Malaria is caused by
parasites that proliferate in the bloodstream. During each replication cycle, some parasites differentiate into gametocytes, the only forms able to infect the mosquito vector and ...transmit malaria. Sexual commitment is triggered by activation of AP2-G, the master transcriptional regulator of gametocytogenesis. Heterochromatin protein 1 (HP1)-dependent silencing of
prevents sexual conversion in proliferating parasites. In this study, we identified
gametocyte development 1 (GDV1) as an upstream activator of sexual commitment. We found that GDV1 targeted heterochromatin and triggered HP1 eviction, thus derepressing
Expression of GDV1 was responsive to environmental triggers of sexual conversion and controlled via a
antisense RNA. Hence, GDV1 appears to act as an effector protein that induces sexual differentiation by antagonizing HP1-dependent gene silencing.
Clonally variant expression of surface antigens allows the malaria parasite Plasmodium falciparum to evade immune recognition during blood stage infection and secure malaria transmission. We ...demonstrate that heterochromatin protein 1 (HP1), an evolutionary conserved regulator of heritable gene silencing, controls expression of numerous P. falciparum virulence genes as well as differentiation into the sexual forms that transmit to mosquitoes. Conditional depletion of P. falciparum HP1 (PfHP1) prevents mitotic proliferation of blood stage parasites and disrupts mutually exclusive expression and antigenic variation of the major virulence factor PfEMP1. Additionally, PfHP1-dependent regulation of PfAP2-G, a transcription factor required for gametocyte conversion, controls the switch from asexual proliferation to sexual differentiation, providing insight into the epigenetic mechanisms underlying gametocyte commitment. These findings show that PfHP1 is centrally involved in clonally variant gene expression and sexual differentiation in P. falciparum and have major implications for developing antidisease and transmission-blocking interventions against malaria.
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•PfHP1 is essential for mitotic proliferation of malaria blood-stage parasites•PfHP1 depletion disrupts singular var gene choice in Plasmodium falciparum•Sexual commitment of malaria parasites is epigenetically regulated by PfHP1
Heterochromatin protein 1 (HP1) is a regulator of heritable gene silencing in eukaryotes. Brancucci et al. demonstrate that the malaria parasite Plasmodium falciparum requires PfHP1 to maintain singular var gene choice and antigenic variation of the major virulence factor PfEMP1. Furthermore, PfHP1 depletion triggers sexual cell-fate decision and differentiation.
Malaria is a life-threatening disease, caused by Apicomplexan parasites of the Plasmodium genus. The Anopheles mosquito is necessary for the sexual replication of these parasites and for their ...transmission to vertebrate hosts, including humans. Imaging of the parasite within the insect vector has been attempted using multiple microscopy methods, most of which are hampered by the presence of the light scattering opaque cuticle of the mosquito. So far, most imaging of the Plasmodium mosquito stages depended on either sectioning or surgical dissection of important anatomical sites, such as the midgut and the salivary glands. Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) enable imaging fields of view in the centimeter scale whilst providing micrometer resolution. In this paper, we compare different optical clearing protocols and present reconstructions of the whole body of Plasmodium-infected, optically cleared Anopheles stephensi mosquitoes and their midguts. The 3D-reconstructions from OPT imaging show detailed features of the mosquito anatomy and enable overall localization of parasites in midguts. Additionally, LSFM imaging of mosquito midguts shows detailed distribution of oocysts in extracted midguts. This work was submitted as a pre-print to bioRxiv, available at https://www.biorxiv.org/content/10.1101/682054v2.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Plasmodium falciparum
accounts for the majority of over 600,000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged and the need for drugs with ...alternative modes of action is thus undoubted. The FK506-binding protein
Pf
FKBP35 has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506 (tacrolimus). Whilst there is considerable interest in targeting
Pf
FKBP35 with small molecules, a genetic validation of this factor as a drug target is missing and its function in parasite biology remains elusive. Here, we show that limiting
Pf
FKBP35 levels are lethal to
P. falciparum
and result in a delayed death-like phenotype that is characterized by defective ribosome homeostasis and stalled protein synthesis. Our data furthermore suggest that FK506, unlike the action of this drug in model organisms, exerts its antiproliferative activity in a
Pf
FKBP35-independent manner and, using cellular thermal shift assays, we identify putative FK506-targets beyond
Pf
FKBP35. In addition to revealing first insights into the function of
Pf
FKBP35, our results show that FKBP-binding drugs can adopt non-canonical modes of action – with major implications for the development of FK506-derived molecules active against
Plasmodium
parasites and other eukaryotic pathogens.
