UC Riverside

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.