Bacteriophage Endolysins: Anti-Infectives and Lysidiagnostics for the Post-Antibiotic ERA

Pathogenic bacteria represent a significant and increasing global health and safety concern. In addition, growing antibiotic resistance poses a serious threat to our ability to prevent and treat bacterial infection and disease. Addressing these threats will require the development and application of novel therapeutics and clinical approaches. The focus of our research is on one such novel approach, the use of bacteriophage endolysins as diagnostic (lysidiagnostics) and therapeutic (enzybiotics) tools for pathogens of human disease. Endolysins are modular proteins that facilitate release of progeny phage by digesting the bacterial host cell wall. Structurally, endolysins are composed of an enzymatically active domain (EAD) and a cell wall binding domain (CBD). It is the variable architecture of these protein domains that confers functionality and specificity toward the bacterial host. This work describes studies of endolysins against two pathogens of human disease, Paenibacillus, primarily Paenibacillus thiaminolyticus (P. thiaminolyticus) and Clostridium, specifically Clostridium perfringens (C. perfringens). Paenibacilli are gram-positive, aerobic or facultatively anaerobic, spore-forming bacilli of the soil microbiome. While not traditionally considered human pathogens, they can cause paenibacilliosis in neonates, an emerging disease presenting with sepsis, meningitis complicated by post-infectious hydrocephalus (PIH), and high mortality. Infections by anaerobic bacterial pathogens are primarily caused by gram-positive, toxinogenic, spore-forming members of the Clostridium genus, all of which can cause life-threatening disease. Their pathogenicity is largely mediated through the production of potent exotoxins, which can result in a wide spectrum of clinical manifestations ranging from gastrointestinal illness and enterotoxemia to neurotoxicity and tissue necrosis. Identification of multi-drug resistant (MDR) strains of this primarily food-borne pathogen serves as a bellwether for potentially widespread disease with high lethality. In this thesis, we identified and characterized endolysins for the selective killing of pathogenic Paenibacillus (Chapter 2) and Clostridium (Chapter 4), employing several existing methods. In addition, it was necessary to develop and optimize additional methods and protocols to complement and advance this research. These included: (1) bioinformatics and protein structural analyses to identify endolysin candidates, (2) mid- and high-throughput techniques for protein expression and purification, (3) screening assays for initial characterization of hydrolytic activity, and (4) modifications of existing methods to optimize in vitro bactericidal activity. For P. thiaminolyticus, we identified the first endolysin to be described for this organism (LysPt9) that could achieve \u3e3-logs of bactericidal activity at endolysin concentrations \u3c 100 μg/mL. This endolysin was active against three environmental P. thiaminolyticus strains, the MbaleIII strain derived from neonatal cerebral spinal fluid (CSF), and several other Paenibacilli. To our knowledge, this is the first described endolysin against P. thiaminolyticus that could be optimized for use in therapeutic development. We also identified and characterized endolysins for C. perfringens that are strong candidates for future use in a murine gnotobiotic gastrointestinal (GI) model of C. perfringens infection. These potent candidates are characterized by high protein yields and specific bactericidal activity in studies comparing C. perfringens strains with commensal bacteria derived from the murine gut. Additional studies assessed the use of endolysin cell binding domains (CBDs) for specific identification and potential diagnostics of P. thiaminolyticus (Chapter 3) and C. perfringens (Chapter 4). For these studies, we generated and purified fluorophore-labeled CBDs and optimized methods for fluorescence staining of their target organisms to image protein binding. Results with our candidates showed highly specific binding of fluorescently labeled CBDs to P. thiaminolyticus under conditions of mixed bacterial cultures. Similarly using labeled CBDs, we were able to specifically identify C. perfringens from other commensals in a murine GI microbiome model (Oligo-MM12). Finally, based on these studies, we performed preliminary experiments to develop a gnotobiotic murine model of C. perfringens infection for oral endolysin delivery (Appendix 7.1). Taken together, our work identifies novel therapeutic candidates and diagnostic tools to control and identify two major pathogenic bacteria, P. thiaminolyticus and C. perfringens

The Influence of DNA Methylation on Both H2A.Z Nucleosome Dynamics and the Zinc Finger and Homeoboxes Transcription Factor Family

