Browsing by Department "Molecular Genetics and Microbiology"

Current standard cancer treatments such as chemotherapeutics, and radiation therapy are nearly as likely to kill the patient as cure the cancer. Therapies that have such a narrow window of efficacy are necessary for the treatment of aggressive diseases, but safer alternatives must be created. By discovering novel therapeutics that target specific disease processes within specific diseased cells, while leaving healthy cells unaltered, we can improve the lives of millions of cancer sufferers and their families. A therapeutic's window of efficacy can be measured by the therapeutic index. For many anti-cancer therapeutics, the therapeutic index is very small, the dose of treatment that kills cancer cells and shrinks tumors is nearly the dose that causes toxicity. In cancer patients, this toxicity causes many serious conditions such as gastrointestinal distress, organ damage, and death.

Recently, the model of cancer treatment has evolved from non-specific cytotoxic agents to more selective therapeutics that target cellular processes necessary for cancer cell survival. If a therapy can be targeted to selectively bind and internalize targeted cells, its toxicity would only impact the targeted cells and healthy cells in the immediate vicinity, which would greatly reduce the toxic effects on the rest of the body. Targeting cancer cells can be done through cancer biomarkers, which are cell surface proteins, expressed exclusively, or are much more abundant on the surface of cancer cells than on somatic cells.

Advances in antibody and aptamer technology have enabled researchers to design those molecules to bind specifically to cancer cells, and deliver drugs that alter specific cellular processes. An aptamer designed to bind PSMA, a prostate cancer biomarker, only bound to a specific subset of cancer cells, and delivered a therapeutic siRNA that prevented a specific survival process from occurring. While this technology is promising, it is currently limited to targeting small subsets of cancer types. To generate an aptamer therapeutic that would have greater utility and efficacy, I have examined the properties of a nucleolin aptamer-mediated delivery system that targets multiple types of cancer cells, and delivers various oligonucleotide therapeutics.

The nucleolin aptamer targeted cancer cells by binding to membrane–associated nucleolin. Nucleolin, a conserved protein found in all eukaryotes, shuttles from the nucleus, through the cytoplasm to the cell membrane. Cancer cells express a far greater amount of membrane–associated nucleolin than somatic cells, making nucleolin an ideal cancer biomarker. The shuttling, and oligonucleotide binding attributes of the protein enable it to deliver aptamer chimeras from the cell surface to the nucleus. Therefore the nucleolin aptamer has unique access to the nuclei of cancer cells, and can deliver therapeutic oligonucleotide cargoes through nucleolin binding.

The nucleolin aptamer delivered splice–switching oligonucleotides, a form of antisense technology, improving their efficacy, and potentially increasing their therapeutic viability. The ability to deliver antisense oligonucleotides to the nuclei of cancer cells has the potential for other therapeutic possibilities including the inhibition of transcription with antisense triplexes.

The nucleolin aptamer can also deliver therapeutic aptamers. The nucleolin aptamer–β–arrestin aptamer chimera prevented the stem cell renewal phenotype necessary for leukemia progression in human patient tissue samples. The ability to effectively deliver therapeutic aptamers may lead to clinical applications for many of the aptamers that have been selected against intracellular targets including transcriptional activators.

Oligonucleotide research continues to advance our understanding of potentially therapeutic oligonucleotides. Long non–coding RNAs for example, may impact epigenetics, and transcription. Additionally, locked nucleic acids have been developed to improve binding affinity, thus increasing the efficacy of antisense oligonucleotides. In order to bring these discoveries into the clinic, they must be safely and specifically delivered to their target cells.

This work demonstrated that the nucleolin aptamer could deliver oligonucleotide therapeutics to specific cancer cells. Nucleolin aptamer chimeras have the potential to develop into safe and effective cancer therapies, thus improving the treatment options for cancer sufferers.

Cancer-specific cytopathogenicity of oncolytic viruses is often defined by viral

sensitivity to innate antiviral immune responses, e.g. type I Interferons (IFNs), limiting

cytotoxicity to cells lacking these responses. However, recent work suggests some cancer

cells inhibit IFN-sensitive oncolytic viruses, preventing efficacy. IFNs are also antiproliferative in cancer and activate anti-tumor immunity.

In this work I show that the recombinant poliovirus PVSRIPO, currently in

clinical trial as a treatment for glioblastoma, induces and evades IFN-β signaling in

cancer cell lines infected at low doses. Likewise, IFN-α treatment of cancer cells

inhibited PVSRIPO less than on the related encephalomyocarditis virus (EMCV).

Antibody blockade of the IFN-α/β receptor had no effect on either virus in IFN-secreting

melanoma cell lines. Depletion of the pattern recognition receptor MDA5 or inhibition of

TBK1/IKKε eliminated IFN responses to PVSRIPO or EMCV and promoted EMCV, but

not PVSRIPO, replication. The Toll-like receptor 3 (TLR3) agonist poly(I:C) suppressed

EMCV (semi-independently of type I IFN signaling) but not PVSRIPO. Thus, MDA5 and

TLR3 provoke type I IFN-dependent and -independent antiviral effects, likely involving

upregulation of genes downstream of TBK1/IKKε. PVSRIPO subverts anti-viral

immunity in cancer cells at low doses and activates type I IFNs through MDA5,

supporting its oncolytic and immunotherapeutic use even in IFN-competent cancers.

