Browsing by Author "Derbyshire, Emily R"

The phylum Apicomplexa encompasses multiple obligate intracellular parasites that pose significant burdens to human health including the causative agents of malaria, toxoplasmosis, and cryptosporidiosis which infect millions of humans and cause hundreds of thousands of deaths each year. During their complex life cycles, apicomplexan parasites coordinate the function of specific proteins to both evade the host immune system and thrive under stressful conditions. Notably, Toxoplasma gondii has been found to harbour multiple polyketide synthase (PKS) genes by bioinformatic analysis, suggesting they can produce secondary metabolite polyketides. While secondary metabolite biosynthetic gene clusters (BGCs) have been known in Apicomplexa for over two decades, limited studies on these enzymes have been completed to date and there have been no characterized products, leaving a void in our understanding of the role of these enzymes in parasite biology. Therefore, characterization of these proteins may aid in our ability to target these biosynthetic enzymes as sources of potential therapeutic candidates in Apicomplexa.While protists are underexplored for biosynthetic potential, research points to this kingdom as an untapped potential for new chemical space. T. gondii for instance possesses multiple putative PKS biosynthetic gene clusters (BGCs) however there have been no secondary metabolite products elucidated thus far. Therefore, our work explores a T. gondii PKS, TgPKS2, and investigates the architecture, predicted structures, and activity of multiple domains within this synthase. Subsequently, Chapters 2 and 3 describes our initial studies on TgPKS2 including hydrolysis activities of acyltransferase (AT) domains, mutagenesis studies, and a first of its kind self-acylation activity of acyl carrier protein (ACP) domains in a modular type I PKS. Chapter 4 further emphasizes the unique attributes of TgPKS2, delving into a never before characterized chain release mechanism, while Chapter 5 compares TgPKS2 transacylation activity to well-characterized bacterial and fungal systems. Combined, these chapters describe our work to biochemically explore TgPKS2, discover the role it plays within the T. gondii life cycle, and further our work to elucidate the metabolite(s) produced by this synthase. Altogether, this research lays the ground work for exploring other apicomplexan and eukaryotic polyketide synthases and significantly increases our knowledge of the biochemical properties of these unique proteins.

Malaria is one of the oldest and deadliest diseases in the world, affecting approximately 200 million people annually. The role of protein phosphorylation in the complex life cycle of the malaria parasite, Plasmodium, as well as the promising therapeutic values of protein kinase inhibitors have sparked increasing interest in understanding the Plasmodium kinome. Although many protein kinases have been shown to be essential for Plasmodium survival, their functions remain unknown. Protein kinase 5 (PK5) is a putative cyclin dependent kinase (CDK)-like protein in the malaria parasite, and it is thought to be essential for blood stage proliferation in P. falciparum. In the present study, biochemical binding assays were used to identify potent and selective inhibitors of PfPK5. Two compounds were found to selectively bind to PfPK5 over the human analog, Homo sapiens CDK2. In addition, a known CDK inhibitor was used in the development of a chemical probe to identify potential macromolecules essential to parasite survival. Here, we report important structural moieties potentially involved in PfPK5 binding. Elucidation of the biological targets through the use of our chemical probe may aid in further understanding of Plasmodium biology.

Malaria is one of the leading causes of mortality attributed to infectious diseases worldwide. Every year, hundreds of thousands of children succumb to the disease and hundreds of millions more suffer the characteristic symptoms of malaria. It is caused by eukaryotic parasites of the genus Plasmodium and is transmitted to the human host via the bite of an Anopheles mosquito. Upon infection, the parasite must travel to the liver where it develops and replicates into merozoites, the parasite form that is able to infect red blood cells. It is only after release back into the blood stream as a merozoite that the parasite invades red blood cells, leading to the manifestation of disease.