DNA methylation represents a widespread and heritable modification that exerts substantial influence on chromatin organization and function in many eukaryotic genomes. The presence of DNA methylation has been strongly linked to transcriptional regulation, in both activating as well as repressive ways, though exactly how a small chemical mark can enact such drastic and seemingly opposing effects on gene expression remains an open question. In my thesis work, I describe the influence DNA methylation has on two classes of chromatin proteins, the histone variant H2A.Z and the Zinc Finger and Homeoboxes (ZHX) family of transcription factors. DNA methylation and H2A.Z preferentially occupy exclusive areas of the genome, biasing against their colocalization in the same genomic space. However, exactly how this mutual exclusion occurs has largely been left a mystery. We find that DNA methylation impacts H2A.Z nucleosome dynamics via both physical destabilization effects and influences on H2A.Z-specific nucleosome remodeler activity. Using single particle cryo-EM, we solved structures of human H2A.Z nucleosomes on methylated and unmethylated DNA, suggesting that the presence of methylated DNA can destabilize histone-DNA contacts and increase linker DNA opening/flexibility. This conclusion was corroborated by that observation that DNA methylation on H2A.Z nucleosomes increases its accessibility to a restriction enzyme, while the influence of DNA methylation on H2A nucleosomes is minimal. However, this change occurred to a much lesser degree than that induced generally by H2A.Z nucleosomes compared to canonical H2A nucleosomes. Through sequencing studies and the use of crude physiological cytoplasmic extract from Xenopus laevis eggs coupled with sperm nuclei, we confirm that the H2A.Z and DNA methylation antagonism is conserved in Xenopus laevis. We further find that the degree of this H2A.Z and DNA methylation antagonism increases, and that the amount of overlap between them decreases, in nuclei from a Xenopus fibroblast cell line compared to egg extract. Using the egg extract system, we show that DNA methylation inhibits the binding of the SRCAP complex, the primary nucleosome remodeler responsible for H2A.Z deposition, and that the loss of SRCAP abolishes H2A.Z’s preference for unmethylated DNA. From these results, we conclude that the SRCAP complex is the primary mediator driving the deposition of H2A.Z on unmethylated regions of the genome. During the course of characterizing proteins whose chromatin binding is affected by DNA methylation and H2A.Z, we identified members of the ZHX transcription factor family, ZHX2 and ZHX3, as preferential methylated DNA binders in Xenopus egg extract. The ZHX proteins are an enigmatic group of homeodomain (HD) proteins characterized by the presence of two N-terminal C2H2 zinc fingers followed by four to five tandem HDs. This family has largely been viewed as repressive factors, preventing gene transcription through inhibition of the pioneer factor, NF-YA. We find that the ZHX family members robustly and specifically bind to methylated DNA substrates over unmethylated. Many HD proteins have come to be recognized as DNA methylation sensors; however, conventional HD proteins typically only contain one HD and mediate methylated DNA binding through conserved residues. We asked whether the unique repetitive HD structure of the ZHX proteins play a role in DNA methylation recognition that differs from classic HDs. Using truncation mutants, we identify the first and second homeodomains as necessary for DNA binding in all ZHX family members (ZHX1, ZHX2, ZHX3). Structural predictions and sequence alignment analysis indicate that homeodomain 2 serves as the primary DNA binding domain and may recognize DNA methylation via a mechanism distinct from currently known methyl-binding homeodomain proteins. Additionally, we find that ZHX members bind chromatin in a cell cycle-dependent manner and that mitotic eviction of ZHX proteins is driven by a highly conserved N-terminal stretch of 13 amino acids which contains several predicted phosphorylation sites. Finally, we show that the ZHX2-ZHX3 heterodimer is the preferential dimer formed between ZHX members and that the ZHX members do not associate with NF-YA in either Xenopus egg extract or fibroblast cell lines. Altogether, our findings on how DNA methylation is able to repel chromatin elements like H2A.Z, which has a well-known regulatory role at promotors of active genes, as well as recruit putative repressive factors like the ZHX protein family help to shed light on the ways DNA methylation shapes the genomic regulatory landscape

Studies into Transcriptional Pausing Regulators and Drug Resistance in Mycobacterium Tuberculosis

