Thesis (Ph. D.)--Case Western Reserve University, 2009. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references.

This thesis reports investigations of the interactions between the intestinal epithelial barrier and the intracellular apicomplexan Eimeria spp., both in vivo and in vitro. Initially, conventional in vivo studies using genetically modified animals were used to investigate the contribution of innate lymphoid cells (ILCs) to immune protection of the intestinal barrier. Additionally, to understand complex epithelial host-pathogen interactions a novel in vitro model of small intestine organoids was developed. Data suggest that immunoprotection against Eimeria vermiformis infections is mediated by T cells. Furthermore, there is an indication that ILCs have a detrimental effect in Eimeria vermiformis-infected immunocompromised animals. However, the role for ILCs in the regulation of the immune response remains unclear. The life cycles of Eimeria vermiformis and Eimeria falciformis are highly complex, comprising multiple schizogonies followed by a gametogony. In vitro life cycle completion has not been achieved to date due to the limitations of monolayer cell line models. It is likely that for a successful parasite development the interaction of the different epithelial cell types present in intestinal organoids is required. The development of intestinal organoids by Sato and colleagues gave rise to a breakthrough in cellular studies, providing the tools to study complex interactions between host tissues and invading pathogens in vitro. I showed that small intestine-derived organoids grow exponentially after passage and that each organoid contains distinct specialised epithelial cell types, such as Paneth, Goblet or enteroendocrine cells, suggesting that the organoid model closely resembles the native intestinal epithelium and that Eimeria spp. benefit from the three-dimensional structure and physiological characteristics of the organoid model. Intestinal organoids were infected with E. vermiformis or E. falciformis sporozoites. These completed several rounds of asexual replication but did not proceed to the final gametogony. Despite the need for the development of sensitive techniques applicable to three-dimensional cell culture models, these results indicate that intestine-derived organoids are a promising model to study host-parasite interactions at the intestinal epithelial barrier at the cellular and molecular levels.

Cellular metabolic and immune pathways can be acted upon by diverse molecular factors. Some examples include small molecules, regulatory proteins or RNAs, intermediary metabolites and hormones. These factors can also be introduced or induced by pathogens during infections. Indeed, it is known that complex interplay exists between metabolism and immunity. However, the ways in which these interactions occur, and the nature of the players are active subjects of research. Herein, three different studies are presented which investigate the roles of three distinct modulators of metabolism and/or immunity. Firstly, a natural product produced by a pathogenic fungus is shown to activate the aryl hydrocarbon receptor and induce the expression of xenobiotic metabolizing enzymes. Secondly, the modulation of lipid metabolism by an immunometabolic antiviral microRNA, microRNA-185, is deconvoluted using activity-based protein profiling (ABPP), transcriptomic and lipidomic analysis. This study also identifies a novel enzymatic target of microRNA-185 which can be targeted pharmacologically to reduce hepatitis C virus infectivity. Finally, a third study investigates the link between a poorly characterized enzyme, lysophospholipase-like 1 (LYPLAL1), and hepatic glucose metabolism using a specific activity-based probe. Overall, the work presented in this thesis makes use of various molecular and chemical biology methods to probe pathways which are acted upon by structurally diverse factors to improve our understanding of host-pathogen interactions and metabolism.

