PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum.

Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain the structural integrity and loading of this complex on mitotic chromosomes. This complex is present in all eukaryotes and has recently been identified in Plasmodium spp. However, how this complex is assembled and whether the kleisin subunit is critical for this complex in these parasites are yet to be explored. To examine the role of PfCAP-H during cell division within erythrocytes, we generated an inducible PfCAP-H knockout parasite. We find that PfCAP-H is dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the mitotic chromosomes. While mitotic spindle assembly is intact in PfCAP-H-deficient parasites, duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis occurs normally. Altogether, our data suggest that PfCAP-H plays a crucial role in maintaining the structural integrity of the condensin I complex on the mitotic chromosomes and is essential for the asexual development of malarial parasites. IMPORTANCE: Mitosis is a fundamental process for Plasmodium parasites, which plays a vital role in their survival within two distinct hosts-human and Anopheles mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in Plasmodium spp., our understanding of how this complex is assembled and its precise functions during the blood stage development of Plasmodium falciparum remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of P. falciparum. Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.

PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum.

Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain the structural integrity and loading of this complex on mitotic chromosomes. This complex is present in all eukaryotes and has recently been identified in Plasmodium spp. However, how this complex is assembled and whether the kleisin subunit is critical for this complex in these parasites is yet to be explored. To examine the role of PfCAP-H during cell division within erythrocytes, we generated an inducible PfCAP-H knockout parasite. We find that PfCAP-H is dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the mitotic chromosomes. While mitotic spindle assembly is intact in PfCAP-H deficient parasites, duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis occurs normally. Altogether, our data suggest that PfCAP-H plays a crucial role in maintaining the structural integrity of the condensin I complex on the mitotic chromosomes and is essential for the asexual development of malarial parasites.

Global Release of Translational Repression Across Plasmodium's Host-to-Vector Transmission Event.

Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5' or 3' end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.

Identification of basal complex protein that is essential for maturation of transmission-stage malaria parasites.

Malaria remains a global driver of morbidity and mortality. To generate new antimalarials, one must elucidate the fundamental cell biology of Plasmodium falciparum, the parasite responsible for the deadliest cases of malaria. A membranous and proteinaceous scaffold called the inner membrane complex (IMC) supports the parasite during morphological changes, including segmentation of daughter cells during asexual replication and formation of transmission-stage gametocytes. The basal complex lines the edge of the IMC during segmentation and likely facilitates IMC expansion. It is unknown, however, what drives IMC expansion during gametocytogenesis. We describe the discovery of a basal complex protein, PfBLEB, which we find to be essential for gametocytogenesis. Parasites lacking PfBLEB harbor defects in IMC expansion and are unable to form mature gametocytes. This article demonstrates a role for a basal complex protein outside of asexual division, and, importantly, highlights a potential molecular target for the ablation of malaria transmission.

Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria.

Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, field-applicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. Our P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (5 P. falciparum and 10 P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections.