VTechWorks Repository :: Browsing by Author "Jutras, Brandon L."

Browsing by Author "Jutras, Brandon L."

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Battling Bacteria: Characterizing the NOD-Like Receptor (NLR) Immune Response to Brucella abortus and Borrelia burgdorferi in Host-pathogen Interactions
Tupik, Juselyn D. (Virginia Tech, 2024-08-19)
The innate immune system is integral for defense against infectious diseases. Characterized by Pattern Recognition Receptors (PRRs), which sense conserved molecular motifs known as Pathogen-Associated Molecular Patterns (PAMPs), the innate immune system sets a system of checks and balances to regulate inflammation in host defense. In this dissertation, we focus on one class of PRRs known as the NOD-like Receptors (NLRs) in response to bacterial diseases. This class consists of pro-inflammatory receptors that form a multi-protein complex termed the inflammasome, as well as regulatory NLRs that modulate inflammation. Here, we investigated the roles of inflammasomes and negative regulatory NLRX1 in response to bacterial diseases. First, we studied brucellosis, a zoonotic, chronic disease often transmitted in unpasteurized dairy products from livestock. Using murine models and bone marrow-derived macrophages (BMDMs) challenged with Brucella abortus, we found that canonical inflammasomes in murine models were protective against brucellosis through the initiation of inflammatory cell death called pyroptosis. In contrast, the inhibition of inflammation by NLRX1 adversely led to increased pathology in the spleen and liver in infected murine models. Despite these contrasting results, Brucella genomic DNA was an effective PAMP for NLR recognition. These results suggest the importance of DNA recognition by NLRs during brucellosis. Second, we investigated NLRX1 regulation of Borrelia burgdorferi in Lyme arthritis using murine models. Characterized by persistent inflammation of the joints, Lyme arthritis is an enigmatic and difficult inflammatory condition to resolve. We found that NLRX1 was protective against arthritis. By characterizing changes in gene and protein expression of infected ankle joints, in addition to in vitro studies in BMDMs and fibroblasts, we found that NLRX1 enhances cell migration and regulates cell metabolism. Our results suggest that NLRX1 may metabolically shift macrophages toward a more favorable wound-healing environment for arthritis resolution. Ultimately, this work emphasizes the importance of balance in NLR signaling, which occurs within NLRs and from crosstalk with other inflammatory pathways. Further, NLR signaling is highly multifaceted and context-specific for the cell type and bacterial disease, showcasing the complexity of host-pathogen interactions when battling bacteria.
Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis
Jutras, Brandon L.; Lochhead, Robert B.; Kloos, Zachary A.; Biboy, Jacob; Strle, Klemen; Booth, Carmen J.; Govers, Sander K.; Gray, Joe; Schumann, Peter; Vollmer, Waldemar; Bockenstedt, Linda K.; Steere, Allen C.; Jacobs-Wagner, Christine (National Academy of Sciences, 2019-06-17)
Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi. A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PGBb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb. In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations.
Borrelia burgdorferi SpoVG DNA- and RNA-Binding Protein Modulates the Physiology of the Lyme Disease Spirochete
Savage, Christina R.; Jutras, Brandon L.; Bestor, Aaron; Tilly, Kit; Rosa, Patricia A.; Tourand, Yvonne; Stewart, Philip E.; Brissette, Catherine A.; Stevenson, Brian (American Society for Microbiology, 2018-06-01)
The SpoVG protein of Borrelia burgdorferi, the Lyme disease spirochete, binds to specific sites of DNA and RNA. The bacterium regulates transcription of spoVG during the natural tick-mammal infectious cycle and in response to some changes in culture conditions. Bacterial levels of spoVG mRNA and SpoVG protein did not necessarily correlate, suggesting that posttranscriptional mechanisms also control protein levels. Consistent with this, SpoVG binds to its own mRNA, adjacent to the ribosome-binding site. SpoVG also binds to two DNA sites in the glpFKD operon and to two RNA sites in glpFKD mRNA; that operon encodes genes necessary for glycerol catabolism and is important for colonization in ticks. In addition, spirochetes engineered to dysregulate spoVG exhibited physiological alterations.
BpaB, a novel protein encoded by the Lyme disease spirochete’s cp32 prophages, binds to erp Operator 2 DNA
Burns, Logan H.; Adams, Claire A.; Riley, Sean P.; Jutras, Brandon L.; Bowman, Amy; Chenail, Alicia M.; Cooley, Anne E.; Haselhorst, Laura A.; Moore, Alisha M.; Babb, Kelly; Fried, Michael G.; Stevenson, Brian (Oxford University Press, 2010)
Borrelia burgdorferi produces Erp outer surface proteins throughout mammalian infection, but represses their synthesis during colonization of vector ticks. A DNA region 50 of the start of erp transcription, Operator 2, was previously shown to be essential for regulation of expression. We now report identification and characterization of a novel erp Operator 2-binding protein, which we named BpaB. erp operons are located on episomal cp32 prophages, and a single bacterium may contain as many as 10 different cp32s. Each cp32 family member encodes a unique BpaB protein, yet the three tested cp32-encoded BpaB alleles all bound to the same DNA sequence. A 20-bp region of erp Operator 2 was determined to be essential for BpaB binding, and initial protein binding to that site was required for binding of additional BpaB molecules. A 36-residue region near the BpaB carboxy terminus was found to be essential for high-affinity DNA-binding. BpaB competed for binding to erp Operator 2 with a second B. burgdorferi DNAbinding protein, EbfC. Thus, cellular levels of free BpaB and EbfC could potentially control erp transcription levels.
