30th Annual Meeting of the German Society for Parasitology

Prof.

Prof. Dr.

A comparative proteomic analysis of Toxoplasma gondii RH wild-type and four SRS29B (SAG1) knock-out clones to understand the impact of sag1 disruption on the gene expression profile during the lytic cycle in vitro

Abstract text The role of the major T. gondii tachyzoite surface antigen 1 (SAG1), has remained elusive. We have previously generated four T. gondii RH SAG1 knock-out (KO) strains by CRISPR-Cas9 and characterized these KOs regarding the number of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker. Accordingly, two clones have a single integration of the mdhfr-ts into the SAG1 gene. In one clone, sag1 was disrupted by insertion of a short DNA sequence unrelated to the resistance marker, but mdhfr-ts was integrated elsewhere in the genome. Another clone exhibited an insertion of mdhfr-ts into sag1, but several other mdhfr-ts copies were found in the genome. In this study, we determined the overall change(s) in protein expression patterns in all four clones by applying a comparative shotgun proteomics approach in relation to wild-type (WT) parasites using biological triplicates of each KO and WT clone. In SAG1 KO strains, the expression levels of 53 proteins were significantly altered. Of these, 12 were upregulated (fold change (FC) ≥ 1.5) and 41 were downregulated (FC ≤ 0.66) as compared to the WT parasites. In KO parasites, the expression of peptidase family M3 protein and putative transmembrane protein were 8- and 7-fold upregulated, respectively, and the expression of CBS domain-containing protein and TBC domain-containing protein was reduced by 90%, compared to WT tachyzoites. 39 SAG1 related sequence (SRS) proteins were found to be expressed in WT tachyzoites, and among those five were upregulated and three were downregulated in SAG1 KO parasites. In addition, depletion of SAG1 expression also impacted dense granule (GRA) protein expression; GRA 9 and GRA2 were upregulated. With regard to the phenotype, scanning electron micrographs did not reveal notable differences in the shape or dimensions of tachyzoites, but the surface of the mutants lacking SAG1 exhibited a higher density of particles compared to the smoother surface of WT tachyzoites. Plaque assays revealed reduced growth of KO tachyzoites compared to WT parasites. In conclusion, this comprehensive analysis shows, for the first time, that the lack of SAG1 expression has a profound impact on the tachyzoite proteome, including the tachyzoite surface composition, and affects the tachyzoite growth characteristics in vitro. This highlights the need for further in vivo studies to elucidate the functional role of SAG1 during infection in the mammalian host.

Molecular Parasitology

Talk

Functional Repurposing of Anionic Phospholipids for Regulating Calcium Homeostasis in Toxoplasma gondii

Abstract text Dimitrios Alexandros Katelas, Rosalba Cruz Miron, Nishith Gupta Department of Molecular Parasitology, Faculty of Life Sciences, Humboldt University, Berlin, Germany Introduction Toxoplasma gondii is a prominent apicomplexan parasite infecting many warm-blood organisms. Its lytic cycle in human host cells requires a range of phospholipids, some of which have emerged as key players in signaling and calcium regulation. Objectives Following our previous research, this work aimed to evaluate the synthesis and roles of anionic phospholipids during the lytic cycle of T. gondii. Materials &amp; Methods We engineered a repertoire of parasite mutants (knockout, conditional knockdown, and overexpression strains) and phenotyped them by standard molecular biology methods, including immunofluorescence, western blot, and lipidomic assays. Results Herein, we show that tachyzoites of T. gondii harbor two similar enzymes in the endoplasmic reticulum, catalyzing the synthesis of phosphatidylserine (PtdSer) and phosphatidylthreonine (PtdThr). Transgenic expression of a gene-encoded biosensor (LactC2-GFP) revealed the distribution of two phospholipids in the parasite ER and inner membrane complex. While tachyzoites could survive deletion of PtdThr synthase (PTS), de novo synthesis of PtdSer is essential for the lytic cycle. Notably, the PTS mutant displayed a defective calcium homeostasis, resulting in impaired gliding motility and concomitant invasion and egress events. Our lipid binding assays disclosed interaction of PtdThr and PtdSer with the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump, and its ectopic overexpression in the PTS mutant restored its lytic cycle. Conversely, auxin-mediated knockdown of SERCA and PtdSer synthase abrogated the parasite development with evident defects in replication and locomotion. Our independent work on P4-ATPases (Chen et al, JBC 2021; CSBJ, in review) has discovered a PtdSer/PtdThr flippase located at the apical end of tachyzoites that contributes to the motility-dependent invasion and egress. Conclusion PtdSer in mammalian cells is known to regulate the calcium release via IP3 channel, which remains to be identified in T. gondii (ongoing work). These data, taken together, strongly suggest evolutionary speciation and functional repurposing of two analogous glycerophospholipids to cater the lytic cycle of T. gondii.

