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Title
MEMBRANE-LOCALIZED TRANSCRIPTION REGULATORS : UNDERSTANDING POST-TRANSLATIONAL REGULATION AND SINGLE-MOLECULE DYNAMICS OF TCPP IN VIBRIO CHOLERAE
Creator
Demey, Lucas Maurice
Date
2022
Collection
Electronic Theses & Dissertations
Description
Vibrio cholerae is a Gram-negative gastrointestinal pathogen that has evolved an elegant regulatory system to precisely time production of essential virulence factors. A key step in this regulatory system is the transcription of a soluble AraC-like transcription factor, ToxT. ToxR and TcpP, two membrane-localized transcription regulators (MLTRs), positively regulate toxT. Much work has contributed to our understanding of TcpP and ToxR regulation, yet major gaps remain in our knowledge of...
Show moreVibrio cholerae is a Gram-negative gastrointestinal pathogen that has evolved an elegant regulatory system to precisely time production of essential virulence factors. A key step in this regulatory system is the transcription of a soluble AraC-like transcription factor, ToxT. ToxR and TcpP, two membrane-localized transcription regulators (MLTRs), positively regulate toxT. Much work has contributed to our understanding of TcpP and ToxR regulation, yet major gaps remain in our knowledge of these MLTRs. MLTRs are unique one-component signal transduction systems because they respond to extracellular stimuli by influencing gene transcription from their location in the cytoplasmic membrane. In Chapter 2, I explore the prevalence and diversity of MLTRs within prokaryotes to enhance our understanding of TcpP and ToxR. I show that MLTRs are far more common among prokaryotes than previously anticipated and that MLTRs are an understudied class of transcription regulators. In Chapter 3, I describe the use of super-resolution single-molecule tracking to investigate how TcpP, a model MLTR, identifies the toxT promoter. I provide evidence that TcpP binds to the toxT promoter independent of ToxR, and TcpP transitions to a specific diffusion state. The data support the first biophysical model for how TcpP-like MLTRs locate their target promoters. TcpP is subject to a form of post-translational regulation known as regulated intramembrane proteolysis (RIP). RIP of TcpP results in its complete inactivation, resulting in loss of virulence factor production. TcpH inhibits RIP of TcpP under certain pH and temperature conditions. In Chapter 4, I describe the mechanism TcpH employs to inhibit TcpP RIP while V. cholerae is present in the mouse gastrointestinal tract. I demonstrate that the dietary fatty acid α-linolenic acid enhances inhibition. I also show that α-linolenic acid promotes TcpH-mediated inhibition of TcpP RIP by increasing association of both proteins with detergent-resistant membrane (DRM) domains. My work provides the first evidence that DRMs influence virulence factor transcription in V. cholerae and that a dietary fatty acid promotes V. cholerae pathogenesis.
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Title
The impact of lactobacillus and bacteriophage on group b streptococcus and the placental membranes
Creator
Shiroda, Megan
Date
2019
Collection
Electronic Theses & Dissertations
Description
The human microbiota encompasses the microbes that live on and in the human body. While some body sites including the vaginal and intestinal tracts have been studied extensively for their role in human health, other body sites have been historically considered sterile and are less studied. One such site is the placental membranes that surround the fetus during pregnancy and serve as an important protective barrier during pregnancy. Several studies have established which bacteria are found in...
