30th Annual Meeting of the German Society for Parasitology

Prof.

Contribution of Actin-like proteins Alp1 and Alp2b in Plasmodium progression

Abstract text Plasmodium requires active motility in order to spread and must traverse and invade mammalian and mosquito organs. Actin-like proteins (Alps) are apicomplexan-specific, highly divergent members of the actin superfamily. Alps are characterised by several unique regions and likely have critical functions during parasite infection. However, the functions and stage specific contributions of Alps in parasite progression are unknown. The corresponding genes of Alp1 and Alp2b were knocked-out in Plasmodium berghei and the phenotypic impacts of these deletions on the parasites were investigated. Remarkably, the absence of Alp1 or Alp2b led to a drastic reduction or complete block of mosquito midgut colonisation at different steps of transmission respectively. Specifically, Alp1 was essential for the motility of ookinetes, which actively colonise mosquito midguts, while Alp2b was crucial for parasite fertilisation. Alp2b function in P. berghei was partially restored by P. falciparum Alp2b complementation, indicating a degree of cross-species conservation. Mutations of selected unique regions of P. berghei Alp2b prevented exflagellation, implicating these regions as critical contributors to Alp2b function and thus potential targeting regions. Our study reveals the importance of Alps in Plasmodium transmission and the stage-specific contributions of these members of apicomplexan actin superfamily.

Drug Development/Target Identification

Molecular Parasitology

Talk

Prof. Dr. apl.

Prof. Dr.

Parasite G6PD is a promising target for drug development against Leishmania (DRUID WS)

Abstract text The enzyme glucose-6-phoshate dehydrogenase (G6PD) of the pentose phosphate pathway is required for redox regulation. As G6PD is found amongst numerous parasites causing neglected tropical diseases (NTD), including Leishmania (L.), identifying a drug to target G6PD in various Leishmania strains is a very promising strategy. In order to genetically validate Leishmania G6PD as a potential drug target, we employed CRISPR/Cas9 to knockout this gene in both cutaneous and visceral Leishmaniasis (CL and VL) causing strains. Knock out of both alleles appeared to be lethal, viable parasites were only obtained while expressing Leishmania G6PD from a rescue plasmid. Loss of this plasmid resulted in parasite death, pointing to an essential role of G6PD for parasite survival. To further evaluate the role of G6PD in Leishmania, we established a CRISPR/Cas9/DiCre inducible knockout system. In this system parasites loose G6PD gene expression as well as G6PD enzymatic activity, over a time period of seve days resulting in both L. donovani and L. major full G6PD knockout strains. Interestingly wild type L. donovani contained a much higher level of G6PD activity as compared to wild type L. major parasites. By flow cytometry analysis, we could show that G6PD knockout decreases the parasites" tolerance against reactive oxygen species in the promastigote life stage of both species. In contrast to the viable L. major G6PD deficient axenic amastigotes, the L. donovani G6PD induced knockout strain was unable to survive as axenic amastigotes. Moreover preliminary data suggest that the L .major G6PD knockout had does not survive inside human macrophages. In summary, our data genetically validate G6PD as a promising drug target in both CL and VL causing Leishmania, and support the strategy to develop drugs for targeting multiple parasites causing NTDs.

