The mRNA-bound proteome of the human malaria parasite Plasmodium falciparum.

BackgroundGene expression is controlled at multiple levels, including transcription, stability, translation, and degradation. Over the years, it has become apparent that Plasmodium falciparum exerts limited transcriptional control of gene expression, while at least part of Plasmodium's genome is controlled by post-transcriptional mechanisms. To generate insights into the mechanisms that regulate gene expression at the post-transcriptional level, we undertook complementary computational, comparative genomics, and experimental approaches to identify and characterize mRNA-binding proteins (mRBPs) in P. falciparum.ResultsClose to 1000 RNA-binding proteins are identified by hidden Markov model searches, of which mRBPs encompass a relatively large proportion of the parasite proteome as compared to other eukaryotes. Several abundant mRNA-binding domains are enriched in apicomplexan parasites, while strong depletion of mRNA-binding domains involved in RNA degradation is observed. Next, we experimentally capture 199 proteins that interact with mRNA during the blood stages, 64 of which with high confidence. These captured mRBPs show a significant overlap with the in silico identified candidate RBPs (p < 0.0001). Among the experimentally validated mRBPs are many known translational regulators active in other stages of the parasite's life cycle, such as DOZI, CITH, PfCELF2, Musashi, and PfAlba1-4. Finally, we also detect several proteins with an RNA-binding domain abundant in Apicomplexans (RAP domain) that is almost exclusively found in apicomplexan parasites.ConclusionsCollectively, our results provide the most complete comparative genomics and experimental analysis of mRBPs in P. falciparum. A better understanding of these regulatory proteins will not only give insight into the intricate parasite life cycle but may also provide targets for novel therapeutic strategies

Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum.

Gene expression in Plasmodium falciparum is tightly regulated to ensure successful propagation of the parasite throughout its complex life cycle. The earliest transcriptomics studies in P. falciparum suggested a cascade of transcriptional activity over the course of the 48-hour intraerythrocytic developmental cycle (IDC); however, the just-in-time transcriptional model has recently been challenged by findings that show the importance of post-transcriptional regulation. To further explore the role of transcriptional regulation, we performed the first genome-wide nascent RNA profiling in P. falciparum. Our findings indicate that the majority of genes are transcribed simultaneously during the trophozoite stage of the IDC and that only a small subset of genes is subject to differential transcriptional timing. RNA polymerase II is engaged with promoter regions prior to this transcriptional burst, suggesting that Pol II pausing plays a dominant role in gene regulation. In addition, we found that the overall transcriptional program during gametocyte differentiation is surprisingly similar to the IDC, with the exception of relatively small subsets of genes. Results from this study suggest that further characterization of the molecular players that regulate stage-specific gene expression and Pol II pausing will contribute to our continuous search for novel antimalarial drug targets

<i>Plasmodium </i>Condensin Core Subunits SMC2/SMC4 Mediate Atypical Mitosis and Are Essential for Parasite Proliferation and Transmission

Condensin is a multi-subunit protein complex regulating chromosome condensation and segregation during cell division. In Plasmodium spp., the causative agent of malaria, cell division is atypical and the role of condensin is unclear. Here we examine the role of SMC2 and SMC4, the core subunits of condensin, during endomitosis in schizogony and endoreduplication in male gametogenesis. During early schizogony, SMC2/SMC4 localize to a distinct focus, identified as the centromeres by NDC80 fluorescence and chromatin immunoprecipitation sequencing (ChIP-seq) analyses, but do not form condensin I or II complexes. In mature schizonts and during male gametogenesis, there is a diffuse SMC2/SMC4 distribution on chromosomes and in the nucleus, and both condensin I and condensin II complexes form at these stages. Knockdown of smc2 and smc4 gene expression reveals essential roles in parasite proliferation and transmission. The condensin core subunits (SMC2/SMC4) form different complexes and may have distinct functions at various stages of the parasite life cycle

Babesia duncani multi-omics identifies virulence factors and drug targets

Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.We thank R. Gao for her contribution to the initial eforts to sequence the B. duncani genome. C.B.M.’s research was supported by grants from the National Institutes of Health (AI097218, GM110506, AI123321 and R43AI136118), the Steven and Alexandra Cohen Foundation (Lyme 62 2020), and the Global Lyme Alliance. S.L.’s research was supported by grants by the US National Science Foundation (IIS 1814359) and the National Institutes of Health (1R01AI169543-01). K.G.L.R.’s research was supported by the National Institutes of Allergy and Infectious Diseases (R01 AI136511, R01 AI142743-01 and R21 AI142506-01), the University of California, Riverside (NIFA-Hatch-225935) and the Health Institute Carlos III (PI20CIII/00037).S

