Browsing by Subject "Plasmodium"

Increased attention has recently been placed on understanding the natural variation of the malaria parasite Plasmodium vivax across the globe, as in 2020 alone, P. vivax caused an estimated 4.5 million malaria cases and lead to over 600,000 deaths around the world. P. vivax infections in central Africa have been of particular interest, as humans in Sub-Saharan Africa frequently possess a P. vivax resistance allele known as the Duffy-negative phenotype that is believed to prevent infection in these individuals. However, new reports of asymptomatic and symptomatic infections in Duffy-negative individuals in Africa raise the possibility that P. vivax is evolving to evade host resistance.Whole genome sequencing has become more common as a means of understanding the population diversity of P. vivax. However, there is still a scarcity of information about P. vivax in central Africa. In this dissertation, I analyze whole genome sequencing data from a new P. vivax sample collected from the Democratic Republic of the Congo in central Africa. By studying P. vivax from central Africa, we can begin to understand the evolutionary history of the pathogen in this part of the world as it relates to the global context of this pathogen. I also investigate the relationship of P. vivax in the DRC with a potential animal reservoir of a closely related species, P. vivax-like, in non-human primates in this region. Due to the scarcity of P. vivax samples in central Africa, I also investigated methods with which to best make use of whole genome sequencing data, particularly in generating phylogenetic trees. While many studies of P. vivax genetic diversity employ whole genome variation data in order to study evolutionary relationships of P. vivax populations, in this dissertation I make use of the P. vivax apicoplast, a non-photosynthetic plastid organelle genome. The apicoplast genome is five times longer than the mitochondrial genome and does not undergo recombination, making it a valuable locus for studying P. vivax evolutionary history using phylogenetic trees.

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.

Hydroxyurea, a mainstay of sickle cell management in the developed world, offers a range of potential benefits to children with sickle cell disease. There is strong evidence that hydroxyurea induces production of fetal hemoglobin (HbF) in red blood cells, which is generally associated with reduced morbidity and fewer incidents of clinical events in sickle cell patients. Based on literature from in vitro investigations, it has also been suggested that hydroxyurea may confer some protection against malaria parasitemia by inhibiting proliferation of the malaria-causing parasite - Plasmodium falciparum.

The purpose of this study was to examine the effects hydroxyurea use on P. falciparum infection, parasite density, HbF and morbidity among children with sickle cell disease living in a malaria endemic setting. We analyzed baseline data of 95 children (aged 1 – 10 years) enrolled in the EPiTOMISE clinical trial (Enhancing Preventive Therapy of Malaria in children with Sickle cell anemia in East Africa) between January 2018 and September 2018.

Our analyses showed no significant difference in the prevalence of P. falciparum infection between hydroxyurea users and non-hydroxyurea users, prevalence ratio [95% CI] = 1.14 [0.76, 1.71]. Among infected children, median (IQR) log parasites densities were also similar between hydroxyurea users, -0.96 (-1.67, 0.41) and hydroxyurea non-users, -0.12 (-1.32, 3.48), p-value=0.146.

We did observe substantial hematological benefits among hydroxyurea users including an approximate 1 unit increase in median hemoglobin concentration and a 2.7-fold increase in median percentage HbF. However, this observation did not translate to any meaningful difference in the prevalence of morbidity events.

In agreement with the few studies on hydroxyurea use in malaria endemic settings, these results suggest that there may be no added risk of P. falciparum infection to sickle cell patients who routinely use hydroxyurea. Furthermore, our results reflect that hydroxyurea use is associated with increased HbF and a better hematological profile in this population. There is need for more research on hydroxyurea use in sub-Saharan Africa to help resolve any existing concerns and conflicting data in the current body of literature.

Malaria is an infectious disease caused by apicomplexan Plasmodium species. These protozoan parasites are transmitted by a mosquito vector to a human host, wherein they undergo an asymptomatic liver stage followed by a symptomatic blood stage of infection. Despite eradication efforts, Plasmodium remain accountable for hundreds of thousands of mortalities per year, mostly caused by P. falciparum. The spreading resistance to the front-line antimalarials that broadly disrupt parasite proteostasis demands further characterization of their adaptive stress responses and novel multi-stage drug targets. This work focuses on the essential P. falciparum molecular chaperone heat shock protein 90 (PfHsp90). Specifically, PfHsp90 is expected to directly interact with a subset of parasite proteins to facilitate their ATP-dependent maturation, stabilization, and regulation. Despite this critical function, the scope of its chaperoning interactions—as well as its consequent contributions to mitigate cellular stress and maintain parasite proteome integrity throughout development—remains largely unresolved. To enable its functional interrogation, we first aimed to establish chemical inhibitors of PfHsp90 with greater affinity to the parasite compared to the conserved human homolog (HsHsp90). In general, our testing supports the particular utility of compounds that bind at the chaperone’s nucleotide-binding domain, as opposed to a putative C-terminal allosteric site, based on their high affinity and resolved mode of ATP-competitive inhibition. From this class of competitive inhibitors, we identified XL888 as exhibiting moderate selectivity to PfHsp90, despite that it was initially developed as a HsHsp90 inhibitor. Subsequent structural evaluation indicated that the PfHsp90 lid subdomain contributes to the parasite chaperone’s higher affinity interaction with XL888’s tropane scaffold. Considering this molecular basis, we were able to develop Tropane 1 as a novel XL888 analog with nanomolar affinity and approximately 10-fold selectivity to PfHsp90, which further demonstrated dual-stage, anti-Plasmodium activity. We next surveyed the PfHsp90-dependent proteome using innovative chemical biology strategies. Based on their thermal stability after chaperone inhibition, we identified 50 candidates as putative PfHsp90 interactors. A significant enrichment of proteasome regulatory particle components was represented in this analysis, from which we subsequently validated that PfHsp90 chaperones the 26S proteasome to support the controlled recycling of cellular proteins. Additionally, we adopted bio-orthogonal labeling with unnatural amino acids to track proteome dynamics in Plasmodium parasites. To date, we have implemented this approach to support that compromised PfHsp90 activity coordinates translation attenuation as a stress response. However, this work sets the foundation to employ such labeling to quantify PfHsp90-coordinated proteome dynamics across multiple parasite life stages. Collectively, these findings broaden our understanding of PfHsp90’s regulation of Plasmodium parasite proteostasis and further establish the potential of this molecular chaperone as a novel, multi-stage antimalarial drug target.

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.