The Role of Gram - Negative Anaerobes in Hidradenitis Suppurativa

Hidradenitis Suppurativa is a chronic autoinflammatory disease with unknown triggers.New evidence in the last five years has begun to clarify two parts of the pathogenesis of the disease. These two parts are the fundamental role of keratinocyte inflammation from early through late-stage disease and the role of the microbiome as an antigenic and infectious trigger of pathogenesis. Gram-negative anaerobic bacteria (GNA) such as Prevotella, Porphyromonas, and Fusobacterium are commonly identified in HS lesions, and their prevalence in lesions is associated with HS disease severity. Additionally, newly epithelialized structures called dermal tunnels are found in severe disease and are associated with biofilm as well as the production of keratinocyte inflammatory cytokines. Our lab has previously defined the effects of gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes and found them to stimulate atopic dermatitis and psoriasis-like transcriptome profiles. In this thesis, using in vitro models of keratinocyte stimulation with heat-killed preparations of both gram-positive and gram-negative bacteria, we have explored a robust inflammatory profile generated in normal human keratinocytes by these GNAs. These GNAs elicit a strong IL17-dependent pathway response not seen in gram-positive bacteria and likely driven by IL-17C production. Overall, the magnitude of production of cytokines is much greater in all GNAs, with Fusobacterium nucleatum (FN) demonstrating the most intense activation that is mimicked to a lesser extent by Prevotella melaninogenica and Prevotella nigrescens. Significant differences in transcriptome profiles were found between all species, including members of the same species. We also find that this inflammatory response in GNAs is both TLR and JAK-dependent and that variation in cytokine production is likely driven by differential activation by TLR4 andTLR2 receptors. We then compare transcriptomes obtained from biopsies samples of HS patient lesional, perilesional, and nonlesional skin and from patients with psoriasis to GNA-keratinocyte profiles and IL17A stimulated keratinocyte profiles. We find that FN is most and significantly associated with HS gene transcriptomics. Our data is the first to provide a comprehensive profile of the effects of GNA stimulation on human keratinocytes that is more broadly applicable to innate immune epithelial defenses against GNAs. The identification of these bacteria as strong antigenic stimuli prioritizes targeting these bacteria in new therapies for HS, potentially through TLR4 or JAK inhibition

Using Small Molecule Tools to Study ATPase Mechanoenzymes

The dysregulation of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and chemical inhibitors and activators can be powerful tools to probe mechanisms and test therapeutic hypotheses. However,the structural conservation across the AAA protein family makes designing selective chemical inhibitors challenging. Additionally, unlike chemical inhibitors that can stabilizea single conformational state of an enzyme, activator binding must be permissive to different conformational states needed for enzyme function, and we do not know how AAA proteins can be activated by small molecules. My thesis work covers the development of a chemical genetics approach to inhibit AAA proteins, starting from atriazolopyridine-based fragment that binds the AAA domain of the microtubule severing protein katanin, and the identification of a druggable site for chemical activators in valosin-containing protein (VCP)/p97, a AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders.For the chemical genetics approach, we designed ASPIRe-1 (Allele-Specific, Proximity-Induced Reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin,ASPIRe-1 treatment increases the accumulation of CAMSAP2 at microtubule minus-ends, confirming specific on-target cellular activity. Importantly, ASPIRe-1 also selectively targets engineered cysteine mutants of VPS4B, FIGL1, and VCP. For the small molecule activator part of my thesis work, from a screen optimized to identify compounds that stimulate VCP ATPase activity as little as 20%, I discovered activators that represent five chemotypes. Minimal modification of the most potent hit, an isoindoline-based compound, resulted in VCP Activator 1 (VA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~3-fold. Cryo-EM studies resulted instructures (~2.9-3.5 Å-resolution) of VCP in apo and ADP-bound states, and revealVA1binding an allosteric pocket near the C-terminus in both states. Finally, I engineered mutations in the VA1 binding site that confer resistance to VA1, and furthermore, modulate VCP ATPase activity to a similar level as VA1-mediated activation. Together, these findings suggest a chemical genetics approach to decipher AAA protein cellular functions and uncover a druggable allosteric site that can also be occupied by VCP\u27s C-terminal tail to control activity

Mechanistic Insights into Direct Interactions that Mediate the Activity of DOT1L Complex in MLL-Rearranged Leukemia