The liver stage is clinically silent, yet an obligatory stage of the Plasmodium life cycle. Our knowledge of this portion of the life cycle is lagging compared to that of the blood stage because of inherent difficulties in experimental design. In particular, very little is known about the host and parasite gene expression during the early hours of infection. This work seeks to gain a greater understanding of the biological processes of host and parasite throughout the liver stage of infection through dual-RNA sequencing. We first utilize next-generation sequencing to map the global transcriptional state of the P. berghei-infected hepatocytes during the entire course of the liver stage infection. We find the most significant changes in gene expression occur early during infection and are primarily related to the host mounting an immune response. During mid to late time points of P. berghei infection of hepatocytes, genes related to host metabolism are enhanced among the differentially expressed genes, indicating a shift in active cellular processes later in infection.

From the host transcriptomic dataset, we identify aquaporin-3 (AQP3), a water and glycerol transporting membrane protein, as significantly induced upon P. berghei infection. Microscopic experiments reveal that the host AQP3 protein is trafficked to the parasitophorous vacuole membrane (PVM), the interface between the parasite and host cytosol. Through molecular genetic and chemical approaches, we show host AQP3 is essential for the proper development of the parasite during the liver and blood stages of the life cycle. Phenotypic studies suggest AQP3 is utilized by the parasite to obtain nutrients for growth.

Lastly, we also utilize target-based screens to identify novel antiplasmodial small molecules that have potential for treating liver stage malaria. We interrogate the species specificity of a panel of Hsp90 small molecules inhibitors and seek to understand the chemical moieties that determine species selectivity. We also utilize cell-based assays to screen for and identify compounds that act synergistically. The work presented herein sheds light on novel host-parasite interactions during the liver stage of Plasmodium infection and explores novel small molecules for malaria treatment.

Plasmodium is the causal agent of malaria, which is a parasitic disease that affects more than 215 million people annually and is endemic in 91 countries worldwide. Unfortunately, the parasites have developed resistance to all current pharmaceuticals used to treat malaria, including the front-line treatments artemisinin and artemisinin combination therapies. Due to the rapidly increasing drug resistance problem, new multi-stage inhibitors of Plasmodium are desirable. Of particular interest as multi-stage drug targets are parasite kinases since they are essential regulators in signaling, cell cycle control, and metabolism. Additionally, kinases play important roles in disease states, including cancer, heart disease, and neurodegenerative disorders. This has encouraged the work described here, which focuses on characterizing the atypical protein kinase 9 (PK9), protein kinase 5 (PK5), and shikimate kinase (SK) in Plasmodium with biochemical and chemical methods.

Specifically, target-based screening with the atypical P. falciparum PK9 revealed that benzimidazole and aminoquinoline compounds are able to bind the parasite kinase with low µM Kd(app) values. Furthermore, the top screening hit, takinib, was able to reduce parasite load in a dose-dependent manner. Takinib is the first reported binder of PfPK9 and was found to increase liver stage parasite size during later stages of infection. This unique phenotype may be the result of takinib influencing nutrient acquisition by the parasite or by modulating a cell-cycle control pathway. Takinib was also found to inhibit the human TAK1 (HsTAK1) kinase, which phosphorylates UBC13 and is involved in K63-linked ubiquitination pathways in the host. PfPK9 phosphorylates a parasite UBC13 and this work supports modulation of K63-linked ubiquitination in live parasites by takinib, suggesting similar functionalities between PfPK9 and HsTAK1.

To identify a more parasite-selective probe, 15 takinib analogs were evaluated for binding to PfPK9. HS220 was identified as an analog with the ability to bind to PfPK9, but without activity against HsTAK1. HS220 was confirmed to increase liver stage parasite size and decrease K63-linked ubiquitin on several parasite proteins, suggesting both takinib and HS220 have the same cellular target. The identification of the K63-linked ubiquitin targets will be essential to elucidating the downstream members of the PfPK9 signaling cascade. Future studies to further optimize a cellular thermal shift assay coupled with mass spectrometry may confirm on-target binding of takinib and HS220 in Plasmodium parasites. Finally, a model of PfPK9 was generated to guide hypotheses about takinib-binding and enable structural comparison with HsTAK1.

