Browsing by Subject "Malaria"

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.

Dengue viruses (DENV) and other mosquito-borne flaviviruses are rapidly emerging human pathogens that threaten nearly half of the world’s population. There is currently no effective vaccine or antiviral therapeutics for the prophylaxis or treatment of DENV. While traditional drug development efforts have focused on inhibitors of viral enzymes, an alternative approach is to target host proteins that support virus replication. In an effort to identify novel human enzymes important for the DENV-2 life-cycle, we conducted a genome-wide RNAi screen and identified ERI3, a putative 3′-5′ exonuclease, as a novel DENV-2 host factor. Cell-free assays confirmed that purified ERI3 is capable of degrading single-stranded RNA in a 3′ to 5′ direction. We conducted a screen for compounds that inhibit ERI3 in vitro and identified small molecules that antagonized both exonuclease activity. In summary, we identified a host exonuclease that is important for DENV-2 replication and is a potential therapeutic target. Our approach illustrates the utility of identifying host enzymatic functions for development of anti-viral drugs.

Large-scale molecular epidemiologic studies of Plasmodium falciparum parasites have provided insights into parasite biology and transmission, can identify the spread of drug resistance, and are useful in assessing vaccine targets. The polyclonal nature infections in high transmission settings is problematic for traditional genotyping approaches. Next-generation sequencing approaches to parasite genotyping allow sensitive detection of minority variants, disaggregation of complex parasite mixtures, and scalable processing of large samples sets. Therefore, we designed, validated, and applied to field parasites a new approach that leverages sequencing of individually barcoded samples in a multiplex manner. We utilize variant barcodes, invariant linker sequences and modular template-specific primers to allow for the simultaneous generation of high-dimensional sequencing data of multiple gene targets. This modularity permits a cost-effective and reproducible way to query many genes at once. In mixtures of reference parasite genomes, we quantitatively detected unique haplotypes comprising as little as 2% of a polyclonal infection. We applied this genotyping approach to field-collected parasites collected in Western Kenya in order to simultaneously obtain parasites genotypes at three unlinked loci. In summary, we present a rapid, scalable, and flexible method for genotyping individual parasites that enables molecular epidemiologic studies of parasite evolution, population structure and transmission.

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.

Background: Despite widespread access and use of insecticide-treated bed nets (ITN) in Bungoma County in Kenya, there has been little reduction in malaria infection rates. It has been hypothesized that this gap between theoretical and actual ITN efficacy is caused by improper use of ITNs, poor physical condition of ITNs, or insecticide resistance in local mosquitos. This study aims to examine potential factors that affect the efficacy of the ITNs in Western Kenya.

Methods: In order to assess the aforementioned aim, a longitudinal observational study was conducted. The study enrolled 9 households and performed weekly data and mosquito collections. Data and sample collection was conducted over an 8-week duration, from June 2016 to July 2016.

Results: The study found high ITN usage in the study households (99.3% coverage), a negative association between the number of mosquitoes collected and time, a high proportion of blood fed mosquitoes (0.409), and statistically significant associations with the proportion of blood fed mosquitos and twelve different predictor variables. Conclusion: This study shows that it is feasible to examine factors reducing ITN efficacy in the area and lays down a potential template to be scaled up to examine these factors more specifically.

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.

Human genetic variation can be leveraged to understand the subtleties of how common variants with small effect sizes can alter cellular phenotypes and ultimately affect susceptibility to pathogenic disease. By combining GWAS of different phenotypic scales and basic cell biology, we can answer how a particular SNP affects a disease. This body of work elucidates the biological mechanism of how a SNP in VAC14, which encodes a human scaffolding protein involved in phosphoinositide metabolism, alters susceptibility to Typhoid Fever and other pathogens.

Using Hi-HOST (High-throughput Human in vitro Susceptibility Testing), a GWAS platform for cellular host-pathogen traits, we discovered that the ‘A’ allele of rs8060947 was associated with decreased VAC14 protein expression and increased Salmonella Typhi invasion. We experimentally confirmed the phenotype using RNAi to transiently decrease VAC14 protein expression in LCLs and Helas and saw increased Salmonella Typhi invasion. Further studies, using genetic and pharmacological manipulations were able to determine how VAC14 affects Salmonella Typhi invasion. CRISPR knockout VAC14 cells had a robust increase in invasion, and had increased cholesterol accumulation in the cell. Salmonella preferentially docks to cholesterol on the host plasma membrane as one of the first steps involved in invasion. Thus, increasing cholesterol at the plasma membrane increased the number of docked bacteria and ultimately caused higher invasion percentages.

To confirm the relevance of cholesterol and Salmonella Typhi beyond cell culture, we infected the swim bladder of Zebrafish with S. Typhi. Fish were pretreated with Ezetimibe, an FDA approved cholesterol-reducing drug, and then subsequently infected with S. Typhi. Fish treated with Ezetimibe, had decreased cholesterol staining by filipin, and had increased survival from S. Typhi infections. Additionally, because of the optically transparent nature of the zebrafish embryo we were able to image the fish 24hrs after infection and show that ezetimibe treated fish had higher bacterial clearance.

In addition to the fish studies, a collaboration with Dr. Sarah Dunstan (University of Melbourne) was able to retrospectively determine that VAC14 had an effect on human susceptibility to typhoid fever. The ‘A’ allele for SNP rs8060947, which we showed had decreased VAC14 protein expression and increased S. Typhi invasion in cell culture, was found to be more common in people with typhoid fever, suggesting the ‘A’ allele increases human susceptibility to this disease. All together, we have shown that decreased VAC14 expression causes an increase in cellular cholesterol, leading to an increase in docking and invasion of Salmonella and ultimately increasing your chances of acquiring typhoid fever.

The central role of cholesterol in entry of multiple pathogens led us to hypothesize that natural variation or experimental manipulation of VAC14 expression could play a role in pathogens beyond Salmonella. Here we show that its effects extend beyond bacteria to parasites. With cholesterol regulating entry of Plasmodium into hepatocytes, we hypothesized that increasing the amount of cellular cholesterol in hepatocytes will increase Plasmodium entry. These ideas are being tested in collaboration with Maria Toro and Dr. Emily Derbyshire (Duke University). However, unpublished human genetic data already support the idea that VAC14 regulates susceptibility to malaria infection. The same SNP associated with Salmonella invasion (rs8060947) is associated with malaria risk in African populations (Gavin Band and the MalariaGEN Consortium, personal communication).

VAC14 may also affect pathogen entry through its role in regulation of endosomal trafficking. VAC14 forms a complex with the FIG4 phosphatase and PIKfyve kinase to modulate endosomal trafficking through the metabolism of PtdIns(3,5)P2. Recently, FIG4 and PIKfyve were found to be necessary for Ebola entry in a somatic cell genetic screen. Using our VAC14 CRISPR knockout cells we determined that cells mutated for VAC14 had a similar phenotype. Ebola virus-like-particle (VLP) entry decreased dramatically in cells lacking VAC14. While we discovered that VAC14 affects cellular cholesterol, its main reported function is to regulate endosomal trafficking. We hypothesize that lack of VAC14 interferes with proper endosomal maturation and thus prevents the Ebola VLP from reaching its intracellular receptor NPC1 and exiting into the cytoplasm.

The common allele (A) that alters VAC14 expression is associated with decreased protein synthesis, and increased susceptibility to both Salmonella and Malaria infection. On the other hand, decreased VAC14 expression inhibits proper endolysosomal trafficking, inhibiting Ebola infection. These two mechanisms of affecting different infectious diseases may provide opposing forces in an example of balancing selection.