Citrobacter rodentium is a gut pathogen, which infects the distal colon of mice. It has many similarities to human Enteropathogenic and Enterohemorrhagic E.coli in terms of mechanisms of pathogenicity and methods of transmission. Like many other gram negative bacteria, C. rodentium has developed a complex and highly specialised protein secretion system, known as type three (T3SS), to deliver bacterial proteins into eukaryotic cells. By injecting effector proteins into host cell cytoplasm, the pathogens are able to modulate host cellular functions to facilitate their own survival and replication. There is growing evidence that Attaching Effacing (AE) pathogens can inject effector proteins into gut epithelial cells, which dampen pro-inflammatory responses. There is also evidence that EPEC, Yersinia and Shigella can inject effectors into immune cells and also modulate their function. The objective of this work was to visualise and identify the host cells targeted for type III secretion by C. rodentium, and consequently determine the effect on host immune responses. The method chosen to detect cells targeted for effector protein delivery was the β-lactamase reporter system, where cells loaded with the fluorogenic substrate CCF2-AM emit a green FRET signal upon excitation by UV light, but emit a blue signal when cleaved by β-lactamase. By creating reporter strain of C.rodentium expressing fusion proteins between NleD effector and β-lactamase, I was able to show that C.rodentium is capable of injecting NleD in a wide variety of murine cell lines including Swiss 3T3 fibroblasts, J774 macrophages, CMT93 epithelial cells and BW715 T cells in a dose and time dependent manner in vitro. In addition, I found that C.rodentium has the ability to inject proteins into the cytoplasm of immune cells isolated from mouse lymphoid tissues including the spleen, mesenteric lymph nodes and Peyer's patches. Detailed analysis of the types of cells injected with effectors in vitro showed that NleD- injected cells represented B cells, dendritic cells and T cells. After inoculation of mice with the reporter strain of CitropACYCnleD, the plasmid encoded reporter fusion remained stable throughout infection and was able to inject cells in vitro after passage through the mouse gut. Unfortunately under the conditions described in this study, we were unable to visualise any gut cells targeted for protein delivery by C. rodentium in vivo, thus highlighting the complex nature of the host pathogen relationships in the gut. Although there is a need to develop better strategies to visualise effector translocation in vivo, our study has demonstrated, for the first time, the ability of C. rodentium to target immune cells for effector injection in vitro.

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

Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

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.

Comparative analysis of multiple genomes presents many challenges ranging from management of information about thousands of local similarities to definition of features by combination of evidence from multiple analyses and experiments. This research represents the development stage of a database-backed pipeline for comparative analysis of multiple genomes. The genomes of three recently sequenced species of Brucella were compared and a superset of known and hypothetical coding sequences was identified to be used in design of a discriminatory genomic cDNA array for comparative functional genomics experiments. Comparisons were made of coding regions from the public, annotated sequence of B. melitensis (GenBank) to the annotated sequence of B. suis (TIGR) and to the newly-sequenced B. abortus (personal communication, S. Halling, National Animal Disease Center, USDA). A systematic approach to analysis of multiple genome sequences is described including a data model for storage of defined features is presented along with necessary descriptive information such as input parameters and scores from the methods used to define features. A collection of adjacency relationships between features is also stored, creating a unified database that can be mined for patterns of features which repeat among or within genomes. The biological utility of the data model was demonstrated by a detailed analysis of the multiple genome comparison used to create the sample data set. This examination of genetic differences between three Brucella species with different virulence patterns and host preferences enabled investigation of the genomic basis of virulence. In the B. suis genome, seventy-one differentiating genes were found, including a contiguous 17.6 kb region unique to the species. Although only one unique species-specific gene was identified in the B. melitensis genome and none in the B. abortus genome, seventy-nine differentiating genes were found to be present in only two of the three Brucella species. These differentiating features may be significant in explaining differences in virulence or host specificity. RT-PCR analysis was performed to determine whether these genes are transcribed in vitro. Detailed comparisons were performed on a putative B. suis pathogenicity island (PAI). An overview of these genomic differences and discussion of their significance in the context of host preference and virulence is presented. / Master of Science

Bovine viral diarrhea virus (BVDV) is a pestivirus that suppresses the innate and adaptive host immune responses. Each of the two classified genotypes (BVDV1 and BVDV2) has two distinct biotypes – cytopathic (cp) and non-cytopathic (ncp) – and evidence has suggested that cytopathic strains may disrupt host interferon (IFN) synthesis and IFN-mediated responses. However, inconsistent results examining ncpBVDV strains have generated controversy regarding whether they also exhibit this capability. The purpose for this study was to determine the occurrence and functionality of IFN-induced responses within the serum cattle infected with ncpBVDV2-1373. Specifically, this involved analysing the changes in both the serum levels of IFN-α and IFN-γ and the expression of genes that are classically regulated by these cytokines. Serum analysis showed that the infected cattle induced both serum IFN-α and IFN-γ during BVDV infection while PBMC analysis showed increased expression of genes that classically respond to IFN-α – Mx-1, OAS-1, and STAT-1 – and IFN-γ – SOCS-1 and SOCS-3. These findings are supported by temporal kinome analysis, which verified activation of the JAK-STAT signalling network within the PBMCs of the virus-infected animals. In addition to establishing evidence for its synthesis, results from this challenge identified IFN-γ as a possible indicator of animal mortality as analysis of its change within the non-surviving, infected animals was statistically greater than the levels of the surviving, infected animals. Collectively, these results demonstrate 1373-mediated induction of, and host cell response to, both IFN-α and IFN–γ, and the potential for IFN-γ to be a predictive marker for mortality during BVDV infection.

<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

Drosophila melanogaster has been extensively used as a model organism to study diverse facets of biology, including host-pathogen interactions and the basic biology of its pathogens. I have used the fruit fly as a model to study elementary aspects of Nora virus biology, such as the role of the different proteins encoded by the virus genome. Nora virus, an enteric virus transmitted via the feca-oral route, does not cause any obvious pathology in the fly, although the infection is persistent. Nora virus genome consists of a positive strand RNA that is translated in four open reading frames (ORF).  Since sequence homology studies did not yield much information about the different Nora virus proteins, I have used the cDNA clone of the virus to construct mutants to identify the specific function of each protein. My results have shown that, 1) The protein(s) encoded by ORF 1 are crucial for the replication of the virus genome. 2) The C-terminus of the ORF 1-encoded protein (VP1), is an inhibitor to the RNAi pathway. 3) The transmembrane domain in the N-terminus of the ORF2-encoded protein (VP2) is important for the formation of Nora virus virions. 4) The ORF 3-encoded protein (VP3) forms α-helical trimers and this protein is essential for the stability of Nora virus capsid.                                                     I have also performed RNA sequencing to investigate the transcriptional response of D. melanogaster in response to Nora virus infection and my results indicate that,                        5) The upregulation of genes related to cellular stress and protein synthesis and the downregulation of basal digestive machinery, together with the induction of upd3, implies major gut epithelium damage and subsequent regeneration.