Malaria, due to parasitic infection by Plasmodium protozoa, is a severe global disease with 300 million annual cases leading to 1-2 million deaths. While a few anti-malarial treatments continue to work, the continual emergence of new drug resistant parasites is an urgent public health concern. Previous studies in the Rathod laboratory suggest that chemical tools can be used to distinguish variations in evolutionary capabilities of different malaria parasite strains. Some strains such as Dd2 display the Accelerated Resistance to Multiple Drugs (ARMD) phenotype, whereas the HB3 strain displays the non-ARMD phenotype. In previous studies, Dd2 strain acquires resistance to several antimalarials at a significantly higher frequency than the HB3 strain. In this experiment, a cyclopropyl carboxamide (GSK2645947), a novel class of antimalarial agent, was used to select for resistant parasites from Dd2 and HB3 with the aim of comparing their relative frequencies of resistance to GSK2645947. A series of selection experiments were carried out utilizing different concentrations of GSK2645947 and different numbers of starting parasites. The parasites acquired resistance to GSK2645947 at similar frequencies in Dd2 and HB3 cells. The extent of resistance to GSK2645947, measured using EC50 assays, was similar in Dd2 and HB3 resistant clones; resistant parasites from neither lines showed cross-resistance to other antimalarial drugs. The present results point to a novel mechanism for rapidly acquiring resistance to GSK2645947, a mechanism that is equally prevalent in Dd2 and HB3 strains of P. falciparum parasites.

Pre-erythrocytic malaria vaccines may need to target numerous sporozoite (spz) and/or liver-stage proteins to be effective. If the protective antigens could be definitively identified, multi-component subunit vaccines could be produced. Using a novel high-throughput T cell screening system, we recently identified a CD8 response against the Plasmodium yoelii L3 ribosomal protein in sporozoite-immunized BALB/c mice. Unlike the CD8 T cell response against the circumsporozoite protein (CSP) that increases with each parasite exposure in our system, the L3-specific response is not boosted by repeated exposures to attenuated sporozoites. We have shown that L3 -specific cells have no cell intrinsic defects that counteract their re-expansion or function but rather that broad anti-sporozoite immune responses in secondary or later exposures eliminate expression of L3, thereby preventing any opportunity for activation of memory L3-specific CD8+ T cells. This T cell outcome following immunization may be emblematic of other T cells with liver-stage targets as well. Here, we tested the L3-specific T cell response in another species (P. berghei) and also employed heterologous (cross-species) prime-boost approaches. We explore the difference in parasite load and L3-specific CD8+ T cell responses between homologous and heterologous immunization regimens. We screened mice against 37 liver-stage proteins and found that cross-species immunization boosted more responses than homologous immunization. The identities of the specific targets are under investigation. Cross-species boosting may be useful as we work to identify antigens that promote an L3-like CD8+ T cell response for inclusion in a novel multivalent malaria vaccine.

Malaria is an infectious disease caused by various species of the Plasmodium parasite which affects an estimated 207 million individuals, resulting in approximately 627,000 deaths per year. While this disease is primarily prevalent in developing nations, over half of the world’s population is at risk for malaria. Among existing malaria drugs, primaquine is uniquely effective against late-stage Plasmodium gametocytes and liver hypnozoites; however, it is dangerous to patients who are G6PD deficient. G6PD deficiency is caused primarily by point mutations in the Glucose-6-Phosphate Dehydrogenase (G6PD) protein—an enzyme that catalyzes the formation of NAPDH (a reducing agent) in the Pentose Phosphate Pathway. This condition affects around 400,000 individuals worldwide, most of whom live in areas at risk for malaria. Screening for G6PD deficiency is not readily available in many of these at risk areas and thus complicates treatment of malaria with primaquine. Therefore, screening for compounds that stabilize mutant forms of G6PD may yield potential candidates that can be used in combination with primaquine for a new therapeutic regimen. In a screen of ~100,000 compounds, 71 compounds were found to have stabilizing effects on mutant G6PD R454H (Andalus). Screening was done with a standard biochemical assay that measured enzyme activity through the detection of reaction product, NADPH (detected by fluorescence). Additional biochemical assays and studies on these 71 hits will follow to assess the mechanism and extent to which these compounds increase mutant G6PD Andalus activity. Candidate compounds that make it through these follow up experiments will then be tested with other G6PD mutants in search of a stabilizer of multiple G6PD mutants.