Tuberculosis (TB) is the leading cause of death worldwide due to an infectious agent. Although this is true globally, the greatest impact of TB is concentrated in regions of the world that are plagued by poverty and scarce resources. Mycobacterium tuberculosis (Mtb) is the bacterium that causes TB infection. Mtb is spread from person-to-person by coughing infectious droplets. These droplets carrying Mtb take residence in the lungs of an infected person, where it can persist even in the absence of active symptoms. Part of what makes TB such a successful pathogen is its ability to evade antibiotics. Currently, drug-sensitive TB is treated by a cocktail of 4 antibiotics over the course of 4- 6 months. However, drug-resistant TB calls for additional second and third-line antituberculars and is prescribed for a total of 6-9 months. These treatments are far from ideal and result in a wide range of unpleasant symptoms, such as gastrointestinal, neurological, and hepatic side effects. For these reasons, it is incredibly difficult for patients to successfully complete TB therapy, which further exacerbates antibiotic resistance. Approximately 400,000 people develop drug-resistant TB each year. Therefore, new drug targets are desperately needed. Considering this, we are studying the transcription cycle of Mtb, which is regulated differently than the well-studied model bacterium Escherichia coli (Eco). While transcription initiation is well characterized in the field, later steps like elongation, pausing, and termination remain underexplored. Here, we use structural, biochemical, and functional approaches to characterize later steps of the Mycobacterial transcription cycle. We aim to shed light on vulnerabilities within the cycle that may be targeted by therapeutics. We are therefore studying RNA polymerase (RNAP), the central enzyme of transcription, and associated transcription factors. We aimed to study pausing, an essential regulatory step occurring during elongation. Elongation is not constant but is instead intermittently interrupted by “pausing” events where the RNAP stops and waits for further instructions from the cell before resuming transcription. Pausing is regulated by transcription factors. One such factor is known as NusG- the only transcription factor conserved across all three domains of life. NusG was identified as an anti-pausing factor in Eco, meaning that it decreases pausing. Our studies have identified the previously unknown function of Mtb NusG. We conducted in vitro transcription assays, structural studies, and in vivo functional studies to reveal that Mtb NusG is a pro-pausing factor, opposite to Eco. This pro-pausing activity is modulated by a conformational change in the RNAP known as “swiveling”, involving the transition of the enzyme into an inactive state that is incompatible with nucleotide catalysis. Using single particle analysis, we show structurally how Mtb NusG stabilizes the swiveled state, promoting pausing, while Eco NusG stabilizes the “anti-swiveled” state, promoting elongation. Following this study, we sought to investigate the clinical relevance of pausing. Rifampicin is the most potent antitubercular used in the clinic. A single point mutation in the β subunit of RNAP, βS450L, accounts for ~70% of all Rifampicin-resistant (RifR) Mtb in the clinic. The presence of the βS450L mutation reduces transcription speed by 4-fold leading to over-pausing and over-termination relative to wild type (WT). This hyper-termination effect results in a fitness defect. However, there are secondary, compensatory mutations existing in the RNAP and other transcription factors that relieve hyper-termination effect and restore fitness to the cell. Using biochemical and functional, in vivo studies, we showed the first mechanism of RifR compensation in NusG. Utilizing a genome-wide association study, we discovered novel compensatory mutations in NusG. These mutations alleviate the fitness cost of βS450L and restore transcription speed close WT levels. Beyond NusG mutants, there are also compensatory mutations in RNAP subunits α and β’, or rpoA and rpoC, respectively that correct for RifR fitness defects. These are in fact the most common type of compensatory mutations. Following up on our work into RifR compensatory evolution in nusG, we are investigating the mechanisms by which the RNAP mutants rescue RifR fitness defects using structural and biochemical approaches. The data presented in this thesis both characterizes the structure and function of the universal transcription factor NusG as a pro-pausing factor in Mtb and also shows how the later transcription steps of pausing and termination could serve as targets for antituberculars. We show the first mechanism of RifR fitness compensation and the importance of maintaining ratios of elongation and termination for optimized bacterial fitness. Targeting pausing and termination and associated factors to enhance swiveling could exacerbate the fitness defect associated with RifR βS450L Mtb, combatting drug resistance. Similar compensatory mechanisms are known to exist in other subunits of RNAP, rpoA and rpoC. Understanding how these mutations work mechanistically could shed light on novel targets for inhibiting drug-resistant TB

