The human malaria parasite remains a major public health burden in developing nations. Despite many years of research, the mechanisms controlling gene expression in the parasite are still poorly understood. While the P. falciparum genome lacks more than fifty percent of the transcription factors anticipated to regulate its 6372 genes, it encodes a large amount of genes involved in RNA metabolism and chromatin remodeling. Furthermore, preliminary data in the laboratory showed extensive nucleosome remodeling during the parasite's asexual cycle. Therefore, we hypothesized that change in chromatin structure plays an important role in controlling parasite development. To understand the role of histone post-translational modifications (PTMs) in transcriptional regulation and histone turnover, we used a shotgun proteomic approach. A total of 246 histone PTMs were identified with 126 being novel. Parasite histones were highly acetylated and methylation marks associated with transcriptional silencing were detected at low levels. To elucidate the mechanism regulating histone turnover, we treated parasite cultures with inhibitors of two distinct pathways that degrade bulk amounts of protein; the ubiquitin-proteasome system and the autophagosome-lysosome pathway. Parasites treated with inhibitors of the autophagy pathway displayed an accumulation of histone protein. The autophagy pathway was overlooked in the parasite; thus, we investigated it at the comparative genomic, cellular, biological and genetic levels. PfATG8, an autophagosome membrane marker, was detected throughout the erythrocytic stages in the apicoplast and the cytoplasm. Proteins associated with PfAtg8 were isolated by immunoprecipitation and identified by mass spectrometry. Gene ontology enrichment showed an enrichment of proteins involved with the digestive food vacuole, the phagolysosome, and the nucleus. In summary, we determined that the autophagy pathway is multifunctional and is likely involved in vesicle traffic, apicoplast biogenesis, and protein catabolism. To further validate its role in histone turnover, we took a cellular approach and colocalized histones and PfATG8 vesicles. Collectively, our work provides key information of mechanism regulating epigenetic and its effects on gene expression in the human malaria parasite.

Malaria is one of the most lethal infectious diseases in many developing countries.

Approximately half of the world’s population is at risk of malaria transmission, and this number can be expected to grow as drug resistant strains continue to develop. Among the human infectious Plasmodium species, Plasmodium falciparum causes the most severe and lethal form of malaria. This parasite has an extreme AT-rich genome and a complex life cycle that is likely to be regulated by coordinate changes in gene expression. However, the mechanisms behind this fine-tuned gene expression and regulation system remain elusive. For instance, only a limited number of transcription factors have been identified. Recent studies suggest that epigenetic and post-transcriptional regulation may be used as alternative regulation strategies to compensate for the lack of transcription factors in this parasite. Therefore, in this dissertation work, we further explored the transcriptome, epigenome, and the proteome to better understand the transcriptional mechanisms in P. falciparum. In chapter 1, we demonstrated that genes are usually defined by unique nucleosomal features and that nucleosome landscape alone could be used to identify novel genes in organisms with a nucleotide bias. Next, we investigated nascent RNA expression profiles and observed that the majority of genes are transcribed at the trophozoite stage in response to the open chromatin structure of that stage. These results helped us link chromatin reorganization events to transcriptional activity and highlighted the importance of epigenetic and post-transcriptional regulation in this parasite. Therefore, in the latter two chapters, we further examined the proteasome and transcriptome isolated from both nuclear and cytoplasmic fractions to identify potential chromatin regulators. As a result, we identified a large number of chromatin-associated proteins and lncRNAs that are likely to have important roles in chromatin regulation and post-transcriptional and translational regulations. Collectively, data and results from these studies will become stepping-stones for future malaria studies and further assist the identification of promising anti-malarial drug targets.