Malaria is a disease that poses a significant global health burden and is caused by the apicomplexan parasite Plasmodium. Plasmodium spp. are transmitted by an Anopheles mosquito and require both host and vector to complete their life cycle. Once in the host, parasites travel to the liver and undergo an obligatory and asymptomatic liver stage. This elusive stage is necessary to produce blood-infective parasites, to which the cyclical blood stage causes symptomatic disease. Understanding the liver stage of the Plasmodium life cycle is crucial for prevention of malaria but due to the technical challenges and asymptomatic nature of this stage, little is currently known about this stage. Crucially, during the liver stage of some Plasmodium spp., like the human-infective P. vivax, have the ability to form dormant parasites, termed hypnozoites, that can persist for weeks, months and even years and cause relapses in symptomatic infection. Elucidating host-parasite interactions during the liver stage, especially in these dormant hypnozoites, will be crucial for development of new therapeutics.To interrogate parasite dynamics within the host hepatocyte we conducted an RNA sequencing analysis throughout the P. berghei liver stage, covering as early as 2 hours post infection (hpi) and extending to 48 hpi. Our data revealed that hundreds of genes are differentially expressed at 2 hpi and multiple genes known to be important for later infection are upregulated as early as 12 hpi. Further, using hierarchical clustering along with coexpression analysis, we identified clusters functionally enriched for key liver stage processes and some of these clusters were highly correlated to the expression of ApiAP2 transcription factors, containing mainly uncharacterized DNA binding motifs. Beyond transcriptional profiling, host cellular structure has been shown to be altered during P. berghei infection, but this has not yet been investigated during P. vivax liver stage infection. While utilizing high-resolution microscopy to investigate parasite dynamics during the liver stage, we also characterized temporal changes of the P. vivax liver stage tubovesicular network (TVN). Our results highlight that host-parasite interactions occur in both dormant and replicating liver stage P. vivax forms and implicate a function for AQP3 in both forms. To elucidate host factors that attribute to Plasmodium liver stage we utilized genetic and chemical approaches to uncover potential mechanisms of various host factors, in particular host AQP3. We demonstrate AQP3 localizes to other apicomplexans, T. gondii and C. parvum. Investigating transcriptional regulation of host genes during infection we demonstrate preliminary evidence of two potential host transcription factors that regulate other host factors shown to be important in Plasmodium liver stage. Overall, these studies investigate host-parasite dynamics in Plasmodium liver stage though transcriptomic, high-resolution microscopy, and chemical and genetic approaches.

The apicomplexan parasite Toxoplasma gondii is one of the most highly distributed parasites on earth. Despite this, very little is known about the chemical mechanisms utilized by the parasite during the stages of its complex life cycle. Although protists have not traditionally been studied as natural product producing organisms, T. gondii possess multiple putative polyketide synthase (PKS) biosynthetic gene clusters (BGCs), indicating their potential to be a unique source of undiscovered secondary metabolites. In other infectious agents like bacteria and fungi, well-established culture conditions, tractable genomes, and often predictable bioinformatics have made these organisms the standard systems for natural product discovery. Research in this area has demonstrated that such pathogens use their metabolome to shape their environment and enhance survival through the production of bioactive natural products, many of which have been utilized as, or have inspired, therapeutic compounds. PKS biosynthetic gene clusters illustrate how microorganisms can utilize simple building blocks from primary metabolism to assemble complex scaffolds with evolutionary advantageous activities. Despite the prolific nature of apicomplexan parasites and the presence of PKS biosynthetic gene clusters in several members of this phylum, there have been no characterized enzymes or natural product secondary metabolites from apicomplexan sources; leaving a potentially rich area unexplored. To address this gap of knowledge, we aimed to investigate both the biosynthetic proteins and the resultant polyketide metabolites from the model apicomplexan T. gondii. To this end, Chapters 2 and 3 describes our sequencing and bioinformatic analyses of TgPKS2, which resolves the complete sequence and domain architecture of this megaenzyme. Additionally, these chapters describe our work to biochemically characterize individual domains from these proteins, as well as our work towards the reconstitution of full protein modules. Both studies further strive to illuminate structural components of the polyketide metabolite from TgPKS2. To expand beyond TgPKS2, Chapter 4 focuses on the second T. gondii PKS, TgPKS1 and its unique fatty acid loading mechanism, and represents the first metabolic study investigating metabolite production in a heterologously expressed PKS from an apicomplexan parasite. Finally, Chapter 5 highlights our investigations into the biological role of theses gene clusters in T. gondii and apicomplexan parasites, confirming the upregulation of TgPKS2 in the bradyzoite cysts forming stage of T. gondii development. To complement this, we describe our efforts towards generation of a CRISPR mediated TgPKS2 knockout strain of T. gondii and optimization of mass spectrometry conditions for differential metabolic analysis of these T. gondii life stages. In all, this work has made significant progress in our understanding of polyketide biosynthesis in T. gondii and has laid the groundwork for future biochemical and metabolomic studies into natural product biosynthesis in this phylum of pathogenic organisms.

