Malaria is caused by protozoan parasites of the genus Plasmodium. Among the five species that infect humans, P. falciparum is most lethal, though its range is restricted to the tropics. P. vivax, responsible for up to 40% of disease episodes, can be found in temperate zones as well and is a serious cause of morbidity and reduced economic well-being. Cumulatively, parasite species precipitate in excess of half a billion malaria episodes each year, a million fatal.

The lifecycle of Plasmodium parasites is complex, encompassing multiple sexual and asexual stages specialized to inhabit a preferred tissue-type in host or vector. Disease symptoms and pathology, however, stem from the asexual intraerythrocytic cycle, during which parasites repeatedly invade and proliferate within red blood cells; these blood-stage parasites are the target of antimalarial chemotherapy, and the development during the 20th century of novel antimalarial drugs reduced the proportion of the world's population at risk of malaria from over 70% in 1900 to 40% today. However, acquisition of resistance to control measures has led to a recent increase in disease incidence and nearly complete loss of efficacy of the former frontline antimalarial chloroquine.

While pathogenic resistance to chemotherapeutics is often conferred by the mutation of their target proteins, chloroquine's toxic effect to Plasmodium parasites stems from its impairment of an essential but immutable process, that of the crystallization of host-derived heme liberated from hemoglobin during amino acid scavenging. In P. falciparum, resistance to the drug arises from selection of mutations in the P. falciparum chloroquine resistance transporter (PFCRT), but other Plasmodium parasites exhibit independent modes of chloroquine resistance acquisition. Plasmodium's genetic intractability precludes comprehensive in vivo examination of the mechanics of chloroquine resistance. We have expressed PFCRT in S. cerevisiae for chemical-genetic profiling in the presence and absence of chloroquine and PfCRT; we have also investigated yeast's short-term transcriptional response to chloroquine treatment. We find that chloroquine impairs yeast vacuolar homeostasis, protein trafficking, and ability to sense nutritional repletion, and that expression of chloroquine-resistant PFCRT can partially suppress these defects.

This graduate work consists of projects concerned with functions of RNA, use of high throughput sequencing technology, and the molecular biology of the malaria parasite Plasmodium falciparum. The intricacies of recognition of mRNA molecules by yeast transport machinery were explored in an unbiased manner, leading to identification of both primary sequence and secondary structural motifs. High throughput sequencing was used to recover a new type of bornavirus associated with proventricular dilatation disease in psittacine birds. High throughput sequencing was again used to find leads as to the genetic determinant of artemisinin resistance in Plasmodium falciparum, finding an amplification on chromosome 10 as the most promising lead. Parasite response to artemisinin was also studied at the transcriptional level using microarrays to document the entry into and recovery from the ring-like dormant state induced in Plasmodium falciparum by artemisinin. Finally, splicing in Plasmodium falciparum was investigated by sequencing mRNA from four timepoints in the blood stages of the parasite. Specific software was written to extract all exon-exon junctions within that dataset and higher order analysis revealed previously unknown splice sites, hundreds of alternative splicing events, and the presence of spliced antisense RNAs in the transcriptome.

The recent advances in genomic sequencing technologies in the past decade have enabled the unprecedented ability to investigate infectious diseases and organisms that were traditionally difficult to study in the laboratory. In particular, the human malaria parasite, Plasmodium falciparum, is a unique challenge because of the difficulty to grow these obligate intracellular parasites during the complex blood stages. Yet despite these challenges, genomic approaches have allowed us to investigate the parasite in an unbiased and comprehensive manner. The work described here uses DNA sequencing, RNA sequencing, high-throughput screening, and bioinformatics tools to understand the fundamental biological processes of the malaria parasite such as mechanisms of drug resistance, regulation of translation, and determinants of translational efficiency. A mechanistic understanding of how the parasite can escape antimalarial drug pressure is valuable for future drug development and the design of new drugs that avoid known parasite resistance mechanisms. To this end, we have developed a bioinformatics pipeline that will identify mutations such as copy number variations or single nucleotide polymorphisms that confer resistance to various drugs in vitro. The process of translation in malaria is poorly understood as the technologies for measuring translation or protein products are limited to measuring only the most abundant proteins. To address this problem, we took a genome-wide approach of ribosome profiling which quantitatively measures transcription and translation simultaneously for the entire genome. This study led to many interesting questions revolving around how the P. falciparum ribosome functions differently from other eukaryotic ribosomes and what cis-acting sequences determine the efficiency of translation. To answer these questions, we developed an in vitro translation system derived from cell free lysates of P. falciparum to both screen for drug compounds that specifically inhibit malaria translation and not other eukaryotic translation and we have employed this assay to find specific sequences found primarily on the 5’ untranslated region of an mRNA that can modulate translation. In total, this work addresses the regulation of gene expression in the asexual blood stages of this medically relevant parasite.