As human skin ages, its ability to both repair wounds and protect them from infection declines. Several factors play major roles in this, including thinning of epidermis and loss of collagen leading to skin fragility, as well as the decline of innate immune function, though the latter has been less distinctly linked to skin aging. Given the rising aging population globally, understanding how the skin responds to injury across the life spectrum is increasingly important. This work attempts to understand specifically how innate antiviral immunity of the skin is downregulated in age. To do so, we leveraged a discovery where we determined that aging skin has a differential circadian clock, a known immunological regulator, compared to that of younger skin. The circadian rhythm is a biological clock that uses a transcriptional-translational feedback loop to set up patterns of activity throughout the body. This loop uses positive transcription factors BMAL1 and CLOCK, which set up their own repressors, including the PER and CRY family of proteins. This rhythm also influences biological functions throughout the body. In the scope of this work, we became interested in the fact that circadian rhythms were found to influence epithelial repair in injury, antiviral immunity and interferon stimulated genes. To begin our study, we asked whether the known repair and immune dysfunctions of aging skin could be possibly tied to a dysfunctional circadian rhythm. Using qRT-PCR, we found that aging murine skin has a decreased circadian transcription when compared to that of younger skin. Such a phenotype was replicated by human keratinocyte studies using serial passaging as an aging surrogate. Having determined that aging does indeed play a role in regulating cutaneous circadian rhythms, we set out to determine what immune mechanisms of the skin are regulated by this aging-circadian axis. Specifically, we tested circadian regulation of antiviral proteins. Antiviral proteins of distinct families and functions all protect the skin from pathogen invasion. Prior work by our lab had shown that antiviral proteins were induced by skin wounding in a pathway dependent on the cytokine IL-27. Using approaches including qRT-PCR, flow cytometry, and immunofluorescence, we determined that aging skin wounds not only have an attenuated antiviral protein response, but also contained reduced numbers of CD301b+ immune cells that produce IL-27. These distinct immune deficiencies lead to an unprotected skin wound microenvironment in aging skin. However, little is understood about the molecular mechanisms responsible for the antiviral immune deficiencies in the aging skin. To address this, we began by probing publicly available datasets, where we found that the expression of antiviral proteins had 24-hour rhythms of expression in murine skin. Similarly, we found that that rhythmic expression of antiviral proteins occurs in human keratinocytes that were synchronized in a circadian fashion. Additional support for a direct line of circadian regulation of antiviral proteins came from circadian siRNA studies, where we knocked down expression of circadian gene CLOCK and saw an associated downregulation in antiviral proteins within human keratinocytes. Further, we demonstrate via qRT-PCR that murine skin harvested at different time points have different antiviral protein mRNA levels. Subsequent computational analysis showed that Bmal1-/- murine skin is deficient in antiviral protein expression, establishing a direct link between circadian factors and antiviral proteins. In order to better understand the effect of circadian rhythms on wound immune responses, we made use of a number of experimental models, including both Bmal1-/- and ClockΔ19 mutant mice, as well as wild type animals. We wounded wild type animals at distinct time-of-day, and found that the level of antiviral proteins display time-of-day responses, peaking at 8pm. Using circadian mutant mice, we found that these animals have attenuated wound responses with respect to antiviral protein induction; specifically, wounded ClockΔ19 mice do not produce antiviral proteins to the same extent as wild type mice. We were able to tie this directly to IL-27 signaling in two distinct manners. Firstly, using flow cytometry, we found that the CD301b+ cells that produce IL-27 in response to wounding are reduced in number in circadian mutant mouse skin, and moreover, produce less IL-27 as measured by median fluorescence intensity. To determine the role of IL-27 in the time-of-day response of wound-induced antiviral protein expression, we wounded IL-27fl/fl-LysM-Cre mice at two distinct times-of-day and measured antiviral protein production. These mice lack IL-27 production from myeloid cell lineages, including CD301b+ cells. We found that loss of IL-27 diminished time-of-day differential expression of antiviral proteins in wounds, further suggesting that the link between circadian rhythms and antiviral proteins was in fact in part mediated by IL-27. To further our understanding of the cytokine milieu of circadian wounds, we also wounded mice that lacked Type I interferon receptor (IFNAR1) expression and found that loss of Type I interferon signaling also blunted time-of-day antiviral protein responses. These data support a role of both interferons and IL-27 in circadian antiviral protein induction. In order to provide a functional aspect to these findings, we infected wildtype and circadian disrupted keratinocytes and human skin with Herpes Simplex Virus Type I (HSV1). We measured HSV expression in the skin by both immunofluorescence and visual characterization as well by qPCR for viral component UL29. We found that circadian disruption of either BMAL1 or CLOCK sensitizes keratinocytes to HSV1 infection in vitro. On the other side of this spectrum, we questioned whether circadian enhancing drugs, including the compounds SR8278 and nobiletin, can activate circadian rhythms in skin cells and improve skin defense against HSV1 infection. Using a BMAL1:Luciferase reporter, we characterized both drugs as having a circadian augmenting effect in keratinocytes. Upon infection with HSV1, both SR8278 and nobiletin protected human skin from viral spread. Further, we found that SR8278’s antiviral effect was predicated on circadian activity, as BMAL1 and CLOCK siRNA knockdown in keratinocytes lessened the drugs effect. To determine mechanism of circadian drug’s antiviral activity, we tested whether our circadian drugs activate canonical antiviral signaling pathways, such as OAS and IFITM. We found that via qPCR, circadian drugs require the presence of these proteins to fight virus effectively. As a study of clinical relevance, we evaluated the role of acyclovir treatments alongside our circadian drugs. We found that circadian drugs SR8278 and nobiletin did not synergize their effects with acyclovir at a variety of doses tested. We found that acyclovir, as expected, broadly suppressed HSV1 activity at even low doses in keratinocytes, an effect that circadian augmentation was unable to potentiate. This could be due to a number of factors, including dosage optimization and viral susceptibility to drug. However, given the rise of acyclovir resistant HSV, our novel approach may be clinically viable. In particular, we believe this may be a viable treatment platform for aging skin infections; to this end, we tested the ability of SR8278 to suppress HSV1 infection in the skin of mice over a year of age. SR8278 significantly reduced viral spread in this model, suggesting that circadian augmentation may be a useful clinical adjunct in aging skin infections. To determine if these findings were applicable to other non-herpes family viruses that infect the skin, we turned to a model of West Nile Virus infection. West Nile Virus is a mosquito borne illness with increasing range and infection number in the United States. Moreover, it is inoculated through the skin before causing neurological infection, a pathway similar to herpes viruses. Also similar to herpes viruses, West Nile virus is a far more pressing clinical issue in aging populations, who fare worse with this viral infection. Most importantly, there are no currently specific treatments for West Nile Virus infections. Using our HSV infection data as a starting point, we found that circadian drug treatments suppressed West Nile Virus levels in infected keratinocytes. Other work conducted over the course of this PhD encompassed aspects of both circadian and IL-27 signaling in the skin. Using human keratinocytes, we endeavored to understand what environmental factors could drive altered circadian rhythms in the skin, for either elderly or younger tissue. While dogmatically, circadian rhythms are patterned from the brain to the whole body, we built on recent work showing a light dependent murine cutaneous clock by showing that mock sunlight can alter circadian expression in human keratinocytes, without other stimuli present. Further study is needed to understand how our skin’s clock responds to sun mechanistically. Overall, my work over the course of this PhD has established a link between aging, circadian rhythms, and antiviral immunity, and underpinned the important role of the cytokine IL-27 and type I interferon on cutaneous wound responses to a variety of pathogens. This work will provide possible therapeutic avenues, particularly for aging skin, in how to address skin wound care in safe, biologically relevant ways via circadian rhythm exploitation.