Show moreThe human microbiota encompasses the microbes that live on and in the human body. While some body sites including the vaginal and intestinal tracts have been studied extensively for their role in human health, other body sites have been historically considered sterile and are less studied. One such site is the placental membranes that surround the fetus during pregnancy and serve as an important protective barrier during pregnancy. Several studies have established which bacteria are found in this site, but few studies have been conducted to characterize their interactions in vitro or to understand their impact on the placental membranes. Further, our knowledge of the viral component of the microbiome in human health remains incomplete.In this dissertation, Lactobacillus, a well-studied probiotic in other body sites, was evaluated for its effect in the placental membranes. As these membranes were previously considered sterile, we sought to assess the ability of Lactobacillus to colonize these cells and evaluate its impact on them. A cell line model of the outermost layer of these membranes, the decidual cells, was used to establish that Lactobacillus can associate and impact a known cell signaling pathway, the Mitogen Activated Protein Kinase (MAPK) pathway, which is associated with inflammation and host cell death. Total protein level of p38, an important upstream protein in this pathway, was found to be significantly lower with Lactobacillus than in mock infection. These data suggest that Lactobacillus could maintain a commensal interaction in the placental membranes as described in other body sites. Lactobacillus is also known to inhibit pathogen invasion. Group B Streptococcus (GBS) can ascend from the vaginal tract to infect placental membranes, triggering premature birth or neonatal infection. Four Lactobacillus strains representing three species were characterized to assess their impact on two GBS strains (colonizing and invasive). We found live Lactobacillus does not affect GBS growth or biofilm production. L. gasseri increased association of both strains of GBS to the decidual cells but did not result in increased invasion of the cells. Instead, co-culture with Lactobacillus reduced host cell death. Secreted products of Lactobacillus drastically reduced growth in 35 GBS strains that broadly represent GBS diversity and could prevent biofilm formation; this inhibition was strain dependent. Unfortunately, increased GBS-induced host cell death with Lactobacillus supernatants was observed. Collectively, these data suggest that both live Lactobacillus and its supernatant could impact GBS interactions with the placental membranes.Bacteriophage are one of the most abundant members of the microbiome but their impact on opportunistic pathogens such as GBS remains unknown. As GBS can be isolated from gastrointestinal tract, we hypothesized fecal phage communities would inhibit the growth of GBS in vitro. Approximately 6% of the tested communities inhibited the growth of 35 GBS strains. To better understand differences in GBS strain inhibition, we examined capsule, sequence and clinical types of the strains. As no significant differences were found with these traits, we next examined Clustered Regularly Interspaced Palindromic Repeats (CRISPR), which serve as an adaptive immune system against invading foreign DNA by the acquisition of spacer sequences. GBS strains with fewer than nine spacer sequences were less likely to be lysed by a phage community than strains with more than sixteen spacers. We further hypothesized that presence of GBS in the corresponding bacterial component of each phage community would correlate to its ability to inhibit GBS growth. While this correlation did not exist across all GBS strains tested, sensitive strains of GBS were significantly more likely to be inhibited by phage communities with a lower abundance of GBS. Collectively, these data suggest that the phage component of the intestinal microbiome could impact GBS colonization. To further characterize these interactions, an individual bacteriophage should be isolated from these communities.
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Title
CHEMICAL BIOLOGY AND GENETIC STUDIES TARGETING THE MYCOBACTERIAL CELL ENVELOPE
Creator
Williams, John Tison
Date
2021
Collection
Electronic Theses & Dissertations
Description
Mycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell...
Show moreMycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell phenotypic screen I identified novel inhibitors of this valued target. A combination of lipid profiling and an innovative competitive binding assay supported MmpL3 as the target of these inhibitors. Cross resistance profiling of MmpL3 inhibitors against twenty-four unique mmpL3 Mtb mutants demonstrated that the level of resistance is associated with the proximity of resistant mutants to essential residues for protein function. Further, these resistance profiles suggested that MmpL3 inhibitors fall into separate clades depending on their chemical scaffolds. The results of this screen led to the development of novel potent analogs for one of the identified MmpL3 inhibitors, HC2099. These analogs were active against clinically relevant drug resistant Mtb strains that cause treatment failure in patients. Active analogs were able to kill Mtb inside of infected macrophages, an infectious niche of Mtb, without inducing cytotoxicity against these important immune cells. One of these analogs, MSU-43085, was orally bioavailable and successfully inhibited Mtb growth in infected mice, supporting further development and highlighting the therapeutic potential of this series. High throughput screens are often used to identify new inhibitors of Mtb growth. However, prioritized hits form these screens often identify