Infectious diseases are a leading cause of morbidity and mortality worldwide. Despite significant advancement in our understanding of infectious disease biology, existing microbiologic diagnostic tests often fail to identify etiologic pathogens in cases of suspected infection. Metagenomic next-generation sequencing (mNGS) offers the potential for a universal pathogen detection method, but analysis and interpretation of findings are challenging. This is especially true for lower respiratory tract infections (LRTIs) where mNGS data interpretation is complicated by the existence of a respiratory microbiome composed of pathobionts present in both health and disease.

To address the need for improved LRTI diagnostics, we first compared two fluid types commonly used for diagnosis of LRTI, showing that despite moderate microbiome differences, both mini-bronchioalveolar lavage (mBAL) and tracheal aspirate (TA) samples are suitable for identification of pathogens in the context of an infection. Then, we evaluated the utility of mNGS as a diagnostic for LRTI in a cohort of 92 TA samples from adults with acute respiratory failure. We developed methods for sifting putative pathogens from commensal microbiota as well as pathogen, microbiome diversity, and host gene expression metrics to identify LRTI-positive patients and differentiate them from critically ill controls with noninfectious acute respiratory illnesses. We applied the models developed for evaluation of LRTI status to several other cohorts and disease contexts to show their broad applicability.

The low sensitivity of existing clinical diagnostics results in an imperfect gold standard, complicating the development of mNGS-based biomarkers. We explored the impact of label noise on host gene expression classifiers and methods for circumventing the issue. First, we tested whether label-noise robust logistic regression approaches could improve classifier performance by enabling the use of a larger training set. Then, we tested whether variational autoencoders, an unsupervised dimensionality reduction approach, could generate novel insight from combined host and microbial mNGS data. Altogether, this work suggests that a single streamlined protocol offering an integrated genomic portrait of pathogen, microbiome, and host transcriptome may hold promise as a tool for diagnosis of infections and contextualization of patient response.

Plasmodium falciparum, the causative agent of the deadliest type of human malaria continues to be a major cause of morbidity and mortality in many parts of the world. Because of the difficult nature of genetic and biochemical manipulations in P. falciparum genomic approaches are an increasingly important tool for studying this organism. We applied genomic approaches to learn about three distinct aspects of P. falciparum biology: mRNA decay during the intraerythrocytic developmental cycle (IDC), the antimalarial mechanism of tetracycline antibiotics, and the relationship between chloroquine resistance and mutations in the P. falciparum chloroquine resistance transporter (PfCRT).

To study mRNA decay in P. falciparum, we used a genome-wide approach to characterize the decay profile of every gene in the genome during the IDC. We found that half-lives for all genes increased during the IDC, indicating that life-cycle is a major determinant of decay rate in P. falciparum. We also found that specific variations in decay patterns were superimposed upon the dominant trend of progressive half-life lengthening. These variations in decay pattern were frequently enriched for genes with specific cellular functions or processes, making gene function a secondary determinant of decay rate.

Next, we used genomic techniques to study the antimalarial mechanism of action of tetracycline antibiotics. Using DNA microarrays to characterize the transcriptome over two generations after treatment with doxycycline, we found that expression of apicoplast genes is specifically inhibited, even if the drug was removed after the first generation. This inhibition is likely caused by a disruption of apicoplast function as evidenced by a block in apicoplast genome replication, a lack of processing of an apicoplast-targeted protein, and failure to elongate and segregate during schizogony.

Lastly, to study chloroquine resistance mediated by mutations in PfCRT we used Saccharomyces cerevisiae as a heterologous system to begin characterizing the structure-function relationship of PfCRT. We used DNA microarrays to find yeast genes which respond in a dose-dependent manner to chloroquine. These genes will be a starting point towards building a platform for characterizing resistance profiles of new 4-amino-quinoline drugs.

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