Dr. med. vet.

Prof., Dr.

Whole genome sequences of isolates across Europe reveal more diversity within European Toxoplasma gondii type II than expected

Abstract text Introduction: Toxoplasma gondii is a highly prevalent protozoan parasite that globally infects a broad range of animals, including humans. A better knowledge of the genetic diversity and population structure of T. gondii may help to understand transmission routes and sources of infection for livestock and humans. Objectives: There is limited data on genome-wide comparisons of field isolates belonging to the same T. gondii clonal lineage. Therefore, the aim of the present study was to assess genome-wide genetic diversity among T. gondii Type II isolates from Europe, where this clonal lineage is predominant. Materials and Methods: The genomes of 75 European T. gondii Type II isolates, were assessed by whole genome sequencing (WGS) and compared to the Type II reference strain ME49. Results: At least 95% of the reads for each T. gondii European field isolate mapped to the reference whole genome sequence. Mapped reads covered over 99% of the reference genome with a read depth of &gt; 70 per base. The total number of SNPs relative to reference varied between ~4000 and ~12000. Affinity propagation clustering (APC) analysis revealed 5 subpopulations among 75 T. gondii Type II isolates from Europe, whereas ADMIXTURE analysis showed the presence of 3 subpopulations. Subpopulations identified by both, APC and ADMIXTURE corresponded to each other. Interestingly, subpopulations were not equally distributed all over Europe but showed clustering in particular European regions. Conclusion: The European T. gondii Type II population is genetically more diverse than expected. Based on our experiences, WGS may be useful to better understand the molecular epidemiology of the parasite in Europe, to trace infection sources in outbreaks and to detect the introduction of exotic or the emergence of recombinant strains. Funding This work was done as part of TOXOSOURCES project, supported by funding from the European Union&quot;s Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme.

One Health/NTD/Zoonoses

From endocytosis to recycling, the plasma membranes dynamics of the apicomplexan parasite Toxoplasma gondii

Abstract text The plasma membrane is at the interphase of the intracellular content and the environment. It plays a critical role in numerous functions from motility to cell communication. This is particularly true for the intracellular parasites, which are switching between drastically different environments during their life cycle. Endocytosis is an important key mechanism involved in the regulation of the plasma membrane surface protein and uptake of environmental molecule. In apicomplexan parasites, endocytosis has mainly been studied in the genus Plasmodium spp. Only recently, endocytosis has been successfully described in Toxoplasma gondii (T.gondii). The two types of endocytic mechanisms have been described in T.gondii: the uptake of proteins from the host cell and the uptake of plasma membrane proteins of the parasite. Previously, we showed the uptake of fluorescent lipids and antibodies in extracellular tachyzoites. We have now designed a new assay, which does not rely on exogenous material and allows us to image not only endocytosis but the whole plasma membrane dynamics. The gene coding for SAG1, one of the major surface antigens, was endogenously tagged with Halo. Using a new dual (spatial and/or temporal) labelling strategy, we were not only able to differentiate between internal and surface proteins, but also from mother and de-novo synthesised material during replication. We observed during replication that the plasma membrane from the mother is shared with the daughters, endocytosed, and recycled. We quantified this dynamics by tracking the changes in the fluorescence signals. Moreover, we identify for the first time that T. gondii generates extra plasma membrane "blebs" prior to the generation of the daughter cells as a reservoir for daughter cell hatching from the mother. In collaboration with the Waller"s laboratory, we have identified an important complex involved in endocytosis. Interestingly, inhibition of endocytosis also leads to a strong alteration of the plasma membrane dynamics. Taken together these data indicate that the reuptake of the plasma membrane is a critical step during replication.