similar targets such as MmpL3, lipid synthesis enzymes, redox cyclers, as well as inhibitors of the electron transport chain. Follow up studies of these inhibitors are often time consuming, costly and result in the rediscovery of previously identified targets. While this is not necessarily detrimental to Mtb drug discovery, as these reoccurring targets have therapeutic potential. The continued prioritization of inhibitors for these common metabolic pathways potentially limits the identification of inhibitors for novel targets. Therefore, additional steps that identify inhibitors of these common pathways could reshape how high throughput screen hits are prioritized. By applying the targeted mutant screen used to identify MmpL3 inhibitors to a non-prioritized library of hits from a high throughput screen, we identified more than fifty new potential MmpL3 inhibitors. Using an iterative strategy of applying additional mutants of commonly identified targets, this strategy promises to lead to parallel follow-up studies of inhibitors with known and unknown mechanisms of action. The ability of Mtb to enter into quiescent states in response to host stresses is one of the leading causes for the extended time to cure and evolution of drug resistance. These states can be induced by several environmental stresses including acidic pH, hypoxia, and others. In an effort to study this adaptation in the rapidly growing mycobacterial species M. smegmatis, we identified a lethal sodium citrate phenotype. Transcriptional profiling and genetic screening of mutants tolerant to sodium citrate indicated that this phenotype was due to the combined action of both chelation and osmotic stresses. Cell viability could be reduced from sodium citrate killing by cation and osmoprotectant supplementation. From these experiments we propose a model that can be applied to study carbon source uptake and probe the role of genes identified from the forward genetic screen with unknown function
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Title
COMPOSITION AND FUNCTION : UNRAVELING THE ROLE OF GROUP B STREPTOCOCCAL MEMBRANE VESICLES
Creator
McCutcheon, Cole Ross
Date
2022
Collection
Electronic Theses & Dissertations
Description
Group B Streptococcus (GBS) is an opportunistic pathogen that asymptomatically colonizes the vaginal tract of approximately 30% of women. In individuals with an altered immune state such as the elderly, pregnant women, and neonates, GBS can cause invasive infections. During pregnancy, GBS frequently contributes to infections in utero that can result in chorioamnionitis, preterm birth, or miscarriage. The bacterial factors that promote these adverse outcomes, however, are under studied. For...
Show moreGroup B Streptococcus (GBS) is an opportunistic pathogen that asymptomatically colonizes the vaginal tract of approximately 30% of women. In individuals with an altered immune state such as the elderly, pregnant women, and neonates, GBS can cause invasive infections. During pregnancy, GBS frequently contributes to infections in utero that can result in chorioamnionitis, preterm birth, or miscarriage. The bacterial factors that promote these adverse outcomes, however, are under studied. For instance, previous reports have demonstrated that GBS produces membrane vesicles (MVs), which have been linked to adverse pregnancy outcomes in a murine model. Nonetheless, little is known about the composition of GBS MVs and their role in pathogenesis and impact on the immune response. Therefore, using genotypically diverse strains of GBS, I sought to examine MV production and composition, as well as their impact on ascending infection in vivo to better understand their role in GBS disease. While MVs contain a variety of immunomodulatory virulence factors, including hyaluronidase, C5a peptidase, and sialidase, the relative abundance of these virulence factors varies in a lineage dependent manner. With this information in hand, I then assessed the host response to GBS derived MVs using human THP-1 derived macrophages. I found that regardless of strain, MVs induce a potent proinflammatory immune response characterized by the production of proinflammatory cytokines and chemokines. Notably, many neutrophil chemokines were highly upregulated in response to MVs, which is consistent with observations seen during murine challenge with MVs. I went on to demonstrate that MVs, in the absence of t, induce a potent IL-1ß response, which is dependent on caspase-1 and NLRP3, suggesting that the NLRP3 inflammasome is a sensor of GBS derived MVs. Previous data had suggested that MVs induce inflammation at the maternal-fetal interface. We have confirmed these findings and identified a potential mechanism for this inflammatory response. Furthermore, others utilized models of intra-amniotic injection that bypasses many immune defenses such as the extraplacental membranes and the cervix, which may prevent this response, while we assessed the role of MVs during vaginal challenge with GBS. However, we found that the addition of exogenous MVs does not promote ascending infection in vivo. While bacteria were able to ascend from the vaginal tract into gestational tissues, similar levels of bacteria were found regardless of the addition of exogenous MVs. Consistent with this observation, using flow cytometry we showed that neither exogenous MVs supplemented at the time of infection nor vaginal supplementation of MVs alone alter inflammatory responses at the maternal-fetal interface, suggesting that these innate barriers are capable of blocking MV mediated effects. The work presented here dramatically alters our understanding of the composition and host-pathogen interactions of GBS MVs, by substantially increasing our knowledge of both their composition and function during infection.