An Angular Working-Memory Signal that Guides Drosophila Navigational Trajectories

The navigational behavior of many species can be guided not just by immediate sensory input, but also by memories of the environment. In fruit flies, for example, neural signals in a well-studied brain region called the central complex help guide navigation. Previous work has shown that “compass neurons” in the central complex track the fly’s heading angle, while a second set of central complex neurons track the fly’s goal angle. The difference between the fly’s heading and goal angles is computed to produce a steering signal. I discovered that a third set of central complex neurons, called hΔA cells, integrate the fly’s recent traveling direction over a window of ~7-10 s, creating a working memory of the fly’s trajectory. This signal feeds into the circuit just described, promoting flies to continue walking in their recent traveling direction, like an inertia term for the steering circuit. We show that this inertia-like term contributes to a memory-based navigation task, complementing the role of the previously described goal signal in that task. Interestingly, the activity of hΔA neurons depends on behavioral context, dropping significantly in a task where trajectory inertia is unlikely to be helpful. The hΔA signal is built through a circuit motif that repeats a few times in the central complex. This recurring architecture might allow the fly brain to construct a range of memories and goals with distinct spatiotemporal properties that can be used during navigation

Antibody-Mediated Modulation of Polyclonal Immune Responses

Upon pathogen exposure, two main lines of defense are sequentially activated: the innate immune response and the adaptive immune response. The innate immune response is the first line of defense, and consists mainly of myeloid cells that are quickly activated within hours to days to engage in a non-specific manner. This broad response is followed by the adaptive immune response, which progresses more slowly but is capable of generating pathogen-specific immunity. Together, the innate and adaptive immune systems coordinate and facilitate pathogen clearance. A novel feature of the adaptive immune response is the formation of immune memory which facilitates a more rapid and efficient response upon re-exposure to the same or similar pathogen. This memory can be cellular, such as memory B & T lymphocytes, or humoral, such as antibodies. Memory B and T lymphocytes have been well-characterized to differentiate into various effector cells upon re-exposure, whereas antibodies are classically known to block and neutralize circulating pathogen via various effect or functions. Additionally, accumulating evidence has suggested that antibodies themselves are capable of enhancing or suppressing endogenous immune responses; however, the exact manner and mechanisms by which antibodies exert this modulation remains unclear. The first part of this thesis focuses on defining how antibodies can modulate the development of a polyclonal immune response. Infusion of a monoclonal antibody prior to immunization with its cognate protein antigen resulted in two key observations. Firstly, antibody can increase the overall magnitude of the germinal center (GC) and plasmablast (PB) response. Secondly, this enhancement is accompanied by a simultaneous decrease in the fraction of these compartments with measurable antigen binding. This modulation capability was conserved with alternative antigen-antibody combinations, suggesting it is a broadly applicable feature of immune responses. Examination of the mechanisms that drive the enhanced magnitude of the GC and PB response elucidated that while T cells are required for this modulation, no statistically significant differences were observed in T cell compartments  themselves, such as early expanding T cells and T follicular helper cells (Tfh). Moreso, further experimentation illuminated that this enhancement capability was largely independent of complement receptors 1 and 2 (CR1/2) but was instead dependent on interactions with Fc gamma receptors (FcγRs). Both immunohistochemistry and flow cytometry revealed that infused antibody generated immune complexes (ICs)are retained across the lymph node on classical IC retaining follicular dendritic cells (FDCs) and hematopoietic cells such as macrophages and dendritic cells, but that this IC retention is significantly reduced in FcγR deficient mice. Altogether, the data suggests that antibody can facilitate enhanced IC retention and larger magnitude GC and PB responses in a FcγR dependent manner. Despite the FcγR dependence of the antibody-mediated enhanced magnitude of the GC and PB response, the reduction in the fraction of detectable antigen-binding cells in these compartments was found to be independent of FcγRs. Notably, this reduction was observed throughout the seeding phase of the response, suggesting that antibody can recruit a more affinity-diverse pool of B cells. Altogether, antibody via FcγR dependent and independent mechanisms can influence both the magnitude and quality of the immune response