Abstract
The malaria parasite
Plasmodium falciparum
replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual ...development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer
P. falciparum
NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.
BACKGROUND: The post-genomic era of malaria research provided unprecedented insights into the biology of Plasmodium parasites. Due to the large evolutionary distance to model eukaryotes, however, we ...lack a profound understanding of many processes in Plasmodium biology. One example is the cell nucleus, which controls the parasite genome in a development- and cell cycle-specific manner through mostly unknown mechanisms. To study this important organelle in detail, we conducted an integrative analysis of the P. falciparum nuclear proteome. RESULTS: We combined high accuracy mass spectrometry and bioinformatic approaches to present for the first time an experimentally determined core nuclear proteome for P. falciparum. Besides a large number of factors implicated in known nuclear processes, one-third of all detected proteins carry no functional annotation, including many phylum- or genus-specific factors. Importantly, extensive experimental validation using 30 transgenic cell lines confirmed the high specificity of this inventory, and revealed distinct nuclear localization patterns of hitherto uncharacterized proteins. Further, our detailed analysis identified novel protein domains potentially implicated in gene transcription pathways, and sheds important new light on nuclear compartments and processes including regulatory complexes, the nucleolus, nuclear pores, and nuclear import pathways. CONCLUSION: Our study provides comprehensive new insight into the biology of the Plasmodium nucleus and will serve as an important platform for dissecting general and parasite-specific nuclear processes in malaria parasites. Moreover, as the first nuclear proteome characterized in any protist organism, it will provide an important resource for studying evolutionary aspects of nuclear biology.
Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating ...the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Clonally variant protein expression in the malaria parasite Plasmodium falciparum generates phenotypic variability and allows isogenic populations to adapt to environmental changes encountered during ...blood stage infection. The underlying regulatory mechanisms are best studied for the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is encoded by the multicopy var gene family and only a single variant is expressed in individual parasites, a concept known as mutual exclusion or singular gene choice. var gene activation occurs in situ and is achieved through the escape of one locus from epigenetic silencing. Singular gene choice is controlled at the level of transcription initiation and var 5' upstream (ups) sequences harbour regulatory information essential for mutually exclusive transcription as well as for the trans-generational inheritance of the var activity profile. An additional level of control has recently been identified for the var2csa gene, where an mRNA element in the 5' untranslated region (5' UTR) is involved in the reversible inhibition of translation of var2csa transcripts. Here, we extend the knowledge on post-transcriptional var gene regulation to the common upsC type. We identified a 5' UTR sequence that inhibits translation of upsC-derived mRNAs. Importantly, this 5' UTR element efficiently inhibits translation even in the context of a heterologous upstream region. Further, we found var 5' UTRs to be significantly enriched in uAUGs which are known to impair the efficiency of protein translation in other eukaryotes. Our findings suggest that regulation at the post-transcriptional level is a common feature in the control of PfEMP1 expression in P. falciparum.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This ...protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production by the addition of conditioned medium, and they are then exposed to the treatment of interest during sexual commitment or at any subsequent stage of early gametocyte development. Flow cytometry is used to measure asexual proliferation and gametocyte production via DNA dye staining and the gametocyte-specific expression of a fluorescent protein, respectively. This screening approach may be used to identify and evaluate potential transmission-blocking compounds and to further investigate the mechanism of sexual conversion in malaria parasites. The full protocol can be completed in 11 d.
Intravital microscopy allows the visualisation of how pathogens interact with host cells and tissues in living animals in real time. This method has enabled key advances in our understanding of ...host–parasite interactions under physiological conditions. A combination of genetics, microscopy techniques, and image analysis have recently facilitated the understanding of biological phenomena in living animals at cellular and subcellular resolution. In this review, we summarise findings achieved by intravital microscopy of the skin and adipose tissues upon infection with various parasites, and we present a view into possible future applications of this method.
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