30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

Introducing the CRISPR Cas9 Cytosine Base Editor toolbox &quot;LeishBASEedit&quot; – Gene editing and high-throughput screening in Leishmania without requiring DNA double-strand breaks, homologous recombination or donor DNA

Abstract text CRISPR Cas9 gene editing has revolutionized loss-of-function experiments in Leishmania, the causative agent of leishmanaisis. But since Leishmania lack a functional non-homologous DNA end joining pathway, obtaining null mutants typically requires the addition of donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. This makes genome-wide loss-of-function screens under different conditions and across multiple Leishmania species unfeasible to-date. Here, we report a CRISPR Cas9 Cytosine Base Editor (CBE) toolbox to overcome these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created www.leishbaseedit.net for CBE primer design in kinetoplastids. Through a series of reporter assays and by targeting single- und multi-copy genes in L. mexicana, L. major, L. donovani and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We also generated a Leishmania optimised CBE version and successfully targeted an essential gene in a small-scale plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA or isolation of clones, we believe that this enables for the first time functional genetic screens across multiple Leishmania species.

Drug Development/Target Identification

Molecular Parasitology

Talk

Initiation of DNA replication in Trypanosoma brucei is regulated by the interaction of the RNaseH2 complex with DOT1 histone methyltransferases

Abstract text Post-translational histone modifications such as lysine methylation influence the structure and the function of chromatin. DOT1 (disruptor of telomeric silencing 1) is a highly-conserved methyltransferase that exclusively methylates histone H3 lysine K79 (H3K79). T. brucei has two homologues, DOT1A and DOT1B, with differences in product specificity and function. While DOT1B mediates mono-, di-, and trimethylation of H3K76, DOT1A only catalyses mono- and di-methylation of this residue (H3K76me1/2). We showed previously that H3K76me1/2 is restricted to G2 phase and mitosis, and that DOT1A is a master regulator of replication initiation. RNAi-mediated depletion of DOT1A abolishes DNA replication completely whereas overexpression of DOT1A causes re-initiation of replication before the cell has completed S phase, implicating DOT1A as a master regulator of replication initiation. Since conserved regulatory domains are absent in these enzymes in T. brucei, the mechanism of cell cycle-dependent activity of DOT1 enzymes has remained elusive. Recently, we discovered that the ribonuclease H2 complex (RNaseH2) interacts with DOT1 enzymes in T. brucei. In vitro studies with recombinant enzymes and reconstituted nucleosomes demonstrated that RNaseH2 is an inhibitor of DOT1A activity. Using x-ray footprinting, we identified the interface region of the DOT1A/RNaseH2 complex and were able to abolish the inhibitory effect of RNaseH2 by introducing site-specific mutations into this site. Further experiments in vivo suggested that RNaseH2 interactions with DOT1 enzymes orchestrates their cell cycle-dependent activity in T. brucei, representing a novel mechanism of replication regulation.

The actomyosin system of bloodstream form Trypanosoma brucei

Abstract text The eukaryotic cytoskeleton consists of three main components: microtubules, actin filaments, and intermediate filaments. African trypanosomes (Trypanosoma brucei) have heavily invested in their microtubule cytoskeleton, but nonetheless retained a rudimentary actomyosin system consisting of one actin gene and two myosin genes. The exact functions of this highly reduced actomyosin system in T. brucei are not well understood. One of the myosins belongs to the ubiquitous class I family; the other to the trypanosomatid-specific class XXI family. The localisation of the class I myosin in the bloodstream form of T. brucei was refined using a combination of expansion microscopy, biochemical fractionation, and immuno-electron microscopy. Unexpectedly, around half of the myosin was cytosolic, while the remainder was strongly associated with the endolysosomal system but apparently not with the secretory pathway. The class XXI myosin was found to be expressed at a very low level, and appears to be dispensable in bloodstream form cells. Use of an anti-actin chromobody enabled the simultaneous detection of actin and myosin I for the first time in trypanosomes. Unexpectedly, there was little sign of any localisation to the flagellar pocket, suggesting a limited contribution to endocytosis. Overall, the results suggest that the actomyosin system is intimately involved in post-endocytic membrane trafficking events in T. brucei.

Prof.

Evolution of catalase in Trypanosomatidae

Abstract text Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The catalase-encoding gene is conspicuously absent from the genome of most representatives of the family Trypanosomatidae. The exceptions are monoxenous relatives of Leishmania spp., and representatives of the genera Blastocrithidia, Obscuromonas, and Vickermania. In this work, we expressed the Leptomonas seymouri-derived catalase from the Leishmania mexicana beta-tubulin locus using a novel bicistronic expression system, which relies on the 2A peptide of Teschovirus A. We demonstrated that catalase-expressing parasites are severely compromised in their ability to develop in insects, to be transmitted and to infect mice, and to cause clinical manifestation in their mammalian host. Taken together, our data support the hypothesis that the presence of catalase is not compatible with the dixenous life cycle of Leishmania, resulting in loss of this gene from the genome during the evolution of these parasites. In addition, we ablated a catalase-encoding gene from the Leptomonas seymouri genome and demonstrated that parasites' development in vivo depends on the expression level of this enzyme. These studies were compelmented by biochemical characterization of three independently-acquired catalases (of Blastocrithidia, Leptomonas, and Vickermania) in vitro.

Parasite-Host Interaction

Looking forward: Imaging the migration motor of Apicomplexa parasites

Abstract text Apicomplexa parasites, a phylum that includes obligate intracellular pathogens such as Plasmodium spp, Toxoplasma gondii or Cryptosporidium spp share a repertoire of homologous structures, such as the conoid and motor proteins that are required for for motility, which is central for parasite survival and dissemination. The current view, on how Apicompexa parasites moves, is described by the linear model, that postulates that, apicomplexan parasite locomotion is a type of adhesion based motility, powered by an actin-myosin motor, located in a space of 30 nm underneath the plasma membrane, and on the outer layer of the saccule like structure called the inner membrane complex (IMC). Although, a cumulative amount of empirical data support this model, the architecture of the motor, its actual localisation and molecular basis of migration are still largely unsolved. Here, we systematically tagged multiple components of the gliding machinery with multifunctional reporters, such as HALO or SNAP that allow imaging of parasites using super-resolution based methods including STED, uExMIC, graphene-energy transfer (GET) biosensors and FRAP functional live imaging methods. Using this combined approach, we have started to comprehensively and objectively test whether our observations fit with the predictions from the linear model. Our preliminary results, shows that distribution of myosin-A, actin, and other linear model components may agree with alternative configurations of the motor and alternative, mechanistic models are plausible.The data suggest a thorough revision of the current model of migration.

Session XI
Molecular Parasitology IV – Protozoa 2

Termin

Vorsitzende

Programm

Session XIMolecular Parasitology IV – Protozoa 2

Termin

Vorsitzende

Programm

Session XI
Molecular Parasitology IV – Protozoa 2