Uncovering New Mechanisms of Type III CRISPR-Cas System Inhibition and Effector Function

Bacteria and their viruses, called bacteriophages (phages), are in a constant arms race for survival. Phages are one of the most abundant biological entities in the world, with an estimated 1031on earth, outnumbering bacteria ten to one. To defend against phages, bacteria have evolved numerous defense mechanisms. One such defense system is the bacterial adaptive immune system called CRISPR-Cas, which consists of clustered, regularly interspaced short palindromic repeats (CRISPRs) and a set of genes that encode CRISPR-associated (Cas) proteins. While there are seven distinct types of CRISPR-Cas systems identified thus far, we focus on specifically the type III CRISPR system which contains a multi-protein effector complex that functions together with its crRNA in target binding and cleavage. During type III CRISPR-Cas immunity in prokaryotes, RNA-guided recognition of viral (phage) transcripts stimulates the Cas10 complex to convert ATP into cyclic oligoadenylates. These act as signaling molecules that bind to CRISPR-associated Rossmann Fold (CARF) proteins and activate their effector domains. The effector part unleashes various activities that are toxic to the host cell, inhibiting cell growth and leading to growth arrest. We are now beginning to uncover that CARF effectors use a wide array of mechanisms of action. In the first part of this study, we report the structure and function of the Cap1 effector, composed of a pair of transmembrane helices (TM1/2), a CARF-like (CARFL) domain and a domain of unknown function (DUF4579). Cryo-EM studies on apo-and ligand-bound states of Cap1 in glyco-diosgenin detergent revealed the formation of tetrameric complexes in both states, with one cyclic tetra-adenylate (cA4) molecule bound in a pocket composed by the four CARFL domains. Binding of cA4 triggers conformational changes that widen an otherwise narrow pore formed by the four TM1/2 domains. In vivo, Cap1 activation results in membrane depolarization, a growth arrest of the bacterial host and the abrogation of the viral lytic cycle. Our findings reveal the mechanistic basis of membrane depolarization mediated by cyclic nucleotide signaling during the type III CRISPR-Cas response. On the other side of the bacteria-phage arms race, phages have evolved mechanisms to counteract CRISPR-Cas; many viruses express anti-CRISPR (Acr) proteins that interact directly with Cas proteins and inactivate them. To date, there are over 40 Acrs of the type II CRISPR-Cas systems, but in comparison, there are only 4 confirmed Acrs of the type III systems. To identify novel inhibitors of the type III-A system, we performed a transformation screen with a type III-A CRISPR system in E.coli and environmental DNA (eDNA). In the second part of this study, we identified a single open reading frame, ORF6, from the MA1 eDNA library. We show that ORF6 inhibits CRISPR immunity against plasmids and phages in vivo and has high sequence and structural homology to known oligoribonucleases. As we continue to elucidate ORF6’s mechanism of action, we hypothesize that, while it may not be phage encoded, ORF6 could reveal an interesting incompatibility with the type III CRISPR-Cas system that will deepen our understanding of the function of type III CRISPR in bacteria