Rotavirus is the main cause of viral diarrhea among young children. There exist licensed vaccines, but rotavirus still kills nearly half a million children yearly. Live, attenuated vaccines quickly lose potency at room temperature. My host lab is assisting a Seattle NGO callednPATH and the Bill and Melinda Gates Foundation with developing temperature-stabilized, inexpensive vaccines. The current industry standard assay for measuring vaccine potency is time-consuming, subjective, and low throughput. My goal is to develop an objective, inexpensive, automatable, and high-throughput assay of the potency of candidate, next-generation, temperature-stable rotavirus vaccines. To develop this assay, susceptible cells are infected with candidate vaccine-strain viruses manufactured in India and undergoing development at PATH. After cell membrane modification to allow antibody access to cytoplasmic viral proteins, an anti-rotavirus antibody and a fluorescent secondary antibody are used to detect viral protein within infected cells. Experimental samples undergo single-cell flow cytometry analysis of individual cells to differentiate infected and uninfected cells. The vaccine industry-standard potency assay is performed in parallel to directly compare assays. Comparison and titration of polyclonal and monoclonal rotavirus-specific antibodies identified the optimal antibody and concentration for specific intracellular recognition of rotavirus protein. Testing of secondary antibodies and fluorochromes resulted in cell fluorescence characteristics yielding a favorable signal-to-noise ratio without causing false-positive results. A direct relationship between the amount of live rotavirus input and the percentage of infected cells with good linearity and acceptable coefficients of variation was observed. Future work is focused on reducing material costs, comparing the flow cytometry assay to the industry standard, and adapting the assay to additional rotavirus vaccine strains. Based on the above results, flow cytometry is a promising candidate assay for quantifying rotavirus vaccine potency to assist the development of novel live rotavirus vaccine formulations and storage formats.

Trypanosoma cruzi is a protozoan parasite prevalent in South and Central America that causes life-long infection in humans. Approximately 30% of infected individuals develop a condition called Chagas disease which usually manifests as life-threatening cardiomyopathy or pathologies in the gastrointestinal system. Over 8 million people are believed to be infected. Unfortunately, current drugs for treating Chagas disease have low antiparasitic activity, are expensive, and are known to cause harmful side effects. As a result, research needs to focus on discovering new drug targets in Trypanosoma cruzi to help lead to improved drugs. Most eukaryotic organisms synthesize sterols such as cholesterol and ergosterol for essential biological functions that can lead to cell death if these sterols were made incorrectly. This research is investigating the effects of blocking the synthesis of ergosterol by deactivating an enzyme called sterol 14-demethylase. This enzyme catalyzes one of the intermediate of 20 steps of ergosterol synthesis. Inhibitors of sterol 14-demethylase, known as azoles, have been shown to be extremely active on T.cruzi in vitro and are now the center of research regarding anti T.cruzi drug discovery. Further evidence shows that blocking sterol 14-demethylase can lead to an accumulation of sterol intermediates that are converted into toxic agents in the presence of another enzyme, ERG3 enzyme, which is normally active in a later step in the biosynthesis of ergosterol. The objective of the research project I am presenting is to analyze the ERG3 homologs of T.cruzi in the context of the ergosterol synthesis. The putative homologs have been cloned and sequenced. The research now is focused on engineering genetic knockouts of the ERG3 genes to test the hypothesis that the knockout parasites may be resistant to treatment with azole drugs.