<p>Malaria, caused by the apicomplexan parasite Plasmodium, is a disease which affects up to 500 million people each year. Historically, malaria infection has been combated both through the control of its vector, the Anopheles mosquito, and use of a variety of drugs, such as quinine (1800s) and chloroquine (1900s). However, with the evolution of resistance to the majority of available anti-malarial drugs, current approaches have settled upon combinatorial therapies. The most effective of these currently are ACTs (Artemisinin Combination Therapies - Artemisinin derivatives combined with a number of other drugs). However reports of Artemisinin resistance are continuing to emerge, suggesting that new approaches and increased understanding of the Plasmodium parasite is required.</p><p> Beginning with the complete sequencing of Plasmodium falciparum genome and continuing with comprehensive profiling of both the parasite's proteome and transcriptome, various genomic approaches applied in the study of malaria have led to significant new insights into the underlying biology of this parasite. While these new findings have greatly increased our understanding of genetic regulation within the malaria parasite, they largely have not yet translated into new therapeutic approaches. For this reason, considerable attention has been paid to the study of human genetic disorders which convey resistance to malaria, in the hopes that elucidating the mechanisms behind these resistances might lead to increased understanding of the parasite's biology and thus novel therapeutic approaches.</p><p> Sickle cell (HbS) erythrocytes are well known to resist malaria infection. However, the molecular basis of this resistance, long been recognized as multifactorial, contains elements which remain poorly understood. Here we show that the dysregulated erythrocytic microRNA composition, present in both HbAS and HbSS erythrocytes, is a significant determinant of resistance against the malaria parasite Plasmodium falciparum. During the intraerythrocytic lifecycle of P. falciparum, a subset of erythrocyte microRNAs translocate into the parasite. Two microRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite mRNAs and, via impaired ribosomal loading, resulted in translational inhibition of the target mRNA. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical microRNA activity which may present a novel host defense strategy against complex eukaryotic pathogens. In addition, the formation of these chimeric transcripts even in normal host erythrocytes illustrates a unique parasitic post-transcriptional adaptation to the host-cell environment.</p> / Dissertation

Infectious diseases from novel viruses have become a major public health concern. Rapid identification of virus–host interactions can reveal mechanistic insights into infectious diseases and shed light on potential treatments. Current computational prediction methods for novel viruses are based mainly on protein sequences. However, it is not clear to what extent other important features, such as the symptoms caused by the viruses, could contribute to a predictor. Disease phenotypes (i.e., signs and symptoms) are readily accessible from clinical diagnosis and we hypothesize that they may act as a potential proxy and an additional source of information for the underlying molecular interactions between the pathogens and hosts. We developed DeepViral, a deep learning based method that predicts protein– protein interactions (PPI) between humans and viruses. Motivated by the potential utility of infectious disease phenotypes, we first embedded human proteins and viruses in a shared space using their associated phenotypes and functions, supported by formalized background knowledge from biomedical ontologies. By jointly learning from protein sequences and phenotype features, DeepViral significantly improves over existing sequence-based methods for intra- and inter-species PPI prediction. Lastly, we propose a novel experimental setup to realistically evaluate prediction methods for novel viruses.

Bacteria inhabit diverse environmental niches and consequently, must modulate their metabolism to adapt to stress. The nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (collectively referred to as (p)ppGpp) are essential for survival during nutrient starvation. (p)ppGpp is synthesized by the RelA-SpoT homologue (RSH) protein family and coordinates the control of cellular metabolism through its combined effect on over 50 proteins. While the role of (p)ppGpp has largely been associated with nutrient limitation, recent studies have shown that (p)ppGpp and related nucleotides have a previously underappreciated effect on different aspects of bacterial physiology, such as regulating bacterial interactions with its host. This thesis focuses on the coordination of virulence gene expression and evasion of host immunity by (p)ppGpp in Salmonella enterica serovar Typhimurium. In the first data chapter, I describe the role of (p)ppGpp in mediating bacterial resistance to killing by the human complement system. I identified that (p)ppGpp activates ppnN, a nucleotide metabolism associated enzyme, and the biosynthesis of lipopolysaccharide O-antigen to protect Salmonella from cell lysis by complement. The second data chapter compares and contrasts the stringent response of an invasive clinical isolate of Salmonella Typhimurium to a strain of Salmonella Typhimurium that causes acute gastroenteritis using RNA-sequencing. Critical analysis of our transcriptomics dataset showed that flagellar-based motility is differentially regulated by (p)ppGpp in the two strains of Salmonella. Together, these findings demonstrate that (p)ppGpp has significant functional roles beyond mediating adaptation to nutrient limitation. / Thesis / Doctor of Philosophy (PhD)