Structural and Functional Studies of the SARS-CoV-2 NiRAN Domain

The COVID-19 pandemic was a dramatically disruptive event, wreaking devastation across the globe and radically impacting nearly every facet of human life. For the first time, a significant amount of global money and attention was directed at coronavirus (CoV) research, propelling the field to the forefront of biomedical research. This allowed for the rapid development of a suite of vaccines and antivirals targeting SARS-CoV-2 that aided in ameliorating the effects of the pandemic. SARS-CoV-2 was the third CoV zoonosis of the 21st century and is now endemic. Global understanding of the threats posed by CoVs circulating among animals in the wild has increased, powering the urgency for new research into CoV biology. We study CoV replicase enzymes to shed light on the mechanisms underpinning viral replication and gene expression in the hope to elucidate biology conserved across viruses and better define therapeutic targets. The enzymatic activity of the SARS-CoV-2 Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5′ cap formation, while the role of NMPylation is unclear. Prior to this work, the structural mechanisms for these diverse enzymatic activities have not been properly delineated, nor have any drugs targeting this domain been clinically approved. In our initial work, we determined high-resolution cryo-electron microscopy (cryo-EM) structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions with their preferred substrates. These data revealed diverse nucleotide binding poses and divalent metal ion coordination sites within the NiRAN domain that promote its repertoire of activities. The deRNAylation/capping structure explained why GDP is the preferred substrate for the capping reaction over GTP. Altogether, these findings enhanced our understanding of the promiscuous activity of the CoV NiRAN domain and provided an accurate structural platform for drug development. Following this study, we sought to address a flawed study published in Cell claiming to have defined the structural basis of GTP-mediated capping by the NiRAN domain. We demonstrated that their model is not supported by their cryo-EM data and is incompatible with fundamental chemical principles. We discuss our failures, and those of the original authors, at reprocessing the data and identifying an authentic intermediate, clarifying that the mechanism remains unknown. Correcting this model was critical to restoring focus and reorienting the field toward supported models of CoV mRNA capping. Additionally, we present work that details a pipeline for evaluating inhibitors against the NiRAN domain. This pipeline includes two high-throughput assays to monitor NiRAN NMPylation and capping activities, respectively, as well as a means of detecting binding to the NiRAN domain. We reveal early data suggesting the existence of a druggable cryptic pocket in the NiRAN domain and present a path to structurally characterizing the pocket. Finally, we also share preliminary efforts at isolating the pore complex that gates the CoV replication organelle, with the ultimate goal of cryo-EM analysis, work that has the potential to address a host of unknowns in CoV replication

Dining in the Dark ATP Sensing in the Female Aedes Aegypti Mosquito

Blood feeding is fundamental for female Aedes aegypti mosquitoes, which require this protein-rich meal for egg development. After being attracted to a human host by sensory cues such as body odor, body heat, and carbon dioxide exhaled in human breath, the mosquito lands and probes under the skin to feed on blood. Probing uses the stylet, a sharp appendage ensheathed in the mosquito labium that punctures the skin and is highly specialized for blood ingestion. Previous work from the lab with GCaMP calcium imaging of stylet neurons showed that approximately 50% of stylet neurons respond to blood, and that these same neurons respond to a mixture of saline and ATP. However, ATP alone activates only a small subset of 3-7 stylet neurons compared to those activated by blood or saline and ATP. Despite this initial view into what activates feeding using the stylet, the mechanism by which ATP stimulates mosquito feeding, whether the stylet encounters ATP in vivo during feeding, and what receptor and what neurons respond to ATP remains unknown. This work addresses each of these questions. We confirmed classic findings that ATP is a potent feeding stimulant and showed that brief exposure is sufficient to cause sustained ingestion of an ATP-free saline solution that is normally not appetitive. This suggests that ATP is a trigger of feeding and is not required for sustained intake. To ask if the stylet encounters ATP in the course of probing for blood under the skin, we adapted an in vivo mouse skin feeding assay in which we visualized the Aedes aegypti mosquito stylet inside the skin, searching for a blood vessel in real time. We found that the female stylet is highly effective in locating subdermal blood capillaries and that most feeding directly targets the capillaries, a process called “capillary feeding.” In some cases, the stylet obtains blood that is released locally from damaged blood vessels, a process called “pool feeding.” We expressed a genetically-encoded ATP sensor in mouse skin and discovered that ATP is released from skin cells in close proximity to where the stylet is probing inside the skin. This suggests that ATP is released in vivo and has the potential to activate the mosquito stylet neurons during probing. This raises the question of what receptor detects ATP in stylet neurons. Since the Aedes aegypti genome does not contain any orthologs of vertebrate extracellular P2X or P2Y ATP receptors, we focused on ionotropic receptors (IRs), a large family of chemosensory receptors in insects that has previously been shown to respond to stimuli ranging from olfactory to taste to thermal cues. IRs form heteromeric ligand-gated ion channels composed of a ligand-selective receptor subunit and a ligand-insensitive co-receptor. We showed that mosquitoes lacking either of the two major IR co-receptors, Ir25 and Ir76b, show profound defects in feeding. Previous GCaMP calcium imaging work was conducted by monitoring activity in all neurons in the stylet. To narrow down the neurons that respond to ATP to smaller subsets of neurons, we carried out GCaMP imaging in strains in which either Ir25a or Ir76b neurons expressed GCaMP. In such experiments, we found that Ir25a neurons respond to ATP, while Ir76b cells show little or no response. This suggests that the ATP receptor is likely comprised of a ligand-selective IR in complex with Ir25a. We next looked for candidate ligand-selective IRs that could be responsible for ATP sensing. With this candidate gene approach, we identified Ir100c as a putative ATP receptor in female stylet neurons. We generated single nucleus RNA-sequencing (snRNA-seq) data from the stylet and found a small number of chemosensory neurons that express Ir100c. To evaluate the expression and distribution of Ir100c in the female stylet, we performed RNA fluorescence in situ hybridization and found 3-7 Ir100c-positive cells, in agreement with the number of cells that respond to ATP in the calcium imaging experiments. Using gene-targeted mosquito lines that express fluorescent markers either in all neurons or only Ir25a or only Ir76b neurons, we found that Ir100c is expressed in neurons and that 96.9% of Ir100c-positive cells co-express Ir25a, hinting that Ir100c may assemble with Ir25a as a functional ion channel. Finally, we worked in collaboration to develop AlphaFold3 structure predictions that are consistent with the model that a heterotetramer composed of Ir100c and Ir25a is a potential extracellular ATP receptor. This work demonstrates the importance of ATP sensing for mosquito blood-feeding behavior, identifies a minimal subset of neurons in the stylet that respond to ATP, provides evidence that Ir100c+Ir25a is a novel candidate ATP receptor, and establishes ATP signaling as a potential pathway for manipulating blood-feeding arthropods to prevent disease transmission