Trypanosoma brucei (T. brucei) is an ideal model system for the study of a diverse range of biological processes, a number of which are unique to the kinetoplastid pathogens. The ability to genetically manipulate these parasites allows us to investigate the molecular machinery and mechanisms underlying these major processes. We have focused specifically on two areas of study: first, the identification and functions of essential T. brucei kinases; second, the functions of a number of uncharacterized proteins that we predict are essential for precise RNA editing in the mitochondrion of T. brucei. We created conditional null cell lines in which both endogenous alleles of a gene of interest are eliminated and a tetracycline-regulatable copy is inserted. Through the presence or absence of tetracycline, we regulated gene expression and assessed the phenotypic effects. Using this method, we were able to identify a set of protein kinases essential for cell viability, potentially functioning in cell cycle regulation and in connection with other cellular processes. RNA editing is an essential process unique to kinetoplastids, in which multi-protein complexes called editosomes insert or delete uridine nucleotides to create functional mitochondrial mRNAs. The major components of editosomes have been identified, though our understanding of the functional and structural organization of these proteins is limited. This is especially true for a number of proteins with limited detectable homology to other known proteins or domains. By generating and analyzing conditional null cell lines, we will assess the functional and structural roles these proteins play within editosomes for precise RNA editing. We anticipate that these proteins are key to understanding why specific sites in mRNAs are edited by insertion, whilst others are edited by deletion of uridine residues, which is a major outstanding question in the field.

Francisella noatunensis is an emerging fish pathogen that causes disease and mortality for economically important fish such as tilapia and Atlantic cod. The Francisella Pathogenicity Island (FPI) is a region of the genome known to govern virulence for the genus, and the pathogenicity determinant protein A (pdpA) is one of the largest genes found in the FPI. A recent study by our laboratory using zebrafish as a model host indicated that pdpA is important for F. noatunensis to cause mortality in infected zebrafish—thus the pdpA protein appears to be essential for conferring virulence. However, our gene expression analyses on zebrafish innate immune response revealed that there was no significant difference between the response against wild type and the response against pdpA knockouts by day 7 post-infection. In order to better “visualize” the infection progress by these pathogens, I would like to simulate the replication history of F. noatunensis by generating a growth curve of the bacteria within the zebrafish cells. My goal is to compare the growth rate between the wild type and the pdpA knockouts to identify how their ability to replicate within the host is altered as a result of losing the pdpA gene. Zebrafish leukocytes isolated from kidneys and spleens will be used to perform an intracellular growth assay, where the zebrafish cells will be infected with wild type F. noatunensis, and the number of bacterial cells will be determined at multiple points in time to generate a standard growth curve of the pathogen. The same procedure will then be repeated with the pdpA knockouts to identify how their growth rate deviates from the standard curve. The results from this study will give us new insights into the functional role of pdpA and its effect on F. noatunensis fitness.

Tumor necrosis factor (TNF) is a key host factor in tuberculosis (TB). In the zebrafish model for TB, excess TNF triggers necrosis of infected macrophages which leads to exuberant extracellular bacterial growth and host susceptibility. In infected macrophages, excess TNF induces production of reactive oxygen species (ROS) which activate two death pathways one of which involves the mitochondrial transition pore, and the other leads to ceramide production and lysosomal permeabilization. In exploring the events downstream of ceramide production we found that the pro-apoptotic factor Bax is required for macrophage necrosis in excess TNF conditions. Bax has been well-characterized for its role during apoptosis, but this result suggested that Bax might also play a role in necrosis. To determine if this was the case, we engineered Bax mutants that lack its different functional domains. We studied the ability of each mutant protein to regulate apoptosis and necrosis in the zebrafish and found the BH3 domain, which is required for Bax oligomerization during apoptosis, not to be required for necrosis under excess TNF conditions. Rather, an C-terminal transmembrane helix was found to be required for necrosis. These findings demonstrate that the pro-apoptotic factor Bax also regulates necrosis but through a distinct functional domain. Since excess TNF-mediated necrosis has been implicated in human TB pathogenesis, this understanding of how Bax regulates necrosis may guide host-derived therapies.