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Download the Abstract Book 1 Exploring the male-induced female reproduction of Schistosoma mansoni in a novel medium Jipeng Wang1, Rui Chen1, James Collins1 1) UT Southwestern Medical Center. Schistosomiasis is a neglected tropical disease caused by schistosome parasites that infect over 200 million people. The prodigious egg output of these parasites is the sole driver of pathology due to infection. Female schistosomes rely on continuous pairing with male worms to fuel the maturation of their reproductive organs, yet our understanding of their sexual reproduction is limited because egg production is not sustained for more than a few days in vitro. Here, we explore the process of male-stimulated female maturation in our newly developed ABC169 medium and demonstrate that physical contact with a male worm, and not insemination, is sufficient to induce female development and the production of viable parthenogenetic haploid embryos. By performing an RNAi screen for genes whose expression was enriched in the female reproductive organs, we identify a single nuclear hormone receptor that is required for differentiation and maturation of germ line stem cells in female gonad. Furthermore, we screen genes in non-reproductive tissues that maybe involved in mediating cell signaling during the male-female interplay and identify a transcription factor gli1 whose knockdown prevents male worms from inducing the female sexual maturation while having no effect on male:female pairing. Using RNA-seq, we characterize the gene expression changes of male worms after gli1 knockdown as well as the female transcriptomic changes after pairing with gli1-knockdown males. We are currently exploring the downstream genes of this transcription factor that may mediate the male stimulus associated with pairing. Taken together, these data provide the platform to study schistosome sexual development in vitro and develop new strategies to control schistosome egg production. 2 Shifting perspectives: a modification to the life cycle of Trypanosoma brucei Jaime Lisack1, Sarah Schuster1, Ines Subota1, Markus Engstler1 1) Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum, Universität Würzburg, Germany. African trypanosomes are the causative agent of Human African Trypanosomiasis (HAT) and the cattle plague, Nagana. As with all vector-borne diseases, transmission is intimately tied to parasite survival and propagation in the vector, the blood-sucking tsetse fly. Two main stages of T. brucei live in the mammalian host, the proliferative long slender form and the cell cycle arrested short stumpy stage. The transition from slender trypanosomes into stumpy occurs via a quorum sensing mechanism, mediated by the parasite-excreted stumpy induction factor (SIF). As slender populations grow, the SIF threshold is reached and stumpy trypanosomes form. Aside from morphological and metabolic changes, stumpy trypanosomes also express the protein associated with differentiation 1 (PAD1) (Matthews, 2009). The switch from slender to stumpy trypanosomes is thought to accomplish two things. First, it auto-regulates parasite density and hence, prolongs survival of the host. Second, stumpy forms are thought to be ‘pre-adapted’ to survival in the tsetse fly vector. It has long been believed that upon uptake from the mammalian blood, only the ‘pre-adapted’ stumpy trypanosomes can survive in the fly midgut, while slender trypanosomes were thought to die. Keeping slender trypanosome populations below the SIF threshold and diluting parasites at different densities for in vivo fly infections, we show that both slender and stumpy trypanosomes can propagate with comparable rates in the tsetse fly. We amassed a large dataset of fly infections and dynamics, further showing that that only one trypanosome, slender or stumpy, is necessary to infect a tsetse fly. Next, we looked at differentiation hallmarks at the early stages of differentiation, both in cell culture and in the fly. Here, we found that upon differentiation, PAD1, thought to indicate stumpy formation in the mammalian host, is expressed during slender trypanosome differentiation in the fly midgut, without cell cycle arrest or morphological transition to the stumpy stage. Thus, both stumpy and slender cells can complete the life and transmission cycle inside the tsetse fly vector. These results not only hold implications regarding the life cycle of T. brucei but also on transmission dynamics. This data could help answer the long-held question of how disease incidence can be sustained in chronic mammalian infections, at low blood parasitemia, where stumpy trypanosomes are characteristically absent. 