Across the tree of life, specialized metabolites mediate ecological interactions and can ultimately drive evolution. Characterization of these small molecules have led scientists to a greater comprehension of ecological niches at the macro- and micro-scale. A group of medicinally important organisms with life cycles that include various ecological niches are apicomplexan parasites. The most notorious parasites belong to the genera Plasmodium and Toxoplasma, which are responsible for malaria and toxoplasmosis, respectively. Traditional efforts to reduce malaria include pharmaceuticals and prevention of mosquito-based transmission through insecticides and bed nets. Despite this, malaria has prevailed. Efforts were devised to understand the chemical ecology surrounding malaria parasites during the mosquito stage to ultimately reduce transmission. Through structure-, bioinformatic-, and coculture-guided approaches, we have uncovered chemical space within the mosquito-microbiome and evaluated how microbial-produced small molecules influence the parasite during its vector stage. We have also expanded our knowledge of parasite-produce specialized metabolites. Within Toxoplasma parasites, we have begun to characterize a polyketide synthase (PKS) with unknown resultant products. Together, this work provides the basis for understanding how specialized metabolites within the mosquito microbiome affect the Plasmodium parasite transmission capacity while also investigating an underexplored area of natural product chemistry within Toxoplasma parasites.

Parasitic diseases caused by pathogens of the Apicomplexan phylum result in hundreds of thousands of deaths per year, in addition to being an immense socioeconomic burden on vulnerable populations. A notorious case is that of Plasmodium parasites, the causative agents of malaria and one of the most ancient and devastating diseases known to humankind. Prior to infecting red blood cells resulting in the symptomatic stage of infection, Plasmodium parasites infect liver cells and undergo one of the most rapid replication events known in eukaryotes. Given the asymptomatic nature and technical challenges associated with studying the liver stage, this portion of the Plasmodium life cycle remains poorly understood, hindering our ability to target this stage of the life cycle for disease prevention. In particular, the host and parasite pathways that are critical to parasite infection during this hepatic phase remain unknown. The lack of effective vaccines coupled with the widespread emergence of drug-resistant parasites necessitates our understanding of host-parasite infection biology to develop improved therapeutics. To elucidate host-parasite interactions in the Plasmodium liver stage, we implemented a forward genetic screen to identify host factors within the human druggable genome that are critical to P. berghei infection in hepatoma cells, as well as RNA-seq approaches to delineate host and Plasmodium gene expression regulation during infection. Through our genetic screen, we identified the knockdown of genes involved in host trafficking pathways to be detrimental to Plasmodium infection. We additionally pursued mechanistic studies using small molecules and imaging approaches and found that both P. berghei and the related apicomplexan parasite Toxoplasma gondii hijack host trafficking by rerouting host vesicles to their parasitophorous vacuole, although with differing specificities. Our extensive RNA-Seq analysis throughout the P. berghei liver stage revealed that hundreds of parasite genes, including some coding for putative exported proteins, are differentially expressed as early as 2 hpi and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using co-expression analyses, we examined potential regulation of gene clusters by ApiAP2 transcription factors and found enrichment of mostly uncharacterized DNA binding motifs. This finding indicates potential liver-stage targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. We further explored regulatory mechanisms in the liver stage by identifying differentially expressed host lncRNAs in P. berghei-infected cells, and novel putative lncRNAs in P. vivax hypnozoites. Overall, our work uncovered critical host and parasite pathways in the Plasmodium liver stage and highlights the use of high-throughput genetic and transcriptomic approaches in combination with chemical biology and classic cell biology studies to uncover host-parasite interactions in challenging infection systems.