Acute leukemia can arise from the translocation-mediated fusion of the N terminus of the Mixed Lineage Leukemia (MLL) protein to various partner proteins. Although more than 60 different translocation-based fusion proteins have been identified, fusion partners such as AF9, AF10, and ENL constitute the majority of the MLL-rearranged leukemia cases; additionally, these fusion partners are also a part of the DOT1L (disruptor of telomeric silencing 1-like) complex. Aberrant histone H3 Lysine 79 (H3K79) methylation catalyzed by DOT1L was shown to be crucial for the maintenance of MLL-rearranged leukemia. DOT1L is the only known H3K79 methyltransferase, and further, a DOT1L inhibitor is currently being evaluated in clinical trials to treat Acute Myeloid Leukemia. Although cell-based studies have implicated the importance of interactions on the N terminus of the MLL fusion protein for leukemogenesis and disease maintenance, there have been very limited corresponding biochemical analyses demonstrating how these interactions of MLL regulate or contribute to abnormal levels of methylation of H3K79 by DOT1L. Additionally, both cell-based and in vitro studies suggested that H3K79 methylation is dependent upon histone H2B ubiquitylation (H2Bub). However, there is a lack of understanding of how these interactions stimulate MLL-fusion target genes. Besides its catalytic activity, DOT1L complex activity has been postulated to regulate transcriptional elongation on the grounds of its co-localization with Pol II, H3K79 methylation mark distribution on gene loci, and its association with subunits of SEC (Super Elongation Complex) such as AF4 (or AFF4), AF9 (or AF9-related ENL), and ELL1. However, any effect of DOT1L on other transcriptional steps is not known. Addressing these gaps, the present study employs a robust biochemical approach to underscore the importance of both MLL N terminus (MLLN) interactions and H2B ubiquitylation in the regulation of aberrant H3K79 methylation by MLL-fusion protein containing DOT1L complex in MLL-rearranged leukemia. Specifically, interactions between the PWWP (LEDGF) domain and histone H3 Lysine 36 tri-methylation; between the CXXC(MLLN) domain and unmethylated CpG of DNA; and between ubiquitylated H2B and DOT1L protein are found to stabilize the intrinsic nucleosome binding property of MLL. AF9, consequently augmenting the H3K79 methyltransferase activity of the MLL-fusion containing DOT1L complex. As a result, the leukemogenic MLL-fusion protein containing the DOT1L complex exhibits inherently higher methylation activity on nucleosome arrays compared to the natural DOT1L complex. Biochemically defined invitro assays helped to show the individual and synergistic effects of these interactions, offering valuable insights into the mechanisms underlying aberrant H3K79 methylation levels in MLL-fusion target loci. These interactions hold potential as therapeutic targets, paving the way for novel therapeutic strategies. Moreover, immobilized template assays reveal a direct interaction between TFIID and the DOT1L complex, with H2Bub and DOT1L protein enhancing the recruitment of TFIID to chromatin. These direct interactions implicate the DOT1L complex in transcriptional initiation. In summary, this study advances our understanding of the critical role played by the DOT1L complex in MLL-rearranged leukemia. By shedding light on the intricate biochemical mechanisms governing MLL fusion protein interactions and their impact on H3K79 methylation, the research opens new avenues for targeted interventions in the treatment of this disease

New Insights into the Evolution of Learned Vocalization from the Australian Zebra Finch, Taeniopygia castanotis

The common goal of my thesis is to understand the causative events in evolution that produced a clade of song learning birds from non-song learning ancestors.This information is important for shedding light on the evolution of spoken language in our own human lineage, where evolutionary analyses are technically limited. The most recent common ancestor of humans and chimpanzees was presumably a vocal non-learning African ape, alive ~6 mya. At some point between this ancestor and the emergence of modern humans 0.5 mya we evolved more dexterous hand control, bipedalism, light colored eyes, larger brains, less hair, weaker muscles, higher intelligence, greater eusociality, novel sweat glands, and spoken language; to name a few of the traits which separate us from other apes. As there are no extant species internal to this branch lacking one or more of these human specific phenotypes, it is much more difficult to ascribe observed human genome variation to the evolution of specific traits, especially behavioral traits. Further, neutral mutations cannot be removed by looking for shared variance across species because we are the only extant species of vocal learning primates. Human language is also difficult to isolate from a neuroanatomical perspective. Our current interpretation of the literature is that the neurons responsible for the learned movements of speech are either directly adjacent to or intermixed with those moving the hand and face in all primates, making them more difficult to localize. None of these limitations apply in oscine songbirds. In oscines, the vocal learning brain system is more segregated from non-vocal movements; There are over 4000 oscines, and most if not all are thought to be vocal learners; their suboscine outgroup clade is also species rich and consists of many vocal non-learners. Since oscine vocal learning neural circuits are discrete, potentially causative genes can be identified based upon their specialized expression within these circuits under various conditions. A subset of these genes have been found to have similar specializations in human speech brain regions, and thus these potentially trait-causative genes from songbirds can then be studied in the context of human evolution for the same trait using comparative genomics, as we begin to do here. I hope that these experiments and analyses can serve as the beginnings of a framework for future experiments seeking to understand our own evolution through the study of human-convergent traits in non-human lineages