Functions of RTF2 in DNA Replication and Checkpoint Control

The composition of the protein machinery that copies DNA, called the replisome, is dynamically regulated across S phase to promote the accurate and complete duplication of the genome before cell division. Work in our laboratory has uncovered one such dynamic process at replication forks experiencing replication stress, by which proteasome shuttle proteins DNA damage inducible 1 and DNA damage inducible 2 (DDI1/2) regulate the level of replication termination factor 2 (RTF2) at the replisome. When RTF2 is retained at stressed forks, the response to replication stress is compromised and replication forks fail to restart efficiently, leading to genome instability. However, the function of RTF2 within an active replisome has remained uncharacterized. Here, we examine the function of RTF2 across an unperturbed mammalian cell cycle, with a focus on RTF2’s activities at the replisome. We find that RTF2 is essential for maintenance of DNA replication elongation rates and control of replication origin firing. By comparing the composition of RTF2-deficient to RTF2-competent replisomes, we find that RTF2 recruits the heterotrimeric enzyme RNase H2 to the replication fork. Downstream of failed RNase H2 recruitment to the replisome, RTF2-deficient cells accumulate genomically embedded ribonucleotides. In DDI1/2-depleted cells, retention of RTF2 and RNase H2 at the replication fork leads to inefficient replication restart after fork stalling. We propose that retained RTF2-RNase H2 at the replication fork leads to inefficient RNA primer retention. Cells lacking RTF2 replicate DNA slowly and fire excess replication origins in S phase. We find that RTF2-deficient cells bypass the intrinsic S/G2 checkpoint and prematurely accumulate mitotic markers while replication proceeds. To understand the mechanism of checkpoint bypass in these cells, we performed an in silico protein interaction screen using AlphaFold-Multimer. We identified a high-confidence interaction between RTF2 and checkpoint kinase 1 (CHK1), an essential kinase that functions downstream of ataxia-telangiectasia and Rad3-related (ATR) in the intrinsic S/G2 checkpoint, which we confirmed in cells. Remarkably, cells lacking RTF2 fail to recruit CHK1 to replication forks. Abrogation of the putative RTF2 and CHK1 interaction leads to diminished, but not absent, recruitment of CHK1 to replication forks and increased accumulation of mitotic markers while replication proceeds. We find that RTF2 interacts with the essential CMG-interacting and CHK1-activating protein CLASPIN. Our findings show that CLASPIN, like RTF2, localizes CHK1 to replication forks and implicate CLASPIN in control of CHK1 activity during an unperturbed S phase. These findings uncover how RTF2 and CLASPIN poise CHK1 at replication forks to facilitate its switch-like activation by ATR, creating fork-localized CHK1 activity that fuels unperturbed S phase progression and promotes genome stability by maintaining the intrinsic S/G2 checkpoint. As ATR and CHK1 function to halt the cell cycle in response to replication stress, we next evaluated whether RTF2 may function in the replication stress response pathway. We find that RTF2-deficient cells fail to activate CHK1 in response to acute treatment with low doses of replication stress-inducing agents, suggesting that CHK1 localization to replication forks facilitates its dynamic activation by ATR in response to stress. We show that RTF2 and CHK1 are removed from replication forks experiencing low levels of replication stress and propose that this removal is necessary for robust, global activation of CHK1-mediated cell cycle arrest. We further identify a putative ATR phosphorylation site within RTF2’s amino acid sequence that is essential for maintaining DNA replication rates. Finally, we uncover a relationship between RTF2 and another checkpoint protein, monopolar spindle kinase 1 (MPS1), which monitors microtubule-kinetochore connection in the spindle association checkpoint (SAC). RTF2 and MPS1 share extensive co-dependency overlap in cancer cell lines and interact by immunoprecipitation. RTF2- deficient cells exhibit exquisite sensitivity to low-dose inhibition of MPS1, suggesting that RTF2 may further function in the SAC to prevent premature cell division