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Title
LISTERIA MONOCYTOGENES INFECTION ALTERS TROPHOBLAST EXTRACELLULAR VESICLES
Creator
Kaletka, Jonathan Matthew
Date
2022
Collection
Electronic Theses & Dissertations
Description
Listeria monocytogenes (Lm) is a bacterial pathogen that utilizes an intracellular lifecycle to spread throughout the body, including the placenta in pregnant individuals. Placental infection and disease can lead to negative fetal outcomes including spontaneous abortion, birth defects, and stillbirths. Extracellular vesicles (EVs) are tiny particles secreted by nearly every cell type in the body and serve as a cellular signaling mechanism. EVs have been implicated in many cellular functions...
Show moreListeria monocytogenes (Lm) is a bacterial pathogen that utilizes an intracellular lifecycle to spread throughout the body, including the placenta in pregnant individuals. Placental infection and disease can lead to negative fetal outcomes including spontaneous abortion, birth defects, and stillbirths. Extracellular vesicles (EVs) are tiny particles secreted by nearly every cell type in the body and serve as a cellular signaling mechanism. EVs have been implicated in many cellular functions and diseases throughout the body, including those involving the placenta. Placental EVs can have immunomodulatory effects, but during placental disease they can also act in a pro-inflammatory manner, leading to disease progression. EVs can also be proinflammatory during intracellular bacterial infection, where they can communicate the infection and coordinate an immune response. In this dissertation, I investigated how Lm infection of trophoblasts alters the EVs produced by the infected cells, and how they can activate an immune response. Chapter 1 of the dissertation details the current literature on the role that EVs play during bacterial infections and placental development and disease. Chapter 2 focuses on establishing a trophoblast stem cell model (TSC) to study placental infections. TSCs are the source of trophoblasts in the placenta, and cultivation of these cells allow for the continual study of placental disease. Here, I found that TSCs are susceptible to Lm infection, although it requires a higher bacterial load and longer time course compared to other cell types. This chapter details ways to model placenta-pathogen interactions in vitro, allowing for the study of these interactions in a laboratory setting. Chapter 3 investigated how Lm infection of TSCs altered the cargo of the tEVs produced. Previous studies into EVs from infected cells found components from the bacterial cells loaded into the EVs, including bacterial DNA, RNA, and proteins. We found many more unique proteins in the tEVs from infected cells. The infection tEVs had a substantial increase in the number of peptides identified of ribosomal, histone, and tubulin proteins, among others. Gene ontology (GO) analysis showed that the proteins seen in the tEVs from infected TSCs primarily belonged to RNA-binding pathways. This result piqued our curiosity as to if Lm infection also changed the RNA loaded into the tEVs. We performed RNA sequencing to determine the host RNA profiles found in the tEVs. We found different RNA profiles in the tEVs from uninfected and Lm-infected cells. GO analysis on the mRNAs overrepresented in the infection tEVs found that they represent genes from vasculogenesis and placental development pathways. Our results in this chapter show that Lm infection can alter the production and contents of tEVs from TSCs. Chapter 4 of this dissertation aimed to determine how tEVs from Lm-infected TSCs affect immune cells. We found that macrophages treated with infection tEVs produced TNF-α, a pro-inflammatory cytokine. Surprisingly, when we subsequently infected tEV treated cells with Lm, some of the cells became more susceptible to Lm infection. Similar results were seen with treatment with macrophage EVs, where infection EVs made the macrophages susceptible to Lm infection. The work in this chapter suggests that tEVs from Lm-infected TSCs can indeed induce a pro-inflammatory response in macrophages, although this makes the cells more susceptible to infection. Overall, the work presented here explores potential mechanisms as to how the placenta communicates bacterial infections.
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Title
Ecological and evolutionary consequences of exometabolites for microbial interactions
Creator
Chodkowski, John Luke
Date
2021
Collection
Electronic Theses & Dissertations
Description
Interspecies interactions have fundamental roles in shaping microbial communities. Microbial community members can produce and release a diverse set of extracellular small molecules, collectively referred to as exometabolites. Interspecies interactions that result from exometabolites can alter the response and behaviors of microbial community members. This dissertation work demonstrates the establishment of a synthetic community system that facilitates the study of exometabolite-mediated...