Salmonella enterica is a facultative intracellular pathogen capable of eliciting severe, systemic disease necessitating antibiotic intervention. Systemic infection is facilitated by intracellular replication within host immune cells, which is enabled by complex regulatory networks governed by two-component systems (TCSs). Intracellular-active TCSs sense antimicrobial chemical cues in the microenvironment and respond adaptively through transcriptional regulation to support intracellular survival. SsrA/SsrB and PhoQ/PhoP are two essential TCSs that elicit a robust defense against host immunity by regulating clusters of virulence genes and integrating novel targets to support regulon expansion and enhance pathogenicity. Metabolic adaptation is critical to bacterial survival and can initiate host-pathogen interactions that influence infection outcome. Further, mitigation of host immunity by manipulation of arginine metabolism has been documented in intracellular pathogens. Herein, I investigated TCS-mediated regulatory evolution pertaining to arginine metabolism, hypothesizing that adaptations to metabolic regulation might confer a fitness advantage to Salmonella replicating intracellularly. I explored intracellular regulation of de novo biosynthesis and extracellular import of arginine, establishing PhoP-mediated regulation of arginine transport. I determined that arginine transport contributes to bacterial fitness in macrophages and began to investigate the mechanism by which arginine importation enriches for intracellular replication. This work informs on evolutionary mechanisms that serve to enhance virulence in Salmonella and provides further insight into our understanding of the intracellular lifestyle of infection. / Thesis / Master of Science (MSc) / Salmonella enterica is an intestinal pathogen that survives within host immune cells and causes systemic disease. These bacteria replicate within antimicrobial cells by using sensory networks to detect harmful immune factors and respond adaptively by eliciting change in gene expression to defend against immune-based killing. The amino acid arginine is an important component of host immunity, as well as bacterial antimicrobial defenses; therefore, I hypothesized that bacterial metabolism might be adapted to the host immune cell environment in order to mitigate arginine-dependent antimicrobial activity. Here, I establish that arginine metabolism is controlled by intracellular-specific sensory networks, and demonstrate that this regulation is important for bacterial survival. This work provides evidence for the importance of this amino acid in Salmonella infection, which informs on our overall understanding of systemic disease.

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An important aspect of systems biology is the elucidation of the protein-protein interactions (PPIs) that control important biological processes within a cell and between organisms. In particular, at the cellular and molecular level, interactions between a pathogen and its host play a vital role in initiating infection and a successful pathogenesis. Despite recent successes in the advancement of the systems biology of model organisms to understand complex diseases, the analysis of infectious diseases at the systems-level has not received as much attention. Since pathogen related disease is responsible for millions of deaths and billions of dollars in damage to crops and livestock, understanding the mechanisms employed by pathogens to infect their hosts is critical in the development of new and effective therapeutic strategies. The research presented here is one of the first computational approaches to studying host-pathogen PPI networks. This dissertation has two main aims. First, we discuss analytical tools for studying host-pathogen networks to identify common pathways perturbed and manipulated by pathogens. We present the first global comparison of the host-pathogen PPI networks of 190 different pathogens and their interactions with human proteins. We also present the construction and analysis of three highly infectious human-bacterial PPI networks: <i>Bacillus anthracis</i>, <i>Francislla tularensis</i>, and <i>Yersinia pestis</i>. The second aim of the research presented here is the development of predictive models for identifying PPIs between host and pathogen proteins. We present two methods: (i) a domain-based approach that uses frequency of domain-pairs in intra-species PPIs, and (ii) a supervised machine learning method that is trained on known inter-species PPIs. The techniques developed in this dissertation, along with the informative datasets presented, will serve as a foundation for the field of computational pathosystems biology. / Ph. D.