Pseudomonas aeruginosa, known as one of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients, secretes a variety of virulence-associated proteases. These enzymes have been shown to contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds and chronic wounds. Our lab has previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is also highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known for their contribution to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. With this in mind, we hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content and resistance to the antibiotic colistin. We recreate this phenotype using a pellicle biofilm model, in which bacteria are grown statically at the culture air-liquid interface, demonstrating that these phenotypes are not dependent on the coculture host cell substrate. We additionally show that OMVs from PaAP expressing strains, but not PaAP alone or in combination with PaAP deletion strain-derived OMVs, could complement this phenotype. Finally, we found that OMVs from PaAP-expressing strains cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. OMVs mediated the detachment of biofilms formed by both non-self P. aeruginosa strains and K. pneumoniae, another respiratory pathogen, showing that this process may also be relevant in polymicrobial communities and acts on non-P. aeruginosa derived substrates. Our findings represent novel roles for OMVs and the PaAP aminopeptidase in the modulation of bacterial biofilm architecture.

Our group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.

Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.

Cells from all levels of life secrete vesicles, which are nanoscale proteoliposomes packaged with a variety of proteins, lipids, and small molecule cargo. Depending on their origin, these extracellular vesicles are termed exosomes, microvesicles, exomeres, and membrane vesicles, to list a few. Vesicles released from Gram-negative bacteria bud from the outer membrane and are, therefore, referred to as outer membrane vesicles (OMVs). In mammalian systems, OMVs facilitate bacterial survival by alleviating membrane stress, serving as a decoy for bacteriophage and antibiotics, and providing a fast membrane remodeling mechanism. OMVs also contribute to virulence by delivering toxins and other soluble and insoluble cargo to the host cell. The role OMVs play in plant systems remains unknown.

Previous studies revealed that plant pathogenic bacterial vesicles contain virulence factors, type III secretion system effectors, plant cell wall-degrading enzymes, and more, suggesting that vesicles may play similar roles to those from mammalian pathogens in host-pathogen interactions. Further, OMVs elicit several markers for pathogen-associated molecular pattern triggered immunity in plants. These responses include increased transcription of defense markers such as FRK1 and production of reactive oxygen species. Building on these findings, here we show that OMVs from the plant pathogen Pseudomonas syringae and the plant beneficial Pseudomonas fluorescens elicit plant immune responses in Arabidopsis thaliana that protect against future pathogen challenge. Intriguingly, protection is independent of salicylic acid plant defense pathways and bacterial type III secretion. OMVs also inhibit seedling growth, another indication of plant immune activation.

Our initial biochemical studies suggested that the immunogenic OMV cargo was larger than 10 kDa and differed between the pathogen and beneficial species despite similar plant immunity outcomes. Interestingly, protective OMV-mediated responses were protein-independent, while the seedling growth inhibition phenotype was entirely protein dependent. Proteomics analysis confirmed that OMV protein cargo differed between P. syringae and P. fluorescens. While media culture conditions did not dramatically impact the immunogenic activity of isolated OMVs from either species, proteomics analysis revealed a significant shift in P. syringae OMV cargo between complete and minimal media conditions. P. fluorescens OMV cargo was largely the same in the two media conditions, with no significantly enriched proteins in minimal or complete media. Further analysis of the proteins enriched in the P. syringae minimal OMV condition identified one set of proteins with the same baseline abundance in P. syringae and P. fluorescens complete OMVs and another set with a lower baseline abundance compared to P. fluorescens OMVs. These two subsets could contribute to virulence and stress tolerance, respectively. Enrichment analysis uncovered particularly interesting protein categories in the subset with the same baseline abundance. Of interest, several lipoprotein and lipid binding categories were enriched, and proteins involved in synthesis of the phytotoxin coronatine were also enriched in this same-baseline subset. These results support our hypothesis that proteins enriched in P. syringae minimal OMVs with the same baseline abundance in P. fluorescens complete OMVs may contribute to OMV-mediated bacterial virulence in plants. Our findings also suggest that our forthcoming OMV metabolomic analyses may reveal non-proteinaceous cargo that is critical for OMV-mediated plant immune activation.

The work presented here lays the groundwork for future exploration of OMV-plant interactions and adds a new layer of complexity to plant-bacteria interactions. Further, these results reveal that OMVs elicit complex plant immune responses that would be difficult for pathogens to adapt to and overcome, supporting a role for bacterial OMVs in agricultural applications to promote durable resistance and revealing a new potential avenue for disease prevention and management.

Mycobacterial infection leads to the formation of characteristic immune cell aggregates called granulomas. In humans and animal models, tuberculous granuloma formation is accompanied by dramatic remodeling of host vasculature which ultimately benefits the infecting mycobacteria, suggesting the bacteria may actively drive this host process. First, we sought to identify bacterial factors that promote granuloma vascularization. Using Mycobacterium marinum transposon mutants in a zebrafish infection model, we revealed the enzyme Proximal Cyclopropane Synthase of alpha-Mycolates (PcaA) as an important bacterial determinant of host angiogenesis. We found that PcaA-modified trehalose dimycolate, an abundant glycolipid in the mycobacterial cell wall, drives activation of host VEGF signaling and subsequent granuloma vascularization. To facilitate our continuing investigation of granuloma dynamics, we next sought to expand and improve upon the transgenic tools for studying macrophages in the zebrafish model. I describe two such tools: i) the macrophage-specific zebrafish mfap4 promoter, which allows long-term in vivo visualization and manipulation of macrophages during mycobacterial infection, and ii) the first zebrafish transgenic line with constitutive, ubiquitous Cas9 expression, as well as a transgene design capable of generating sgRNAs using macrophage-specific promoters. These tools allow CRISPR/Cas9 gene editing in vivo in the zebrafish in a macrophage-restricted manner.