3 Critical Role for Isoprenoids in Apicoplast Biogenesis by Malaria Parasites Megan Okada1, Russell Swift2, Krithika Rajaram2, Hans Liu2, John Maschek1, Sean Prigge2, Paul Sigala1 1) University of Utah School of Medicine; 2) Johns Hopkins School of Public Health. Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle in Plasmodium malaria parasites and is required for prenylation-dependent vesicular trafficking between discrete subcellular compartments. We have elucidated a critical and previously uncharacterized role for IPP in the biogenesis of the apicoplast organelle itself. Inhibiting IPP synthesis blocks apicoplast elongation and inheritance by daughter merozoites. Exogenous IPP and polyprenols rescue these defects. Knockout of the only known isoprenoid-dependent apicoplast pathway, tRNA prenylation by MiaA, does not affect blood- stage parasites and cannot explain apicoplast reliance on IPP. However, we have localized a previously annotated, but uncharacterized, polyprenyl synthase (PPS, PF3D7_0202700) to the apicoplast lumen. PPS knockdown is lethal to parasites, rescued by IPP, and blocks apicoplast biogenesis, and these observations are sufficient to explain the reliance of apicoplast biogenesis on IPP synthesis. PPS was previously proposed to additionally catalyze the initial phytoene synthase (PSY) step in carotenoid synthesis. However, we found no evidence for PSY function by PF3D7_0202700 or de novo carotenoid synthesis by blood-stage P. falciparum. We hypothesize that PPS synthesizes polyprenols critical for membrane expansion during apicoplast biogenesis. This work provides a new paradigm for isoprenoid utilization in malaria parasites and identifies a novel essential branch of apicoplast metabolism suitable for therapeutic targeting. 4 The Cryptosporidium single-cell atlas reveals key life cycle stages and a commitment to male and female development Katelyn Walzer1, Jayesh Tandel1, Jodi Gullicksrud1, Stephen Carro1, Eoin Whelan1, Elise Krespan1, Daniel Beiting1, Boris Striepen1 1) University of Pennsylvania, Philadelphia, PA. The apicomplexan parasite Cryptosporidium is a leading global cause of diarrheal disease and infects millions of people each year, with a particularly high prevalence in south Asia and sub-Saharan Africa. The current treatment, nitazoxanide, is ineffective in immunocompromised patients and malnourished children, and there is no vaccine. Therefore, a great need exists for new and more effective therapeutics against Cryptosporidium. Transmission of the parasite occurs via the fecal-oral route, and the entire life cycle takes place in a single host: asexual growth, replication, and division take place in intestinal epithelial cells, followed by transition to a male or female form and sexual reproduction. Yet while a few molecular markers have been identified to demarcate this life cycle progression, the signaling pathways and gene expression changes involved in development remain largely unknown. Here, we used single-cell RNA sequencing of infected cultures and mice to determine the complete life cycle transcriptome of Cryptosporidium in vitro and in vivo. Analysis of 9,310 individual parasite transcriptomes revealed clear asexual cycle progression with an abrupt switch to either male or female development during the trophozoite stage. We find no transcriptional evidence for a type II meront, as gene expression dramatically changes only when transition to male or female occurs and none is noted in the prior asexual cycle. In asexual parasites, gene expression was driven by cell cycle progression dominated early by ribosomal biogenesis, processing, and assembly followed by protein folding and then DNA replication. Later asexual stages expressed many secreted proteins, including an abrupt transition to invasion related organelles. While females arrest in vitro, in vivo they progress to sporogony, and sporozoite and merozoite gene expression is highly similar. Importantly, single- cell transcriptional profiling revealed stage-specific and sex-specific expression of AP2 and Myb transcription factors, including distinct expression in early males, outlining a pathway for sex-specific commitment. Future work will focus on determining the functional roles of these transcriptional regulators. Overall, our work provides the first comprehensive view of Cryptosporidium gene expression over the entire life cycle and identifies the key genes in replicative, invasive, and sexual stages and the regulatory networks that control them. 5 N6-methyladenosine in poly(A) tails stabilize VSG transcripts Idálio Viegas1, Juan Macedo1, Mariana De Niz1, João Rodrigues2, Francisco Aresta-Branco3, Samie R. Jaffrey4, Luisa M. Figueiredo1 1) Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa; 2) Clarify Analytical, Rua dos Mercadores 128A, 7000-872 Évora, Portugal; 3) Division
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