Malaria remains a major public health challenge that causes 219 million cases and 435,000 deaths in 2017. During their complex life cycle, Plasmodium parasites (the causative agents of malaria) encounter different cellular stresses due to the changes in the microenvironment, host immune responses and cellular metabolism during rapid parasite growth and expansion. Understanding how Plasmodium reacts to the stresses will provide an opportunity to better control malaria.

Current evidence shows that phosphatidylinositol 3-phosphate (PI(3)P) levels in Plasmodium falciparum correlate with tolerance to cellular stresses caused by artemisinin, a first-line malaria treatment, and environmental factors. However, the functional role of PI(3)P in the Plasmodium stress response and a possible mechanism of protection were unknown. In Chapters 2 and 3, we used multiple chemical probes including PI3K inhibitors and known antimalarial drugs to examine the importance of PI(3)P under thermal conditions that recapitulate malaria fever. Live cell microscopy using both chemical and genetic reporters revealed that PI(3)P stabilizes the acidic and proteolytic digestive vacuole (DV) under heat stress. We demonstrate that heat-induced DV destabilization in PI(3)P-deficient P. falciparum precedes cell death and is reversible after withdrawal of the stress condition and the PI3K inhibitor. These phenotypes are not observed with an inactive structural analog of the PI3K inhibitor. A chemoproteomic and biochemical approach identified PfHsp70-1 as a parasite PI(3)P-binding protein. Targeting PfHsp70-1 with a small molecule inhibitor phenocopied PI(3)P-deficient parasites under heat shock. Furthermore, tunable knockdown of PfHsp70-1 showed that PfHsp70-1 downregulation causes DV destabilization and hypersensitizes parasites to heat shock and PI3K inhibitors. Our findings underscore a mechanistic link between PI(3)P and PfHsp70-1, and present a novel PI(3)P function in stabilizing the DV compartment during heat stress.

In addition to PI(3)P and Hsp70s, parasite’s tolerance against artemisinin also correlates with the expression of the Plasmodium TCP-1 ring complex or chaperonin containing TCP-1 (TRiC/CCT), an essential hetero-oligomeric complex required for de novo cytoskeletal protein folding. In Chapter 4 to 6, we found that the P. falciparum TRiC can be targeted by the antihistamine clemastine by utilizing parallel chemoproteomic platforms. Clemastine destabilized all eight P. falciparum TRiC subunits based on thermal proteome profiling (TPP). Further analysis using stability of proteins from rates of oxidation (SPROX) revealed a clemastine-induced thermodynamic stabilization of the Plasmodium TRiC delta subunit, suggesting an interaction with this protein subunit. We demonstrate that clemastine reduces levels of the major TRiC substrate tubulin in P. falciparum parasites. In addition, clemastine treatment leads to disorientation of Plasmodium mitotic spindles during the asexual reproduction and results in aberrant tubulin morphology suggesting protein aggregation. This clemastine-induced disruption of TRiC function is not observed in human host cells, demonstrating a species selectivity required for targeting an intracellular human pathogen. Our findings encourage larger efforts to apply chemoproteomic methods to assist in target identification of antimalarial drugs, and highlight the potential to selectively target Plasmodium TRiC-mediated protein folding for malaria intervention.