My thesis work has focused on characterizing mechanisms by which human fungal pathogens regulate their adaptive cellular responses in order to survive and cause disease in the human host. Unlike most microbial fungi found in the environment, Cryptococcus neoformans has become a successful human pathogen due to two intrinsic abilities: 1) to survive and grow at human body temperature and 2) to employ virulence factors to combat host immune defenses. Over the past two decades, the fungal pathogenesis field has made enormous progress in identifying and characterizing C. neoformans proteins responsible for these adaptive cellular responses with a particular focus on enzymes, like those involved in cell cycle progression or those responsible for synthesizing components of the fungal cell surface. Although we know a substantial amount about the functions of these enzymes and their implications on fungal pathogenesis, the mechanisms by which these enzymes are regulated are less clear. I have attempted to address this gap in knowledge by focusing my thesis work on the identification and characterization of C. neoformans non-enzymatic proteins that regulate enzymes important for adaptive cellular responses. I have identified and characterized the C. neoformans arrestin proteins as regulators of enzyme ubiquitination, and likely enzyme function, in response to specific extracellular stressors (Chapters 2 & 3). I have also characterized a Cryptococcus-specific protein, Mar1, as an important modulator of host-fungal interactions due to its regulation of cell surface remodeling through maintenance of mitochondrial metabolic activity and homeostasis in response to cellular stress (Chapters 4 & 5). Furthermore, I also performed a comprehensive comparative analysis of different RNA enrichment methods for RNA sequencing applications and long non-coding RNA identification in C. neoformans, which can help researchers select appropriate tools for studying adaptive cellular responses from the RNA level (Chapter 6). These studies collectively have demonstrated that non-enzymatic proteins are important “cellular coordinators” in human fungal pathogens; they regulate the activity of many different enzymes in response to distinct extracellular signals, and as a result are required for both fungal growth and virulence factor employment in response to host-relevant stressors.

The physiology of the tumor microenvironment is characterized by lower oxygen (hypoxia), higher lactate, extracellular acidosis and glucose starvation. We examined the global, transcriptional cellular responses to each of these microenvironmental stresses in vitro, projected them onto clinical breast cancer patients' samples in vivo, and returned to perform further in vitro experiments to investigate the potential mechanisms involved in these stress responses. The reciprocal exchange of information was critical and advanced our understanding of the potential clinical relevance of cellular responses.

Our expression array result showed that lactic acidosis induces a strong response, distinct from that of hypoxia in human mammalian epithelial cells (HMECs), indicating lactic acidosis is not only a by-product of hypoxia but has a unique role as a stimulant to cells in the tumor microenvironment. Cellular responses to lactosis and acidosis further demonstrated that acidosis was the main driving force in the lactic acidosis response. These responding gene signatures were then statistically projected into clinical breast cancer patients' expression data sets. The hypoxia response, as reported previously, was associated with bad prognosis, where as the lactic acidosis and acidosis responses, were associated with good prognosis. Additionally, the acidosis response could be used to separate breast tumors with high versus low aggressiveness based on its inversed correlation with metastatic character. We further discovered that lactic acidosis, in contrast to hypoxia, abolished Akt signaling. Moreover, it downregulated glycolysis and shifted energy utilization towards aerobic respiration.

We continued to examine the cellular response to lactic acidosis temporally in MCF7 cells, a breast cancer cell line. The lactic acidosis response of MCF7 cells also showed the prognostic result of better clinical outcomes in datasets of breast cancer patients. The lactic acidosis responses of HMEC and MCF cells were highly correlated. Strikingly in MCF7 cells, lactic acidosis and glucose deprivation actually induced similar transcriptional profiles, with only a few genes being oppositely regulated. Furthermore, lactic acidosis, similar to glucose starvation, induced AMPK signaling and abolished mTOR. However, lactic acidosis and glucose deprivation induced opposite glucose uptake phenotypes. Lactic acidosis significantly repressed glucose uptake whereas glucose deprivation significantly induced it. Among the genes differentially regulated by these two stresses, thioredoxin-interacting protein (TXNIP) was among the most different. The negative regulatory role of TXNIP on glucose uptake has been demonstrated previously. In the cancer research field, TXNIP is recognized as a tumor suppressor gene. We observed that lactic acidosis induced TXNIP strongly and most importantly, TXNIP played a critical role in regulating glucose uptake in cells under lactic acidosis. Furthermore, MondoA, the transcription factor and glucose sensor previously reported to regulate TXNIP induction upon glucose exposure, was also responsible for regulating TXNIP under lactic acidosis. We demonstrated that TXNIP not only plays an important role in the lactic acidosis response but also has strong prognostic power to separate breast cancer patients based on survival.

Pathological angiogenesis is a widespread biological phenomenon that influences the progression of various diseases, including autoimmune conditions, cancers, and microbial infections. One infection in particular, tuberculosis, is associated with the induction of a potent pro-angiogenic signaling cascade that facilitates bacterial growth and accelerates disease progression. A synthesis of early studies on bacterial factors that drive host angiogenesis with modern genetic findings identified the mycobacterial glycolipid trehalose 6-6'-dimycolate (TDM) as a critical factor driving vascular endothelial growth factor (VEGFA) production and angiogenesis during mycobacterial infection. Despite these recent findings, many of the underlying host response mechanisms remain unknown. The introductory chapter will serve to introduce the reader to the major concepts addressed in this work: Mycobacterium tuberculosis and the disease it causes, the role of macrophages in health and disease, the function of pattern recognition receptors in detecting microbial ligands, the specific downstream intracellular signaling pathway of interest for this work (mediated by the transcription factor, nuclear factor of activated T cells, NFAT), the contributions of angiogenesis to diverse contexts and pathologies, and the promise of host-directed therapies to overcome challenges associated with traditional treatment approaches in infectious disease. Chapter 2 describes the new and existing methodological approaches that were required to complete this work. This work utilizes the zebrafish-Mycobacterium marinum model of tuberculosis infection to facilitate in depth in vivo observation and quantitation of these phenomena. Using this model in tandem with human macrophage cell culture, I was able to model major aspects of the host-pathogen interface, enabling me to identify a critical role for a macrophage-C-type lectin receptor-NFATC2-VEGFA signaling axis required for the angiogenic response to mycobacterial infection and TDM, findings that comprise the core of this work and are detailed at length in Chapter 3. The analysis of the large amounts of data generated in this work required creative approaches to data processing and analysis. To this end, I have developed a set of novel processing modalities in Python and R that are capable of the rapid and reproducible processing of images as well as certain aspects of automated data collection therefrom. These macros, many written for the FIJI/ImageJ programming environment, serve as the infrastructure on which the rest of this work has been built. These will be detailed in Chapter 4. Finally, this body of work leaves many questions as yet unanswered. While it is clear that NFAT signaling is required for VEGFA production, the precise mechanism by which this may work is unclear and could be mediated by either direct DNA binding or indirect activation or cooperative binding with some other transcriptional activator. There also exist a variety of other potential NFAT- and angiogenesis-related phenotypes worthy of exploring using the tools and approaches I have developed. It is my hope that the findings herein stimulate further study on the contributions of NFAT signaling to the host immune response to mycobacterial infection and evaluation of the potential of NFAT inhibition as host-directed therapy to tuberculosis.