Investigating the Supracellular Processes Underlying Emergent Material Phase Properties During Embryonic Skin Morphogenesis

During embryonic development, vertebrate tissues are sculpted by the coordinated behaviors of cells.Vertebrate organs are complex, typically requiring the orchestrated dynamics of hundreds to thousands of cells. Yet, due to their functional significance, the mechanisms underlying organ formation are robust.This implies the need for mechanisms to constrain critical processes during embryogenesis. While much of developmental biology has focused on the aspects of self-organization that relate to patterns of chemicals and molecules across a tissue and their relationship to gene regulatory networks, less attention has been given to the physical mechanisms at play. In particular,it is poorly understood how, in complex tissues, cells self-organize in a robust manner. This line of inquiry has been expounded recently in a classic system for studying periodic pattern formation: the embryonic chicken skin. The formation of feather follicle primordia was shown to be mechanically driven by the developing dermis. Cellular pulling forces, balanced by tissue stiffness, generate periodically spaced multicellular aggregates of dermal progenitors. As the aggregates form, they compress epidermal cells, triggering molecular changes that initiate the feather follicle primordium gene expression program. Yet, two questions remained. How do cells coordinate and tune their mechanical forces across a field of cells in order to generate the correct pattern? Once the dermal condensate forms, what role do molecular signals from the epidermis have in three-dimensionally sculpting the feather follicle primordium? This thesis addresses these questions by examining tissue morphogenesis through the lens of regulatory processes that occur at the multicellular length scale. In Chapter Two, we develop an assay to reconstitute the initiation of follicle patterning ex vivo. We show that contractile cells rearrange and align the extracellular matrix (ECM). Reciprocal interactions between the cells and ECM, mediated by calcium signaling, progressively align the cell-ECM layer. This exchange transforms a mechanically unlinked collective of dermal cells into a continuum with coherent, long-range order. Combining theory with experiment, we show that this ordered cell-ECM layer behaves as an active contractile fluid that spontaneously forms regular patterns. In Chapter Three, we examine how molecular signals—BMPs and FGFs—enable transformation from a flat dermal condensate to a feather follicle primordium with three-dimensional architecture. By analyzing cellular and molecular patterns as the feather follicle primordium forms and matures, we show that distinct multicellular domains emerge during budding that spatially correlate with BMP and FGF activity. By reconstituting these BMP and FGF domains ex vivo, we show that FGF promotes solidification whereas BMP retains fluidity but enhances contractility. Furthermore, we show that a biphasic supracellular complex is sufficient to drive tissue budding, resulting in symmetry breaking in a new spatial dimension. Together, these results present a paradigm for how supracellular-scale material properties generate robust changes in tissue patterning and architecture

Use of Genetically Encoded Tools to Interrogate Mechanisms of Glutathione Homeostasis from Mitochondria to Mice