Late Embryogenesis Abundant Proteins Contribute to Toxoplasma gondii Oocyst Resilience to Environmental Stresses

Abstract text Introduction Toxoplasma gondii oocysts are highly infectious, shed in large quantities by infected felines, are extremely stable in the environment and resistant to most inactivation procedures. Despite being a critical life stage for T. gondii transmission, the genetic basis for the environmental resistance of the oocysts and the contained sporozoites is largely unknown. Objectives Our objective was to test whether a set of sporozoite-specific proteins could determine T. gondii oocyst resilience to environmental stressors. Among them, we decided to investigate Late Embryogenesis Abundant (LEA)-related proteins, since LEAs are known to provide resistance to environmental stresses such as drought, salinity, and freezing in a variety of organisms. All four T. gondii LEA genes are located in a cluster (LEAc) on chromosome XII. Materials &amp; methods We studied the cryoprotective effect of the four LEAs on T. gondii lactate dehydrogenase in vitro using recombinant proteins and assessing aggregation after repeated freeze-thaw cycles. Using an inducible expression system in E. coli, we tested whether the proteins confer resistance to low temperatures. Through biochemical and bioinformatic approaches, we analyzed the LEAs for features of intrinsically disordered proteins. By applying the CRISPR/Cas9 technology, we generated a LEAc knockout in a cat-compatible ME49 strain, and assessed the oocyst sensitivity to environmental stresses compared to that from wild-type parasites. Results Recombinantly expressed LEA proteins showed cryoprotective effects on lactate dehydrogenase in vitro and induced expression in E. coli improved viability at low temperatures. T. gondii LEAs showed features of intrinsically disordered proteins and amphoteric repeats, explaining some of the protein&quot;s properties. Remarkably, LEAc knockout oocysts were more susceptible to high salinity, freezing and desiccation compared to oocysts from wild-type parasites. Conclusion We show for the first time that LEA proteins are important for the environmental resistance of T. gondii oocysts. Future studies using knockout strains for each of the LEA genes alone or in combination are now warranted to elucidate which LEAs are needed for the resistance phenotype.

Parasite-Host Interaction

Dual use of Toxoplasma gondii mitochondrial DNA sequence block boundaries gives rise to short non-coding RNAs

Abstract text 1. Introduction Mitochondrial genomes of apicomplexan parasites have only three open reading frames (ORFs) encoding subunits of the mitochondrial electron transport chain and in addition several genes for highly fragmented ribosomal RNAs. How Apicomplexan mitochondrial RNAs are expressed and processed, in particular how rRNA fragments are produced from precursor RNAs, is largely unknown. Toxoplasma gondii displays one of the most striking and poorly understood Apicomplexan mitogenome organization, built of 21 sequence blocks arranged in different combinations. How these blocks are combined has not been quantitatively analyzed so far. 2. Objectives Generate a full-genome map of the mitochondrial genome of T. gondii. Use the map to allow a comprehensive analysis of short RNAs in T. gondii – the first Apicomplexan transcriptome-wide sRNA analysis outside of Plasmodium. 3. Materials &amp; methods We established a protocol based on differential membrane permeabilization that allows purification of organellar DNA of T. gondii. Using single molecule Nanopore sequencing of mitochondrial DNA, we generated a comprehensive genome map of the variety of existing mitochondrial DNA molecules. Illumina-based short RNA (sRNA) sequencing was used to determine the first mitochondrial transcriptome of T. gondii. 4. Results Our Nanopore sequencing efforts lead to a quantitative understanding of sequence block combinations in the T. gondii mitochondrial genome. We show that there are striking differences in the frequencies of block combinations and that 5&quot;-ends of coding regions are linked to each other within the genome. sRNA sequencing data confirmed the transcription of previously predicted rRNAs and in addition identified a large number of novel noncoding short RNAs. Several of the newly discovered transcripts are spanning the borders of sequence blocks. This leads to the intriguing situation that - depending on the different DNA block combinations - chimeric sRNAs are generated at block borders. sRNAs with fusions of coding sequences with rRNA sequences are observed. 5. Conclusion T. gondii is using block borders to produce short RNAs with overlapping but not identical sequences. This may lead to dual use of sequence elements as coding region and at the same time also for ribosome production.

Session VIII
Molecular Parasitology III – Protozoa 1

Appointment

Chair

Programme

Session VIIIMolecular Parasitology III – Protozoa 1

Appointment

Chair

Programme

Session VIII
Molecular Parasitology III – Protozoa 1