Exploring Chromatin Organization and Regulation in Human Malaria Parasites

The human malaria parasite, one of the deadliest infectious agents in the world, still contributes significantly to the global burden of disease. In 2017, an estimated 214 million cases of infection and over 400,000 malaria-related deaths were reported, a majority of which are caused by the most lethal human malaria parasite, Plasmodium falciparum. Given the absence of an FDA-approved vaccine and parasite resistance to all current antimalarial drugs there is a desperate need for new therapeutic approaches. Plasmodium falciparum has a complex life cycle that requires coordinated gene expression regulation to allow host cell invasion, transmission and immune evasion. However, this cascade of transcripts is unlikely to be regulated by the limited number of identified parasite-specific transcription factors. Increasing evidence now suggests a major role for epigenetic mechanisms in gene expression in the parasite. Therefore, in this dissertation work, we further explore genome architecture, epigenome, proteome and transcriptome including long-non-coding RNAs (lncRNAs) to better understand the relationship between chromatin structure, genome organization and transcriptional regulation in malaria parasites.In the first chapter, we explore genome organization in human Plasmodium parasite stages including the transmission stages from human to mosquito (gametocytes) and from mosquito to human (sporozoites). Our work demonstrates that genome organization is an important regulator for several parasite-specific gene families involved in pathogenesis and immune evasion, erythrocyte and liver cell invasion, sexual differentiation, and master regulators of gene expression. In the second chapter, we investigated genome organization in five malaria parasites and two related apicomplexan parasites with the goal to identify common features of genome organization and possible connections between genome architecture and pathogenicity. We show that in all malaria parasites, genome organization is dominated by the clustering of Plasmodium-specific gene families in 3D space. Our data highlight the importance of spatial genome organization in gene regulation and control of virulence in malaria parasites.In the subsequent chapters, we aim to identify molecular components, specifically proteins and lncRNAs, that maintain and regulate chromatin structure in the malaria parasite. To investigate parasite proteins and protein complexes maintaining and regulating nuclear architecture, we undertook comparative genomics analysis using twelve distinct eukaryotic genomes. We identified conserved and apicomplexan parasite-specific chromatin-associated domains (CADs) and proteins (CAPs). We validated two of our candidate proteins including a novel plant-related protein that is functionally analogous to animal nuclear lamina proteins and might have a role in heterochromatin organization. Finally, we also explore the role of lncRNAs in P. falciparum. In eukaryotes, lncRNAs have been shown to be pivotal regulators of genome structure and gene expression. To investigate the regulatory roles of lncRNAs in P. falciparum, we first explored the intergenic distribution of lncRNA using deep sequencing in nuclear and cytoplasmic subcellular locations. We then validate the subcellular localization and stage-specific expression of several putative lncRNAs at single cell resolution using fluorescence in situ hybridization (FISH) technology. Additionally, we explore the genome-wide occupancy of several candidate nuclear lncRNAs using Chromatin Isolation by RNA Purification followed by deep sequencing (ChIRP-seq) technology. Data analysis revealed that lncRNA occupancy sites within the parasite genome are focal and sequence-specific with a particular enrichment for several parasite-specific gene families, including those involved in pathogenesis, erythrocyte remodeling, and regulation of sexual differentiation. Discovery of these proteins and lncRNAs are the starting point for further exploration of mechanisms regulating chromatin structure and genome architecture in these deadly parasites.Collectively, our data highlight the importance of spatial genome organization as a mechanism of transcriptional regulation in malaria parasites, and our work directly addresses one of the central outstanding questions in Plasmodium biology, namely, how a parasite with approximately 6,000 genes manages to control gene expression in a coordinated fashion using a limited number of transcription factors

The Role of Chromatin Structure in Gene Regulation of the Human Malaria Parasite

The human malaria parasite, Plasmodium falciparum, depends on a coordinated regulation of gene expression for development and propagation within the human host. Recent developments suggest that gene regulation in the parasite is largely controlled by epigenetic mechanisms. Here, we discuss recent advancements contributing to our understanding of the mechanisms controlling gene regulation in the parasite, including nucleosome landscape, histone modifications, and nuclear architecture. In addition, various processes involved in regulation of parasite-specific genes and gene families are examined. Finally, we address the use of epigenetic processes as targets for novel antimalarial therapies. Collectively, these topics highlight the unique biology of P. falciparum, and contribute to our understanding of mechanisms regulating gene expression in this deadly parasite