Glutathione is an evolutionarily ancient molecule that underlies many critical biological processes across all categories of life. It acts as a cofactor in some metabolic reactions, modifies proteins to modulate their activity, and participates in detoxification of xenobiotic compounds. Despite these wide-ranging functions, glutathione is best known as an antioxidant. Some consider it akin to the fountain of youth because it decreases throughout the course of aging while oxidative damage accumulates.In addition to aging, glutathione dysregulation occurs in a plethora of diseases, including neurodegenerative diseases like Alzheimer\u27s and Parkinson\u27s Disease, liver disease, and metabolic disease, among others. However, the specific roles of glutathione that contribute to disease progression remain poorly understood. In part, this is because the function of glutathione in cellular metabolism is still not fully characterized. Glutathione is thought to be critical for mitochondrial function, but it is not known how glutathione gets into mitochondria. Because of this, it has been very difficult to specifically deplete mitochondrial glutathione, resulting in circumstantial evidence that maintenance of glutathione abundance is critical for mitochondrial function. We set out to expand the tools available to characterize the function of compartmentalized glutathione pools. In the first half of this work, I engineered a bacterial enzyme to synthesize glutathione in mitochondria, effectively bypassing both endogenous glutathione synthesis and transport into mitochondria. Using this tool, I tried to identify the mitochondrial glutathione transporter by performing CRISPR-Cas9 screens that relied upon the assumption that the mitochondrial glutathione transporter(s) is/are essential for cell proliferation, and that enabling mitochondrial glutathione synthesis would enable identification of these no-longer essential genes. While this limited approach failed to identify the mitochondrial glutathione transporters, we identified new roles for several genes, including a mechanism of resistance to the most prevalently used glutathione synthesis inhibitor. To identify alternative molecules that may inhibit glutathione synthesis, I designed and performed a high-throughput chemical screen. I was able to identify several candidate molecules, although they are not direct inhibitors of glutathione synthesis. Further identification of the targets of these inhibitors may provide additional insight into the function of glutathione in cellular metabolism. Ultimately, in collaboration with several of my colleagues, we used alternative approaches to identify two putative mitochondrial transporters required for mitochondrial glutathione import. We found that, as expected, mitochondrial glutathione is required for cell viability. However, we found that this was due to glutathione\u27s role as a critical cofactor in iron sulfur biosynthesis, not due to its role as an antioxidant, emphasizing the importance of considering unbiased approaches to determine the function of glutathione in disease progression. In the second half of this work, I developed two mouse models with unregulated glutathione availability. I was surprised to find that that constitutively high glutathione is in compatible with embryonic development, demonstrating that homeostatic mechanisms of maintaining glutathione levels are critical for viability. However, unregulated glutathione synthesis was compatible with life in adult animals and resulted in an increase of GSH up to 5-fold in some tissues. Interestingly, this increase in GSH was compatible with normal tissue metabolism, underscoring that regulation of glutathione synthesis is required for some essential embryonic processes. Future studies with these mouse models with high glutathione will enable interrogation of glutathione sufficiency in disease progression

Fate, Phases, and Form in Vertebrate Organ Morphogenesis-Uncovering a Role of Morphogenesis at the Supracellular Scale

How morphology unfolds from fertilization to birth is one of the most fundamental questions in the life sciences. Self-organization in embryonic development, whereby organs robustly adopt forms through intrinsic processes, is a central feature that remains enigmatic. To achieve greater clarity of such processes, new conceptual and experimental approaches may be needed. Much of work in developmental biology in the past three to four decades has focused on cell and subcellular levels of organization. In line with work of some key conceptual thinkers, we revisit the notions of epigenetics. Rather than confining it to the molecular mechanisms of chromatin remodeling, we propose a broader understanding that includes processes beyond the cellular scale that possess their own generative power. Among the various scales at play, we propose to shift our focus to the mechanical and material processes at the supracellular scale. The hair or feather follicle in the skin represents one of the most apparent morphological features in development, and its spacing serves as a classic model system to study pattern formation. Mesenchymal-ECM relations are a key regulatory niche that remains poorly understood. By developing an ex vivo essay that reconstitutes the initiation of feather follicle pattern, we demonstrated that mesenchymal cell-ECM interplay can create supracellular structures independent of any morphogen activities. This challenges the classic chemical model where a morphogen pre-pattern dictates follicle patterning, prompting the question of what functional roles morphogens serve. found that morphogens enable the creation of membrane-less tissue compartments within the mesenchyme of the follicle with distinct biophysical properties, such as elasticity, viscosity and contractility. Specifically, fibroblast growth factor (FGF) promotes a stiff and solid hemispheric core compartment, whereas bone morphogenetic protein (BMP) promotes a surrounding fluid and active contractile margin compartment. Through their geometric arrangement, the two compartments are mechanically primed to break tissue symmetry and result in follicle budding. We also identified morphogen-enabled supracellular material property differences that were minimal or lost at cellular scales, which highlights the importance of shifting our focus to the supracellular scale in order to understand morphogenetic processes in development and diseases. This new paradigm redefines the role of morphogens, which requires distinguishing between the proximal effects of morphogens, such as gene regulation at the molecular and cellular scale, and their ultimate functional effects, which emerge at the supracellular scale