Disease caused by the Gram-negative Haemophilus cryptic genospecies begins with colonization of the maternal genital or neonatal respiratory tract. The primary goal of this work was to identify and characterize the molecular determinant(s) of Haemophilus cryptic genospecies adherence as a means to better understand the specific adaptation of this species to the urogenital tract and neonatal respiratory tract. Using transposon mutagenesis of prototype strain 1595, we identified a locus that is essential for Haemophilus cryptic genospecies adherence to a variety of epithelial cell lines of both genital and respiratory origin. This locus encodes a protein called Cha that shares homology with trimeric autotransporters. Trimeric autotransporters are composed of an N-terminal signal peptide, an internal passenger domain that harbors adhesive activity, and a short C-terminal membrane anchor domain and are classically characterized by head-stalk-anchor domain architecture. By generating chimeric proteins, we demonstrated that the C-terminus of Cha trimerizes in the bacterial outer membrane and is capable presenting a heterologous passenger domain (Hia) in a functional form, thus confirming that Cha is a trimeric autotransporter. Southern analysis revealed that cha is unique to the Haemophilus cryptic genospecies and is ubiquitous among these strains.

Similar to a number of trimeric autotransporters, the passenger domain of Cha contains scattered clusters of YadA-like head domains associated with head-to-stalk neck adaptor motifs, predicted coiled-coil stalks and a series of identical tandem coding repeats which are not required for adherence. By evaluating the adherence capacity of H. influenzae expressing Cha deletion derivatives, we established that the N-terminal 473 residues of Cha harbor the binding domains responsible for Cha-mediated adherence to epithelial cells. In additional studies, we demonstrated that this same N-terminal region mediates bacterial aggregation through inter-bacterial Cha-Cha binding.

Further analysis revealed that variable Cha-mediated adherence is linked to spontaneous changes in the number of identical tandem repeats predicted to comprise a coiled-coil stalk domain. Variation in repeat copy number has a direct effect on Cha adhesive and aggregative activity, independent of an impact on transcription of the cha locus or surface localization of Cha protein. Moreover, length of Cha surface fibers correlates with repeat copy number expansion. We propose two hypotheses to explain how repeat expansion inhibits bacterial aggregation and host cell binding: 1) Variation in the number of 28-amino acid repeats may influence the conformation of Cha, thus changing the surface accessibility of the Cha binding pocket. 2) Repeat expansion results in the formation of long, flexible Cha fibers on the bacterial cell surface that may have a greater propensity to interact with neighboring Cha trimers at the N-terminus, thereby precluding adherence to other bacteria or host epithelial cells.

In additional studies screening adherent cryptic genospecies isolates for expression of Cha protein, we identified an additional, antigenically-divergent Cha variant that we refer to as Cha2. Amino acid sequence and domain comparison of Cha2 with Cha (now Cha1) revealed that the structurally undefined N-terminal sequences (encompassing the Cha1 adhesive and aggregative domain) are strikingly divergent. Inspite of this, Cha2 mediates efficient adherence to human epithelial cells, similar to Cha1.

Identification of Cha offers insight into the apparent tissue tropism associated with the Haemophilus cryptic genospecies. We speculate that the unique regulation of Cha adhesive activity enhances the adaptive capability of this pathogenic organism in the human host.

Radial glial cells (RGCs) are essential for the generation and organization of neurons in the cerebral cortex. RGCs have an elongated bipolar morphology with basal and apical endfeet that reside in distinct niches. Yet, how this subcellular compartmentalization of RGCs controls cortical development is largely unknown. Here, we employ in vivo proximity labeling, in the mouse, using unfused BirA to generate the first subcellular proteome of RGCs and uncover new principles governing local control of cortical development. We discover a cohort of proteins that are significantly enriched in RGC basal endfeet, with MYH9 and MYH10 among the most abundant. Myh9 and Myh10 transcripts also localize to endfeet with distinct temporal dynamics. Although they each encode isoforms of non-muscle myosin II heavy chain, Myh9 and Myh10 have drastically different requirements for RGC integrity. Myh9 loss from RGCs decreases branching complexity and causes endfoot protrusion through the basement membrane. In contrast, Myh10 controls endfoot adhesion, as mutants have unattached apical and basal endfeet. Finally, we show that Myh9- and Myh10-mediated regulation of RGC complexity and endfoot position non-cell autonomously controls interneuron number and organization in the marginal zone. The first part of this study demonstrates the utility of in vivo proximity labeling for dissecting local control of complex systems, and reveals new mechanisms for dictating RGC integrity and cortical architecture. In the second portion of this work, we have developed a method for purification of endfeet from the embryonic mouse brain and employed it to discover the first global transcriptome of RGC endfeet. Analysis at E15.5 revealed that the network of localized mRNAs is much more extensive than previously appreciated. There are over 3,000 transcripts localized to RGC endfeet and 870 of them are highly enriched in the endfeet compared to the cell body. These data uncovered hundreds of new genes in endfeet and also reinforced our previous findings that cytoskeletal regulators and ECM components are especially important in endfeet. Exploration of the newly discovered localized transcripts will provide valuable insights into additional RGC functions and allow us to assess potential signaling interactions between endfeet and surrounding cells. We also propose a method for subcellular gene knockdown in which we can modulate mRNA levels of a gene of interest in the cell body and endfeet independently in vivo. Through these studies we have discovered vital roles for subcellular gene regulation in RGCs and developed tools to facilitate future studies.