Parasite G6PD is a promising target for drug development against Leishmania

Parasite-Host Interaction

The Fasciola TRPMPZQ channel, a new druggable target

Abstract text Introduction Milestones have recently been achieved in clarifying the mechanism of action of praziquantel (PZQ), the drug of choice to fight schistosomes. PZQ acts as an agonist of a transient receptor potential (TRP) channel within the melastatin family (SmTRPMPZQ), which leads to rapid contraction of the worms and death. Mutagenesis of residues within the binding pocket of SmTRPMPZQ identified an asparagine (Asn) residue as crucial factor for PZQ-sensitivity. While the TRPM channel of other PZQ-sensitive helminths harbor the same Asn, natural variation at this residue renders TRPMPZQ of the liver fluke Fasciola hepatica (FhTRPMPZQ) resistant to PZQ (Park et al. 2021). Exploring FhTRPMPZQ as drug target opens new avenues to find new fasciolicidal compounds. This is particularly pressing against the wide spread of triclabendazole resistance. Objectives In this study, we aim for a de novo discovery of agonists for the F. hepatica TRPMPZQ channel and to test if such agonists have fasciolicidal activity. Materials and Methods A high throughput drug screen using a Ca2+-based reporter assay (Chulkov et al. 2021) was used to identify FhTRPMPZQ-activating chemotypes. One of these chemotypes was tested against FhTRPMPZQ and different stages of F. hepatica in vitro. Further characterization of FhTRPMPZQ is envisaged by RNA interference and gene expression analysis. Results The reporter assay identified several chemotypes triggering Ca2+ influx in FhTRPMPZQ-transfected cells. One series was selected and optimized to yield a ligand that activated FhTRPMPZQ with high sensitivity in vitro (EC50=855±151nM). The same chemotype killed adult and immature F. hepatica at concentrations of 6.25-12.5 µM within 24 h-72 h. The TRPM agonists, therefore, outcompeted triclabendazole, which was lethal at 50 µM within 48 h. Contraction of the worms resembled the phenotype observed for PZQ-treated schistosomes. Finally, our single-cell transcriptomics data suggest a neuronal expression of FhTRPM. Conclusion Our study proves that TRPMPZQ is a druggable target also in a PZQ-resistant helminth. The successful screening approach may be translated to other PZQ-resistant helminths in the future. Park et al. Mechanism of praziquantel action at a parasitic flatworm ion channel. Sci Transl Med. 2021;13(625):eabj5832. Chulkov et al. Identification of novel modulators of a schistosome transient receptor potential channel targeted by praziquantel. PLoS Negl Trop Dis. 2021;15(11):e0009898.

Drug Resistance

Schistosomal helicase eIF4A – A potential target of plant-derived rocaglates? (DRUID-WS)

Abstract text Introduction: Schistosomiasis is caused by an infection with blood flukes of the genus Schistosoma, with S. mansoni being one of several species infectious for humans. No vaccine exists, and treatment of schistosomiasis relies on a single drug - Praziquantel. There is justified fear for the development of resistance, encouraging the search for new targets and drugs. One potential new drug target is the eukaryotic translation initiation factor 4A (eIF4A), an RNA helicase that unwinds stable RNA secondary structures in the 5'-untranslated region of mRNAs. Once the secondary structures are dissolved, ribosomes can access the mRNA and initiate protein synthesis. Rocaglates, a compound class isolated from plants of the genus Aglaia, prevent ribosome binding and thus protein synthesis by inhibiting eIF4A. Thereby, rocaglates clamp the mRNA on the surface of eIF4A by interacting with a specific phenylalanine (Phe163) of human eIF4A. Objectives: We aim to investigate the functions of schistosomal eIF4A and to clarify whether targeting eIF4A of S. mansoni by rocaglates could be a therapeutic option in the future. Material &amp; Methods: Adult S. mansoni worms were treated with rocaglates in vitro. Effects of the treatment on stem cell proliferation were analyzed by an EdU assay. Two schistosomal eIF4A isoforms, one with the specific Phe and one without, were cloned and recombinantly expressed in Escherichia coli, purified by affinity chromatography, and used for a thermal shift assay to validate the interaction of rocaglates and schistosomal eIF4A. Results: Treatment of adult S. mansoni with rocaglates resulted in a reduced vitality and decreased numbers of proliferating EdU-positive cells. First thermal shift assays indicate that only the Phe-containing isoform can be bound by natural and synthetic rocaglates. Conclusion: Schistosomal eIF4A may play a role for proliferating cells in adult S. mansoni. The interaction of rocaglates and the Phe-containing isoform as well as the vitality effects of these plant-derived products on S. mansoni motivate for further evaluation of schistosome helicases as potential targets.

Schistosomal helicase eIF4A – A potential target of plant-derived rocaglates?

Untargeted molecular imaging by mass spectrometry: uptake and fate of anthelmintic drugs

HS I (GF)

Workshop III

DRUID

Workshop

Workshop III
DRUID

Appointment

Chairs

Programme

Workshop IIIDRUID

Appointment

Chairs

Programme

Workshop III
DRUID