Towards a Better Understanding of Facial Movements: Computational Models for Perception, Characterization, and Neural Production

Facial movements are the primary medium for non-verbal communication and involve a complex orchestration of muscles controlled by the brain. The ability to interpret and produce these movements enables the expression of a wide range of emotions and social cues, all essential for social interactions. Despite their significance, the neural mechanisms governing facial movements remain poorly understood, hindered by the complexity of muscle coordination and the limitations of traditional, subjective, and labor-intensive analysis methods. To objectively understand facial motor control, this research adopts a three-pronged approach: 1) Developing and interpreting computational models within a novel multi-task training framework to simultaneously distinguish between facial expression and identity recognition, 2) Introducing a novel self-supervised Person-Specific Model (PSM) framework that extracts person-specific facial movements independently of other facial characteristics, enhancing facial muscle action characterization by leveraging individual differences, and 3) Utilizing data-driven computational models to analyze a unique dataset of single-cell recordings from sensorimotor cortex regions and behavioral video recordings of spontaneous, unconstrained, and naturalistic facial movements of macaques.This research first focused on developing computational models capable of separating facial movements from other characteristics like identity, which is challenging for computational models due to individual variations in facial movements and shapes. Utilizing Convolutional Neural Networks in a novel training framework, networks were trained simultaneously for facial identity and expression recognition, mirroring the human visual system\u27s face processing multi-tasking capability. This approach revealed functional segregation within the network, enabling the differentiation between a person\u27s identity and their expressions by dedicating different facial zones to solve each task and identifying task-specific facial features emerging from the network\u27s latest layers. Building on this, I introduced Person-Specific Model (PSM), an innovative self-supervised learning approach, which successfully extracts individual-specific facial movements independently of other facial characteristics. PSM stands out by leveraging individual differences, improving facial muscle action characterization. Its dual learning approach uniquely reveals a repertoire of facial movement primitives, capturing both universal patterns shared across individuals, and more complex, nuanced movements unique to each individual missed by other traditional methods. In parallel, this research explored the neural basis of facial movements, from larger movements like threats and chewing, to subtle, spontaneous movements in monkeys using naturalistic facial video recordings and single-cell neural data from sensorimotor cortex regions. A flexible computational framework was developed to analyze unconstrained continuous behavior. The analysis revealed that specialized neural patterns were linked to various movements; for example sensory area S1 was active during lipsmack, and primary motor cortex M1 was involved in actions like chewing and lipsmack. Distinct neural subspaces and neurons were associated with different social behaviors. The findings also revealed parallels between the neural dynamics of facial and well-studied arm motor behaviors; for example, threat expressions\u27 neural dynamics resembled reaching and sensory cortical areas like S1 exhibited unique dynamics during these expressions. Transitioning to continuous unconstrained behavior, this framework confirmed S1 and M1\u27s role in lower face movements, and importantly detected subtle neural-behavioral temporal patterns, like the role of anterior primary motor F4 in nose movements and ventral premotor PMV in eye movements, which traditional techniques failed to capture due to minimal movement variance in these facial locations. Particularly, distinct neural control strategies were identified, with regions like S1and M1 more active in larger expressive movements and PMV involved in more subtle movements, highlighting a sophisticated level of neural segregation between these different movement scales. Taken together, these results unveil a comprehensive picture of the intricate cortical control underlying facial movements, distinguishing between larger expressive motions and smaller, subtle actions. Crucially, this work underscores the need for standardized tools capable of analyzing spontaneous, unconstrained behaviors, beyond labeled expressions. I address this challenge in this analysis, promising a deeper understanding of natural behavior and its neural underpinnings

Molecular Mechanisms of Sensing and Synergy by Anti-Bacteriophage Immune Systems in Staphylococcus