Our ability to convert basic knowledge into robust anti-viral therapeutics requires discovery of novel host-virus interactions as well as an informed anti-viral discovery pipeline. We used a genome-scale RNAi-based screen followed by a chemical screen of FDA-approved therapeutics to identify scores of novel dengue virus (DENV) human host dependency factors (HDF) and identified more than 20 potential anti-Zika virus (ZIKV) therapeutics.

Two genes in particular, TTC35 and TMEM111, strongly inhibited DENV infection and, based on comparisons with published literature, implicated a larger protein, the ER Membrane Protein Complex (EMC), as a pan-flavivirus HDF. The EMC is a poorly characterized multiprotein complex that may function in ER-associated protein biogenesis and/or lipid metabolism. Based on our screen data, we hypothesized that the EMC is an uncharacterized HDF that functions through a common mechanism to promote replication of flaviviruses. We report that DENV, ZIKV, and yellow fever virus (YFV) infections were impressively inhibited, while West Nile Virus (WNV) infection was unchanged, in cell lines engineered to lack EMC subunit 4 (EMC4). Furthermore, targeted depletion of EMC subunits in live mosquitos significantly reduced DENV-2 propagation in vivo. In addition, the accumulation of DENV proteins shortly after infection in EMC4 knockout cells was significantly reduced, suggesting that the EMC promotes viral protein biogenesis.

We interrogated a library of FDA-approved drugs for their ability to block infection of human HuH-7 cells by a newly isolated ZIKV strain. Selected compounds were further validated for inhibition of ZIKV infection in human cervical, placental, and neural stem cell lines, as well as primary human amnion cells. Established anti-flaviviral drugs (e.g., bortezomib and mycophenolic acid) and others that had no previously known antiviral activity (e.g., daptomycin) were identified as inhibitors of ZIKV infection. Several drugs reduced ZIKV infection across multiple cell types.

We propose that the EMC may be exploited as a novel therapeutic target for multiple flaviviruses in the future. Also we identified drugs that could be tested in clinical studies of ZIKV infection and provides a resource of small molecules to study ZIKV pathogenesis.

The intracellular innate immune response to viral infection is among the first lines of defense against these pathogens. For the early establishment of an antiviral cellular state and initiation of inflammatory responses, type I interferons (IFNs) are particularly important, as they potently induce the production of hundreds of IFN-stimulated genes (ISGs), many of which have antiviral functions. The type I IFN response requires tight molecular coordination to achieve both efficient production of antiviral proteins and controlled shutoff of inflammatory responses to avoid tissue damage and autoimmunity. Despite the importance of regulation of this antiviral response, current knowledge of the molecular controls governing its activation and suppression remains incomplete. Further, although ISGs have diverse functions and are induced to differing potencies, our understanding of regulatory controls governing the expression of individual or subclasses of ISGs is limited. Current knowledge of type I IFN response regulation is predominantly centered on transcriptional and post-translational regulatory controls. However, post-transcriptional regulation of antiviral responses has begun to emerge as an important layer of control. An example of these post-transcriptional regulatory controls is the RNA base modification N6-methyladenosine (m6A), which regulates many aspects of mRNA metabolism through transcript-specific effects. m6A deposition is mediated by a cellular complex of proteins including METTL3 and METTL14 (METTL3/14) and other cofactors, and m6A can also be removed from RNA by the demethylase proteins FTO and ALKBH5. The presence of m6A on viral and host RNAs has been shown to influence the outcome of infection by diverse viruses. However, the role of m6A in the response to type I IFNs has not been explored. To investigate the role of m6A in the type I IFN response, we began by manipulating m6A levels in the transcriptome through perturbation of the expression of the cellular m6A machinery and measuring the induction of ISGs after IFN treatment. We found that depletion of the m6A methyltransferase proteins METTL3 and METTL14 (METTL3/14) resulted in less protein production of a subset of ISGs, including the antiviral genes IFITM1 and MX1, after IFN treatment. However, the expression of other ISGs and the overall activation of the IFN responses were unchanged. Using methyl RNA immunoprecipitation and sequencing (meRIP-seq), we found that the transcripts of many ISGs are modified by m6A, and these included the METTL3/14-regulated ISGs IFITM1 and MX1 that we had identified. Using polysome profiling and ribosome profiling, we determined that METTL3/14-regulated ISGs are translationally enhanced by METTL3/14. Additionally, ablation of putative m6A sites within the 3’UTR of IFITM1 decreased the translation of a reporter molecule. Overexpression of the m6A reader protein YTHDF1, which has known roles in promoting translation, enhanced the expression of IFITM1 in an m6A binding-dependent fashion. These experiments characterized METTL3/14 and m6A as novel enhancers of the type I IFN response. To determine whether m6A contributes to type I IFN-mediated viral restriction, we depleted or overexpressed METTL3/14 and pretreated cells with a low dose of IFN-β prior to infection with vesicular stomatitis virus (VSV). Interestingly, METTL3/14 depletion decreased the expression of ISGs and allowed increased VSV infection, while METTL3/14 overexpression had the opposite effect. Together, these studies demonstrate that METTL3/14 and m6A enhance the antiviral effect of type I IFN by promoting the translation of ISGs to support the establishment of an antiviral cellular state. Having discovered a role for m6A in the type I IFN response, we also investigated the role of an m6A demethylase protein, FTO. FTO polymorphisms can have profound effects on human health. Certain polymorphisms are associated with fat mass and obesity, cardiovascular disease, while others can cause growth retardation or embryonic lethality. However, the molecular functions of FTO and the cellular pathways that it affects are still not well characterized. We depleted FTO and measured the production of ISGs following IFN-β treatment and found that the production of m6A-regulated ISGs was increased, as expected. However, unexpectedly, we found that FTO depletion increased the mRNA levels of a subset of ISGs. Pulse labeling of nascent transcripts revealed that FTO suppresses the transcription of these ISGs and that FTO-depleted cells are primed for the production of certain ISGs in response to IFN. We then used cells lacking PCIF1, the writer of 2’-O-N6-dimethyladenosine (m6Am), an RNA modification that FTO can also remove, and found that FTO-mediated regulation of ISGs occurs independently of the m6Am modification. These results identify FTO as a transcriptional regulator of a subset of ISGs, which will add an important dimension to our understanding of the molecular functions of FTO and its contributions to inflammatory disease. Future research revealing the mechanisms by which FTO suppresses ISG transcription will be of great interest. Together, these data identify novel functions of m6A and its related cellular machinery in both positive and negative regulation of the type I IFN response and antiviral gene expression.