Investigation of Diverse CARF Effectors in Type III CRISPR - Cas Immunity

All domains of life can be infected by viruses. The simplest forms of life, bacteria, are outnumbered by their viruses, called bacteriophage or phage, by 10 to 1. It is estimated there are more phages on Earth than stars in the sky. Phage act as parasites to bacteria by ejecting their DNA into the cell and hijacking host machinery to produce more viral particles. Once new phage particles are assembled inside the cell, the bacterium will lyse, leading to not only to the death of the bacterium but also the proliferation of more viruses. Bacteria encode a range of strategies to mitigate viral infection. One such example is an adaptive immune system encoded in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) loci. DNA repeats are separated by short sequences, called spacers, which are acquired from invading genetic elements and incorporated into the CRISPR array by CRISPR-associated (Cas) proteins Cas1 and Cas2.The CRISPR locus also encodes either a multi-protein complex or single protein with nuclease activity. Upon infection via phage or plasmid, the host will transcribe the CRISPR array, generating guide RNA sequences called crRNA. These crRNA will associate with a CRISPR nuclease to create an RNA-guided nuclease that mediates site-specific degradation of invader DNA or RNA that is complementary to the crRNA. The type III CRISPR-Cas system is also able to synthesize a cyclic small molecule from ATP through the cyclase domain of Cas10. This molecule, termed cyclicoligoadenylate (cOA), typically contains 3, 4, or 6 cyclized AMP molecules to produce cA3, cA4, or cA6, respectively. This signaling molecule can, in turn, activate an accessory protein of type III CRISPR. Several accessory proteins have been characterized to date. They often contain a CARF domain to sense cOA and an additional effector domain. Binding of cOA leads to a conformational change in the effector domain, which then provides immunity on a community level by inducing a growth arrest of individual infected cells. In my doctoral studies, I have characterized novel CARF effectors and also investigated the role of canonical CARF effector, Csm6, in mediating immunity against mutant targets. The first proteins I characterized are closely related homologs Chp1 and Chp2 (CRISPR-associated HAD phosphatase). These proteins were first identified in a deep search bioinformatics study.1I determined that these proteins function by inducing a growth arrest in the cell through depletion of essential NTPs. Next, I characterized a novel CARF effector containing a PIN domain, which was bioinformatically identified by another graduate student in the lab, Christian Baca. I demonstrated that the PIN-CARF effector depletes RNA upon activation and alone is sufficient to provide antiviral immunity. Lastly, I studied the role of canonical CARF effector Csm6. I genetically dissected the nuclease activity of Cas10 and the RNase activity of Csm6 in the type III CRISPR response and demonstrated that Csm6 is more often required for immunity when a target sequence contains mismatches against the crRNA. From my work, I have contributed to the growing arsenal of diverse CARF effectors and demonstrated, in the case of Csm6, an advantage accessory proteins yield by leading to efficient targeting of mutated target sequences