Viruses parasitize every known life form on the planet for their propagation and spread. To deal with this constant assault, organisms across all domains of life ave evolved immune strategies in the form of genetically encoded systems to defend themselves. Immunity can be conceptualized as occurring in three fundamental stages. First, the immune system must identify an invading pathogen—either directly via recognition of a pathogen-associated molecular pattern (PAMP), or indirectly by sensing the perturbation of homeostasis during the process of infection( guard hypothesis ). Second, this information must be converted into molecular signals to be transmitted to the appropriate compartment within the cell or organism and to amplify downstream immune responses. Third,effect or programs must been acted to interfere with the virus\u27 ability to replicate and parasitize the host.Arising from these fundamental properties, immune systems are composed of multiple distinct components with specialized functions(e.g. the innate and adaptive arms of immunity in vertebrates)and involve a complex network of interactions. A viable immune response must therefore be able to optimally integrate multiple branches within this network in order to operate effectively—that is, to simultaneously enable robust immunity against any invading pathogen while limiting autoimmunity. Bacteriophages (phages)are viruses infecting bacteria that are thought to outnumber their hosts by a factor of ten to one in most environments. They are by far the most ubiquitous biological entities,estimated to exceed a staggering quantity of 1031 phage particles on the planet. As a result of this host-parasite conflict over billions of years, bacteria have evolved many diverse mechanisms of anti-bacteriophage defense, including many different innate systems as well as the adaptive CRISPR-Cas systems. In the last five years, dozens of new genes that confer bacterial immunity against bacteriophages have been identified through bioinformatic searches and more recently, functional screens. Many of these anti-bacteriophage defense systems have subsequently been validated and the details of their modes of activity have been revealed through experimental approaches.Although the mechanistic details of how these newly discovered defense genes interfere with bacteriophage propagation are rapidly being uncovered, there is a major gap in our understanding of how these systems sense bacteriophage infection in order to initiate immunity. Furthermore, it has only recently been recognized that within a single cell,bacteria commonly harbor many different defense systems—on average,five to seven distinct systems.Therefore, our understanding of how different defense systems interact with one another and the consequences of this crosstalk for the immune response and bacterial evolution remains in its infancy.In this thesis, I investigate these two fundamental, but nascent areas of the burgeoning field of bacterial immunology using staphylococci and staphylococcal phages as a powerful model system to study host-virus interactions. In the first part of my thesis (Chapters 1 and 2), I provide a broad introduction to the evolutionary conflict between bacteria and their phages, with a particular focus on staphylococci, a widespread taxon of bacteria with the utmost medical importance.I also discuss a conceptual framework for understanding how a successful immune response is generated in response to infection, with a focus on the current state of understanding of how bacterial defense systems are triggered by invading phages.In the second part of my thesis (Chapters 3 and 4), I demonstrate that a newly discovered defense system called CBASS, the bacterial homolog to the cGAS-STING innate immune pathway in eukaryotes, confers anti-viral defense in staphylococci.Specifically, a minimal type I CBASS operon from Staphylococcus schleiferi that is also broadly present in other species,including Staphylococcus aureus,can provide immunity against some, but not all, staphylococcal phages. I then detail our discovery and characterization of the CBASS-activating bacteriophage RNA (cabRNA), which we propose to be a phage-specific cue during infection that triggers the initiation of CBASS-mediated immunity.In the third part of my thesis (Chapters 5, 6, and 7), I focus on the current state of knowledge on immune crosstalk in prokaryotes, with a focus on how these interactions impact the strength and durability of immunity. Specifically, I explore the complex tripartite interactions between bacteriophages,S. aureus pathogenicity islands (SaPIs), and CRISPR-Cas systems and detail our discovery that SaPI-mediated parasitism of phages can stimulate the development of CRISPR-Cas adaptive immunity through the production of immunizing defective viral particles. In the last part of my thesis (Chapter 8), I discuss the many unanswered questions that remain and the exciting avenues of inquiry that extend from my research. I further discuss the mechanistic insights gleaned from these experimental studies and the implications that this work has on understanding the interactions between bacteria and bacteriophages.In conclusion,the research described in this thesis collectively reveals novel molecular mechanisms underlying the sensing o fbacteriophage infection by CBASS—the bacterial counterpart to cGAS—and the synergistic interactions between S. aureus pathogenicity islands and adaptive CRISPR-Cas immune systems in staphylococci.This work touches upon two fundamental features of every immune response across all domains of life: sensing and synergy. Furthermore, this work provides a foundation for future studies that will continue to uncover the details of how various anti-bacteriophage defense systems are activated during viral infection and additional modes of immune crosstalk in bacteria