As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt to changes and survive stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved a myriad of virulence factors including the production of an exopolysaccharide capsule, as well as secretion of degradative proteases and lipases that also function as defense mechanisms. Outer membrane vesicles (OMVs) acts as a secretion system to disseminate virulence factors and function as a general bacterial stress response to remove accumulated periplasmic waste. Despite the growing insights of the field into the potential functions of OMVs, the mechanism for formation remains to be fully elucidated. The three proposed mechanisms for OMV formation in P. aeruginosa are mediated by the Pseudomonas quinolone signal PQS, the AlgU envelope stress response pathway, and the periplasmic chaperone MucD. This report investigates how P. aeruginosa responds to sublethal physiological stressors with regards to OMV production levels and whether the proposed mechanisms for OMV formation are required for stress-induced OMV formation. We concluded that exposure to cell wall directed stressors increased OMV production and activity of the sigma factor that controls MucD expression, AlgU. AlgU was shown to be sufficient to induced OMV production upon overexpression; however, stress-induced OMV production was not dependent on activation of AlgU as vesiculation could be induced in strains lacking AlgU. Furthermore, MucD levels were not inversely proportional to OMV production under acute stress, and the ability to produce PQS was not required for OMV production. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that hydrogen peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. We also demonstrated that the ability for P. aeruginosa to sense oxidative stress via OxyR, was important for hydrogen peroxide-induced OMV production, by a yet to be determined method. Together these results demonstrate that current proposed mechanisms for OMV formation do not universally apply under all stress conditions, and that additional mechanisms for OMV formation are still to be identified and fully elucidated during acute stress in P. aeruginosa.

The intestine is a multifunctional organ that must perform dichotomous roles in order to maintain health. While it is the primary site of absorption of dietary nutrients, it must also serve as a barrier to both the multitude of microorganisms which reside in the intestinal lumen (the microbiota) and foreign compounds (xenobiotics) which can be toxic to the host. Moreover, the microbiota are required for normal physiology, regulating immunological development, metabolism and behavior. Understanding how the intestine maintains homeostasis and responds to insult in the face of a chemically and microbially complex and dynamic environment is not only a fundamental question of biology, but has important implications for human health. We used zebrafish in order to better understand how the intestine responds to xenobiotics (Chapter 2) and transduces signals from the microbiota to the immune system (Chapter 3).

In Chapter 1, I introduce the complex and reciprocal interactions between xenobiotics, the microbiota, and the host. I highlight examples whereby the microbiota modulates the activity and toxicity of pharmaceuticals, with relevance to diseases of different organ systems. I also describe mechanisms by which the intestine responds to xenobiotic toxicity, and finally advocate for the use of novel model organisms to improve our understanding of these complex interactions.

In Chapter 2, I present our work using the NSAID Glafenine to explore how the intestine responds to xenobiotic challenge. Using transgenic zebrafish and high resolution in vivo imaging, we demonstrate loss epithelial cells in a live animal following xenobiotic challenge. Moreover, Glafenine causes intestinal inflammation, which is potentiated by microbial dysbiosis. We also show that Glafenine can directly alter microbiota composition. Glafenine treatment resulted in activation of the unfolded protein response (UPR), and while pharmacological inhibition of the UPR sensor Ire1a suppressed Glafenine-induced IEC loss, this was associated with increased inflammation and mortality. Ultimately, we demonstrate that Glafenine-induced intestinal toxicity is likely due to off-target inhibition of multidrug resistance (MDR) efflux pumps, as other MDR inhibitors were able to elicit similar phenotypes. Collectively, our findings revealed that (i) MDRs serve an evolutionarily conserved role in maintenance of intestinal homeostasis and (ii) IEC delamination is a protective mechanism which serves to limit inflammation and promote animal survival.

While studies in gnotobiotic mice and zebrafish have demonstrated that the microbiota are required for normal development of the innate immune system, the underlying host and microbial signals which mediate these effects remain largely unknown. We had previously demonstrated that motility of gut commensal bacteria in zebrafish was important for successful colonization of some strains and stimulation of the normal host innate immune response to colonization. In Chapter 3, we describe how microbiota colonization is associated with changes in the PMN transcriptome in addition to promoting systemic abundance and distribution of myeloid cells. Intriguingly, the only pattern recognition receptors found to be differentially expressed in PMNs were the Flagellin receptors tlr5a and tlr5b. Colonization of zebrafish larvae with bacteria lacking Flagellin resulted in attenuated PMN transcriptional activation compared to larvae colonized with isogenic wild type (WT) bacteria. We subsequently demonstrated that direct exposure to purified Flagellin can potently induce transcriptional activation in zebrafish PMNs. These findings identify how the presence of the microbe associated molecular pattern (MAMP) Flagellin serves as a bacterial cue from the microbiota which promotes PMN activation. In Chapter 4, I offer perspectives as to how the Glafenine-zebrafish model system can be used to more deeply investigate host-microbiota-xenobiotic interactions, and genetic, biochemical and computational analyses can help delineate mechanisms by which MDR efflux pumps function in the maintenance of intestinal homeostasis. Moreover, I propose the use of bacterial screens as well as inflammatory and infectious challenge assays in order to better understand the functional outcomes of PMN transcriptional activation elicited by microbiota-derived signals such as Flagellin.