Show moreInterspecies interactions have fundamental roles in shaping microbial communities. Microbial community members can produce and release a diverse set of extracellular small molecules, collectively referred to as exometabolites. Interspecies interactions that result from exometabolites can alter the response and behaviors of microbial community members. This dissertation work demonstrates the establishment of a synthetic community system that facilitates the study of exometabolite-mediated interspecies interactions. This system was then used to understand the consequences of exometabolite-mediated interactions in a 3-member synthetic microbial community over stationary phase using a multi-omics approach. Lastly, an experimental evolution showed the consequences of long-term exometabolite interspecies interactions on the evolution of antibiotic resistance. This work advances knowledge on the dynamic response and behaviors of microbial community members' during non-growth states. This work also demonstrates how bacterial-bacterial interactions in a simple environment can lead to the emergence of antibiotic resistance.
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Title
THE IDENTIFICATION OF NOVEL AMINOGLYCOSIDE ADJUVANTS FOR THE ERADICATION OF PSEUDOMONAS AERUGINOSA BIOFILMS
Creator
Maiden, Michael M.
Date
2018
Collection
Electronic Theses & Dissertations
Description
The Infectious Disease Society of America has named antimicrobial resistance the greatest global threat to human health. More than half of all infections are due to bacteria growing as biofilms, which are a community of cells enmeshed in a self-made matrix that can be up to 1000x more resistant conventional antimicrobials. Pseudomonas aeruginosa in particular, due to its numerous resistance mechanisms is a formable threat that often forms biofilms. Few new therapies have been developed to...
Show moreThe Infectious Disease Society of America has named antimicrobial resistance the greatest global threat to human health. More than half of all infections are due to bacteria growing as biofilms, which are a community of cells enmeshed in a self-made matrix that can be up to 1000x more resistant conventional antimicrobials. Pseudomonas aeruginosa in particular, due to its numerous resistance mechanisms is a formable threat that often forms biofilms. Few new therapies have been developed to combat P. aeruginosa, and our antibacterial arsenal continues to decline. One solution to this daunting problem are anti-resistance compounds or adjuvants, which enhance conventional antimicrobials, extending and improving their utility. Here, we describe three adjuvants, triclosan, oxyclozanide and melittin. We demonstrate that each synergizes with tobramycin against mature P. aeruginosa biofilms. We also define the mechanism of action of triclosan and oxyclozanide, as protonophores that inhibit efflux pump activity, rendering cells susceptible to tobramycin killing. These adjuvants could be used in conjunction with current therapies to both improve their effectiveness, extend their lifespan, and target cells in biofilms
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Title
DEVELOPMENT OF 3D BIOACTIVE AND ANTIBACTERIAL SILICATE-BASED SCAFFOLDS FOR BONE TISSUE REGENERATION IN LOAD-BEARING APPLICATIONS
Creator
Marsh, Adam Christoph
Date
2022
Collection
Electronic Theses & Dissertations
Description
Current gold-standard approaches to addressing the needs of bone defects in load-bearing applications entail the use of either autographs or allographs. Both solutions, however, are imperfect as both autographs and allographs carry the risk of additional trauma, threat of disease transmission, and potential donor rejection respectively. Porous 3D scaffolds are attractive alternatives, illuminating a potential path towards achieving the ideal scaffold for targeting bone tissue regeneration in...
Show moreCurrent gold-standard approaches to addressing the needs of bone defects in load-bearing applications entail the use of either autographs or allographs. Both solutions, however, are imperfect as both autographs and allographs carry the risk of additional trauma, threat of disease transmission, and potential donor rejection respectively. Porous 3D scaffolds are attractive alternatives, illuminating a potential path towards achieving the ideal scaffold for targeting bone tissue regeneration in load-bearing applications, usurping autographs to become the new gold-standard. To unlock the full healing potential of 3D scaffolds, such scaffolds must be multifunctional such that (1) their mechanical performance meets the requisite requirements as dictated by the mechanical performance characteristics of interest for native bone tissue, (2) they stimulate the necessary biological responses for bone tissue regeneration, and (3) they exhibit antibacterial characteristics to combat the threat of infection. To date, no reports document 3D scaffolds exhibiting all three performance characteristics. The aim of this dissertation, therefore, is to deliver 3D scaffolds that are mechanically competent, possess and exhibit inherent and advanced antibacterial characteristics, and are successful at providing the needed biological characteristics for bone tissue regeneration. To achieve