbers have been steadily declining and have returned to one hundred cases in recent years. The reasons for the decrease in the incidence of toxoplasmosis in the Czech Republic could be the gradual improvement of social and hygiene standards and education, control and supervision of food safety. The process of urbanisation has resulted in fewer outdoor activities. On the other hand, the growing popularity of organic food, organic farming and unconventional cooking practices could theoretically contribute to the spread of toxoplasmosis, as well as travelling to southern countries rich in stray cats. However, seroprevalence data do not match such a significant decrease in toxoplasmosis. According to 37 studies performed on different groups of persons in the years 2002-2018, seropositivity in men can be found between 7.9-40.2% (average: 23,9%) and between 15.9-45,9% (average 28,9%) in women. This is not much less than the 26.3% of positive men and 34.1% of positive women found in surveys from 1971 to 1996. The declining reporting discipline associated with the skyrocketing administration can also be a problem. Clinical toxoplasmosis in the past and present is reported twice as often in women as in men. The younger age categories predominate - men 10-24 years, women 20-34 years. The most common clinical forms are lymphadenitis (72.8%), ocular toxoplasmosis (6.0%), primary infection during pregnancy (5.4%), and congenital infection accounts for 0.6%. The incidence is highly locally variable. The highest is in some districts of West Bohemia and Moravia, while the lowest number of cases is reported from Prague and the surrounding area. Supported by MH CZ - DRO (The National Institute of Public Health – NIPH, 75010330)

T Lymphocyte-Dependent IL-10 Protects Against a Lethal IL-12 Driven Cytokine Storm Initiated by Toxoplasma gondii GRA24

A model organism in the study of immunity to infection, Toxoplasma gondii has been instrumental in establishing many key principles of host anti-microbial defense and its regulation. Here, we employed an attenuated uracil auxotroph strain of Type I Toxoplasma designated OMP to further untangle the early immune response to this parasitic pathogen. Experiments using T cell-deficient TCRβ-/- mice unexpectedly revealed that an intact αβ T lymphocyte compartment was essential to survive infection with this uracil auxotroph. Subsequent antibody depletion and knockout mouse experiments demonstrated contributions from CD4+ T cells and most predominantly CD8+ T cells in resistance to OMP. Using transgenic knockout mice, we found only a partial requirement for IFN-γ, and a lack of requirement for Toll-like receptor (TLR) adaptor MyD88 in resistance. In stark contrast to other studies on Toxoplasma, resistance to OMP did not require IL-12p40. Surprisingly, given the attenuated nature of OMP, T cell-dependent IL-10 was found to be critical for survival and deficiency of this cytokine triggered an abnormally high systemic inflammatory response. We also found that parasite molecule GRA24, a dense granule protein that triggers TLR-independent IL-12 production, acts as a virulence factor contributing to death of OMP-infected TCRβ-/- mice. Further, resistance against OMP was restored in TCRβ-/- mice via monoclonal depletion of IL-12p40, suggesting that GRA24-induced IL-12 underlies the fatal immunopathology observed. Collectively, our studies provide insight into a novel and rapidly arising T lymphocyte-dependent anti-inflammatory response to T. gondii which operates independently of MyD88 and IL-12, and that provides protection against the proinflammatory effects of GRA24.

The T. gondii mitoribosome - build from tiny rRNA fragments expressed from mitochondrial genome recombination sites.

Genomics

Apicomplexan mitochondrial genomes are highly reduced comprising only three open readings frames (ORFs) encoding essential subunits of the oxidative phosphorylation machinery along with genes for ribosomal RNAs (rRNA). Intriguingly, the rRNA genes are not organized as continuous long units, as observed in most other organisms, rather, they are scattered as multiple small fragments throughout the mitochondrial genome. The organization of the Toxoplasma gondii mitochondrial genome is even more striking consisting of 23 sequence blocks that undergo non-random recombination with each other. Recent studies in our lab have unveiled the expression of 34 small RNAs in T. gondii mitochondria with majority of them having homology to rRNA fragments previously described in other Apicomplexans. Surprisingly, several of these small RNAs are encoded at genomic recombination sites leading to the intriguing situation that the sequence at block borders is dually used for different RNAs. We found several instances where small RNAs partially share sequence. In some even more remarkable cases sequence is dually used for a small RNAs and a messenger RNA. We wondered whether these peculiar small RNAs could function as ribosomal RNA, being integrated into mitochondrial ribosomes together with canonical rRNA fragments and ribosomal proteins. Thus, we established a density-based centrifugation approach to study translation in T. gondii mitochondria. For several mitochondrial small RNAs we observed migration into deep fractions of sucrose density gradients. Additionally, putative mitoribosomal proteins of the large and small ribosomal subunit as well as the three mitochondrial mRNAs have shown comparable migration patterns providing the first strong evidence for the presence of polysomes in T. gondii mitochondria. Among the small RNAs found in polysomal fractions are several RNAs that are encoded at genome recombination sites. Outstanding examples are RNA3 and RNA19 which partially share sequence with coxI and coxIII mRNA, respectively. Whether the dual sequence usage for rRNA fragments and mRNAs could be relevant for regulatory processes remains unclear and will be addressed in future experiments. Moreover, our density gradient centrifugation protocol offers a powerful tool for comprehensive studies on the composition and assembly of the T. gondii mitoribosome.