The Chlamydiaceae are Gram-negative, obligate intracellular bacteria that are significant pathogens of humans and animals. Intracellularly, the bacteria reside in a membrane-bound vacuole, called the inclusion, from which they manipulate host processes to create a niche optimal for survival and propagation. Acquisition of host-derived lipids is essential for chlamydial growth, yet the source of lipids and mechanisms of trafficking to the inclusion are not well-established. The inclusion avoids interaction with several classical membrane and lipid transport pathways. In a functional genomic screen to identify host modulating chlamydial proteins, our lab identified cytosolic lipid droplets (LDs) as potential target organelles of Chlamydia. LDs are postulated to function in many cellular processes, such as lipid metabolism and transport, membrane trafficking, and cell signaling; therefore, we hypothesized that LDs may be important for Chlamydia pathogenesis as a source of lipids or as a platform for regulating other cellular functions. Here, we characterize the interaction between eukaryotic LDs and the chlamydial inclusion.

We find that LDs are recruited to the Chlamydia inclusion, chlamydial infection disrupts neutral lipid homeostasis, and pharmacological prevention of LD formation inhibits chlamydial replication. Chlamydia produces proteins (Ldas) that localize with LDs in yeast and mammalian cells when transiently expressed and are exported out of the inclusion to peripheral lipid-rich structures during infection. By electron microscopy and live cell imaging, we observe the translocation of intact LDs into the Chlamydia inclusion lumen. Biochemical and microscopic analysis of LDs from infected cells reveals that LD translocation may occur at specialized subregions of the inclusion membrane. The Chlamydia Lda3 protein is implicated in LD tethering to the inclusion membrane, and displacement of the protective coat protein, ADRP, from LD surfaces. This phenomenon could provide access for lipases to the LD core for utilization by the replicating bacteria. Additionally, the functional domains of Lda3 involved in binding to LD and inclusion membranes are identified.

In these studies, we identify eukaryotic lipid droplets (LDs) as a novel target organelle important for Chlamydia pathogenesis and describe a unique mechanism of whole organelle sequestration not previously observed for bacterial pathogens. These results represent a fundamental shift in our understanding of host interactions with the chlamydial inclusion, and may represent a new area for research in the field of cellular microbiology.

Despite a largely static DNA sequence, our genomes are incredibly malleable. Comparative studies of chromatin features between different cell types, tissues, and species have revealed tremendous differences in how the genome is accessed, transcribed, and replicated. However, how the dynamics of chromatin accessibility contribute to development, environmental response, and disease status has only begun to be appreciated. In this work we identified chromatin accessibility changes by DNase-seq in three diverse processes: in granule neurons of the developing cerebellum, with intestinal epithelial cells in the absence of a normal microbiota, and with myelogenous leukemia cells in response to histone deacetylase inhibitor treatments. In all cases, we coupled these analyses with RNA-seq assays to identify concurrent transcriptional changes. By mapping the changes to these genome-wide signals we defined the contribution of local chromatin structure to the transcriptional programs underlying these processes, and improved our understanding of their relation to other chromatin changes like histone modifications. Furthermore we demonstrated use of the strongest accessibility changes to identify transcription factors critical for these processes by finding enrichment of their binding motifs. For a few of these key factors, depletion or overexpression of the protein was sufficient to regulate the expression of predicted target genes or exert limited chromatin accessibility changes, demonstrating the functional significance of these proteins in these processes. Together these studies have informed our understanding of the role chromatin accessibility changes play in development and environmental responses while also proving their utility for key regulator identification.

In the 21st century, the world has already experienced two pandemics caused by respiratory RNA viruses, the 2009 H1N1 “swine-flu” pandemic, and the ongoing COVID-19 pandemic. The 2009 pandemic was, thankfully, mild in terms of casualties; however, it revealed certain failings in existing systems. As a circulating virus, we had approved, in-use antiviral treatments for influenza, but the 2009 H1N1 viruses were resistant to an entire class, adamantanes. Also, the requirement for annually updated influenza vaccines meant there were systems in place for large-scale vaccine production, yet reliance on eggs dramatically limited the ability to scale-up a new vaccine quickly. Finally, global vaccine inequities meant many countries remained relatively unvaccinated against the H1N1 virus, even as the pandemic was declared over in August 2010. The COVID-19 pandemic has been a very different story on some fronts, most notably the death toll, which exceeds 6 million globally. Also, the decade of progress in vaccine research since 2009 has been evident in the rapid development of many highly effective COVID-19 vaccines using different platforms, while vaccine distribution issues remain. Finally, an entire lack of approved coronavirus antivirals required swift action to identify existing drugs and treatments to alleviate disease. The research and clinical response to the COVID-19 pandemic took advantage of existing technologies, pharmaceuticals, and systems and adapted them to solve the present crisis. The problem of this dissertation is to extend these efforts by investing in adaptable research tools, methods, and subjects to prepare for potential future respiratory RNA virus pandemics.In Chapters 2 and 3, we generated virus-specific research tools that are rapidly adaptable to new viral strains. Chapter 2 describes a fluorescent reporter of coronavirus protease activity that may be used to screen antiviral drugs and is compatible with diverse coronavirus protease proteins. Chapter 3 discusses a novel method for generating influenza reporter viruses with wide research applications, notably this manner of introducing exogenous proteins requires minimal genome rearrangements increasing transferability to newly identified strains. These projects demonstrate that even research tools requiring updates with the emergence of new strains can be designed to prioritize rapid adaptability. In Chapters 4 and 5, we interrogate the innate immune responses that determine the outcomes of viral infections. Chapter 4 identifies ETV7 as a negative regulator of the antiviral type I interferon response using CRISPR activation screening. ETV7 was previously known to be induced by interferon, but its role during the response to viral infection remained undetermined. We found ETV7 limits transcription of interferon stimulated gene expression, influencing the antiviral state of a responding cell. Chapter 5 reviews the impact of influenza viral disease outside the site of infection, the respiratory tract, and established methods of studying these effects using animal models. Many of the circulating cytokines implicated in non-respiratory influenza disease from these models, IL-6, IFNs, and TNF-alpha, are known to play a role in influenza and COVID-19 patient disease severity. These investigations show that our understanding of how innate immunity is regulated, and dysregulated, continues to require updating even as the main pathway members and downstream effectors have been identified.