this, this dissertation implements a multidisciplinary approach, utilizing comprehensive structural characterization across a wide range of scales to elucidate process – performance relationships to execute scientifically driven modifications to engineer and deliver a 3D scaffold to successfully target bone tissue regeneration in load-bearing applications. A silver-doped bioactive glass-ceramic (Ag-BG) composition was selected as the material for scaffold synthesis due to its inherent and attractive antibacterial and biological performance characteristics. Two fundamentally different processing approaches were utilized for synthesizing Ag-BG scaffolds: the polymer foam replication technique and fused filament fabrication (FFF). The Ag-BG scaffolds studied herein were found to exhibit advanced antibacterial performance characteristics against methicillin-resistant Staphylococcus aureus (MRSA), a common pathogen implicated in osteomyelitis development, able to combat MRSA both in planktonic and biofilm forms. Ag-BG scaffolds demonstrated the ability to form an apatite-like layer when immersed in simulated body fluid (SBF), an indicator that Ag-BG scaffolds will induce the necessary mineralization for bone tissue regeneration, in addition to exhibiting attractive cell viability, proliferation, and differentiation characteristics when studied in vitro. The mechanical performance of Ag-BG scaffolds reported herein saw progressive improvements in each iteration of Ag-BG scaffold synthesis, achieving desirable mechanical competency and reliability as a result of the multidisciplinary approach formulated. In addition to the exploration of developing 3D antibacterial and biological silicate-based scaffolds capable of targeting bone tissue regeneration in load-bearing applications, foundational work towards the development of class II hybrid scaffolds comprised of gelatin methacryloyl (GelMA) and Ag-BG for targeting softer tissue regeneration. The novel syntheses applied to the successful molecular coupling of GelMA and Ag-BG presents an attractive class II hydrogel showing great promise as a compatible ink for 3D bioprinting cell-laden scaffolds capable of targeting tissue regeneration of more sophisticated systems.
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Title
DEFINING THE FUNCTION OF AN UNCHARACTERIZED CBASS GENE IN THE REGULATION OF 3’3’-CGAMP SIGNALING IN VIBRIO CHOLERAE
Creator
yoon, Soo hun
Date
2022
Collection
Electronic Theses & Dissertations
Description
Bacteria compete with phages in a myriad of environments for survival. This constant arms race has led to the acquisition of cyclic oligonucleotide based antiphage signaling system (CBASS) throughout all bacterial phyla. Phage infection activates CBASS which results in altruistic suicide of the host to save the clonal community, a process termed abortive infection. In Vibrio cholerae El Tor, CBASS is comprised of DncV, CapV, Vc0180, and Vc0181. DncV is activated following phage infection to...
Show moreBacteria compete with phages in a myriad of environments for survival. This constant arms race has led to the acquisition of cyclic oligonucleotide based antiphage signaling system (CBASS) throughout all bacterial phyla. Phage infection activates CBASS which results in altruistic suicide of the host to save the clonal community, a process termed abortive infection. In Vibrio cholerae El Tor, CBASS is comprised of DncV, CapV, Vc0180, and Vc0181. DncV is activated following phage infection to synthesize 3’3’-cGAMP which activates CapV, a phospholipase that degrades the cellular membrane. Though evidence suggests VC0180 and VC0181 allow for response against broader range of phages, their function in relation to DncV remains undefined. To determine their role, we investigate the effect of VC0180 and VC0181 on the stability of DncV in vivo. During this pursuit, we discovered a novel protein which we named Bumo, for Bacterial Ubiquitin Modifier, encoded upstream of capV that is a component of the CBASS operon. We show a novel regulatory network of DncV, in which Bumo protects DncV from degradation and that VC0181 is a protease that degrades DncV. We also show evidence that VC0180 interacts with DncV. To expand our knowledge of CBASS, we also explored the function of CBASS systems encoding HNH-SAVED effectors in other Gram-negative bacteria. We discovered E. coli EDEC13E and P. fluorescens SRM1 have active CBASS systems that affect the growth capacity of heterologous hosts. We also show their nucleotide substrate specificity. Interestingly, the E. coli EDEC13E HNH-SAVED effector is inactivated following the addition of nucleotide signals, which is distinct from all previously described CBASS systems. In conclusion, Bumo is a newly discovered component of the CBASS operon that protects DncV from degradation by Vc0181. VC0180 and VC0181 both regulate DncV in separate ways. Moreover, the function of CBASS is conserved in other Gram-negative species, even though they may have adapted to respond differentially to cyclic oligonucleotide signals. The results of my thesis work lead to a better appreciation of the complexity in the regulation of CBASS signaling and the diversity in the evolution of CBASS across species.
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