Determination of Toxoplasma gondii"s genetic variability in cases of maternal seroconversion during pregnancy from Uruguay

The paradigm for transmission and manifestations of human toxoplasmosis arose based on clinical data and experimentation in mice using strains of Toxoplasma gondii circulating in Europe and North America known as "typical." However, we now understand that the severity of human toxoplasmosis depends on the gestational period upon which the infection is acquired, the parasitic load and genotype of the parasite, which is closely linked to its geographic origin. Worldwide genotyping studies describe a higher incidence of atypical strains in South America, but this concept is mainly based on data obtained in Brazil, Colombia, and Argentina. Pioneering work in Uruguay described the existence of "atypical" serotypes in patients and the isolation of a genetically atypical strain of Uruguayan origin. We hypothesize that there is ample genetic variability of circulating T. gondii strains in humans in our country. To address this, we genotyped T. gondii strains in women who acquired the infection during pregnancy, as determined by the patient's seroconversion, detectable during routine pregnancy checkups. We amplified parasite DNA from peripheral blood, placental tissue, umbilical cord blood and newborn babies peripheral blood. T. gondii DNA was detectable in 17 of 32 patients. Positive samples were genotypes using in silico PCR-RFLP and sequence analyses, of nine polymorphic genetic markers. The presence of "atypical" strains was evidenced in 7 samples. We are currently advancing in their genetic characterization and pursuing the isolation of circulating strains with the aim of correlating genotype with phenotype. Preliminary data suggests that the severity of ocular toxoplasmosis is linked to the genotype. In addition, we updated the epidemiological data on T. gondii prevalence for the general population in the country, extrapolating from serological data obtained from pregnant women, and determined the seroconversion rates during pregnancy for the studied population.

The Role of Purine Auxotrophy in Toxoplasma-Host Interaction

Metabolism, Biochemistry & Drug Development

Studying cellular survival strategies in resource-scarce environments is vital for revealing novel starvation responses across diverse life forms. Substantial advancements have been achieved in understanding purine metabolism in higher eukaryotes. Nevertheless, our understanding of these processes in protozoans is incomplete. This study presents a comprehensive analysis of Toxoplasma's purine dependency due to the absence of purine synthesis pathways 1,2. We developed a model to study purine starvation in Toxoplasma, revealing significant transcriptional changes in response to purine scarcity. This includes the upregulation of histone modifiers during starvation and the downregulation of rhoptry transcripts, which are crucial for host cell invasion 3. Additionally, the study delineates the stage-specific roles of TgENTs, notably TgENT1 and TgENT2, in the parasite lifecycle. Notably, the deletion of TgENT1 led to a reduced number of tissue cysts in infected mice, underscoring its importance in chronic infection. Furthermore, this study investigated the essential nature of TgENT3, whose deletion results in complete growth defects. The outcomes of this project are anticipated to significantly enhance our understanding of purine acquisition and metabolism in intracellular parasites, shedding light on alternative evolutionary pathways that lead to distinct purine-sensing and scavenging strategies. This has broader implications for understanding metabolic adaptations in other intracellular parasites. Kloehn, J., Hammoudi, P.-M. &amp; Soldati-Favre, D. Metabolite salvage and restriction during infection — a tug of war between Toxoplasma gondii and its host. Syst. Biol. Nanobiotechnology 68, 104–114 (2021). Olson, W. J. et al. Dual metabolomic profiling uncovers Toxoplasma manipulation of the host metabolome and the discovery of a novel parasite metabolic capability. PLOS Pathog. 16, e1008432 (2020). Dubremetz, J. F. Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction. Cell. Microbiol. 9, 841–848 (2007).

Turbo Talks for Poster Session I

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