Mosquitoes harbor and transmit a variety of pathogens which are dangerous to humans and incur a constant and significant public health cost. Gaining more detailed knowledge about these pathogens, their interactions with the mosquito, and their molecular biology and genetics will allow for new techniques to be developed to prevent harmful effects on humans. These pathogens vary in complexity from viruses like West Nile virus to eukaryotic apicomplexan parasites like Plasmodium falciparum, the human malaria parasite.Culex mosquitoes routinely transfer viruses like West Nile virus in the United States representing a predominant health threat there. These mosquitoes are also routinely infected by a wide variety of other viruses, which have received very little research attention, yet could affect the transmission of pathogens like West Nile virus and St. Louis encephalitis virus. Therefore, in the first chapter of this dissertation work, we performed small RNA sequencing to examine the full virome of field-caught Culex mosquitoes in multiple geographical regions of southern California. These data also allowed us to analyze the interactions between viruses and identify potential pairs of co-infecting or mutually excluding viruses, as well as closely look at the small RNA immune response of Culex mosquitoes against these viruses, expanding the known role of the antiviral piRNA response in Culex. We also identified mosquito miRNAs that may be involved in antiviral immunity or virus infection by comparing highly infected mosquito pools against lowly infected ones. Apart from viruses, single-celled eukaryotic parasites are also transmitted from mosquitoes to humans. One of the deadliest of these pathogens is the malaria parasite, Plasmodium falciparum. Important regulators that propel the life cycle of this parasite also represent potential drug targets, that when identified could lead to new treatment options to lessen the impact of malaria in Africa and across the globe. We performed a wide variety of experiments and studies in this vein. In the second chapter, we examine the role of long non-coding RNAs (lncRNAs) on parasite gene regulation and predict lncRNAs across the genome, categorizing their properties and their essentiality for parasite survival. We also use experimental techniques to determine the binding sites of several of these predicted lncRNAs, and look at the effect of one in particular, lncRNA-14, by knocking it out and using transcriptomic and phenotypic analysis. The third chapter, on the other hand, uses Plasmodium berghei, a mouse malaria parasite, as a model for human malaria and examines parasites proteins SMC2 and SMC4, the homologs of proteins that make up the condensin complex in model eukaryotes. We determine that, as in other eukaryotes, Plasmodium SMC2 and SMC4 form a condensin complex and are key in cell division, particularly in the mosquito stages of the parasite life cycle. ChIP-seq determined that these proteins bind at the centromeres, and transcriptomic and phenotypic analysis revealed the exact roles of these proteins and their importance. Finally, the remaining two chapters examine RNA-dependent and RNA-binding proteins in Plasmodium falciparum, key elements in post-transcriptional regulation, another important aspect of gene regulation in the life cycle. In the fourth chapter, we performed a screen for RNA-dependent proteins using the R-DeeP protocol, obtaining a list of likely proteins and examining RNA-dependent complexes. We also characterized one of the proteins found as an RNA-binding protein using various techniques including enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq). In the fifth chapter, this technique and others were also used to determine the binding sites and functions of two other predicted RNA-binding proteins with RAP (RNA-binding domain abundant in apicomplexans) domains. These RAP proteins were determined to regulate the parasite mitochondrial rRNAs (mitoribosome). All in all, this dissertation work reveals new insights into mosquito-associated pathogens, their interactions with mosquitoes and mosquito immunity, and important molecular components of their life cycles which could be targeted to reduce their impact on humans.

Toxoplasma gondii is a prolific parasite known to infect a wide variety of warm-blooded animals, including humans. The parasite can interconvert between a dormant form of the parasite (bradyzoites), and an acute form of the parasite (tachyzoites), which manifests as Toxoplasmosis, in a process called recrudescence. This process is tightly controlled through a series of changes in gene expression in response to environmental pressure. Histone modifications are effective post-translational modifications, influencing the parasite’s gene expression through different stages of its life cycle. Despite knowing that recrudescence is, in part, epigenetically regulated, little is known about how environmental factors, such as the type of host cell, affect this process. This study uses histone modifications to observe changes in the epigenomic profile of the parasite as they develop within two different host cells, Astrocytes (ASTs) and Human Foreskin Fibroblasts (HFFs). Using Chromatin Immunoprecipitation followed by deep Sequencing (ChIP–seq) against three different histone marks, we revealed a higher abundance of histone H3K4me3 activating marks within ASTs. This observation was supported by an increased binding affinity of H3K4me3 in ASTs and a decline in the number of affinity binding sites in HFF as recrudescence progressed. Of the annotated genes that were identified to have a high affinity with H3K4me3, several had potential roles in invasion, chromatin remodeling, and cell cycle progression.

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