Characterization of Peptidoglycan, and the Enzymes that Synthesize it, in Borrelia burgdorferi and Insights into the Peptidoglycan of Other Pathogenic Borrelia
DeHart, Tanner Gage (Virginia Tech, 2021-06-03)
Peptidoglycan (PG) is an essential cell-wall biopolymer in virtually all bacteria. It is composed of glycan strands of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) crosslinked by peptide chains of alternating D- and L- amino acids and diamines. PG plays an important role in 1) cell elongation and division, 2) cell strength and morphology, 3) antibiotic susceptibility, and 4) host immune detection and modulation. While differences in peptide chains are common, deviations in glycan strand composition were not previously known to occur. Here, we provide characterization of the first known deviation to bacterial glycan strand composition — GlcNAc-GlcNAc-anhMurNAc (G-G- anhM) in Borrelia burgdorferi, the causative agent of Lyme disease. B. burgdorferi with less G-G-anhM were found to be significantly less motile, flexible, and stress-tolerant while possessing gross morphological defects and less overall PG. Our studies also characterized the muropeptide profile of Borrelia afzelii, Borrelia garinii, and Borrelia hermsii — species of Borrelia associated with causing different disease manifestations of Lyme disease, and relapsing fever, respectively. These species were found to incorporate appreciable amounts of G-G-anhM into their PG, suggesting an evolutionary adaptation to life inside a tick that predates the differentiation of Lyme disease and relapsing fever Borrelia. Finally, we provide partial characterization of a putative penicillin-binding protein in B. burgdorferi — a class of highly conserved PG synthesis enzymes present in the vast majority of bacteria. Collectively, the work in this thesis furthers our understanding of the structure, function, and synthesis of PG in Borrelia.
Elucidating the role of peptidoglycan from Borrelia burgdorferi in Lyme disease pathogenesis
McClune, Mecaila Elizabeth (Virginia Tech, 2024-05-23)
As of 2024, more than 50,000 people suffer from Lyme arthritis — a debilitating late-stage symptom of Lyme disease. Symptoms remain even after the completion of antibiotic therapy and when there is no longer any indication of an active infection. Studies have found that a portion of the bacterial cell wall from the causative agent, Borrelia burgdorferi, is a persistent antigen in Lyme arthritis patients, lingering within the synovial fluid. This antigen, peptidoglycan, is recognized by the immune system in numerous ways. Multiple publications have shown that peptidoglycan is proinflammatory and can cause arthritis when injected in vivo. The same was found to be true for B. burgdorferi peptidoglycan. Studies focused on the structure of peptidoglycan from B. burgdorferi have shown atypical differences in both glycan and peptide chemistry that likely alter immune recognition. Due to a lack of necessary enzymes and transporters B. burgdorferi are unable to recycle their peptidoglycan as they elongate and produce daughter cells. This leads to a 45% reduction of their total cell wall that is released into the environment. The work detailed below focuses on this antigen to further our knowledge as to its in vivo biodistribution pattern, half-life, and ability to induce arthritis. For these experiments B. burgdorferi peptidoglycan (pBb PG) was purified, fluorescently labeled, and tracked in vivo to study its clearance pattern and rate. Three different mouse models for Lyme arthritis were utilized in these studies and all experienced persistence of B. burgdorferi peptidoglycan in their liver for upward of 20 days. There were differences in the rate of clearance between types of mice, suggesting the involvement of host genetics. Serum collected weekly throughout this experiment showed over a log fold change in the abundance of ALT and AST levels, which indicates liver dysfunction. Proteomic analysis of the livers of mice post pBb PG injection showed altered levels of proteins important for mitochondrial function and iron homeostasis. When human PBMCs were stimulated with PG from various bacteria it was found that at 12 h pBb PG differentially expressed genes involved in energy metabolic pathways, including oxidative phosphorylation and the citric acid cycle. A subset of Lyme disease patients continue to experience symptomology even after completion of multiple rounds of antibiotics. These patients are termed to have post treatment Lyme disease syndrome and typically experience fatigue as their most common symptom. This symptom in combination with the findings of this dissertation regarding the link between pBb PG and energy metabolism warrants further investigation. Especially since this biopolymer has been found to persist in the synovial fluid of Lyme arthritis patients. Better understanding how these processes are connected could allow for the eventual development of a way to target this material for clearance, or ways to inactivate it. Both options have the potential to help alleviate the devastating symptomology experienced by patients.
Eubacterial SpoVG Homologs Constitute a New Family of Site-Specific DNA-Binding Proteins
Jutras, Brandon L.; Chenail, Alicia M.; Rowland, Christi L.; Carroll, Dustin W.; Miller, M. Clarke; Bykowski, Tomasz; Stevenson, Brian (PLOS, 2013-06-20)
A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG a-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent b-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins.
Exploring the Forces Underlying the Dynamics and Energetics of G-quadruplexes with Polarizable Molecular Dynamics Simulations
Salsbury, Alexa Marie (Virginia Tech, 2021-05-24)
G-quadruplexes (GQs) are highly stable noncanonical nucleic acid structures that form in the DNA of human cells and play fundamental roles in maintaining genomic stability and regulating gene expression. These unique structures exert broad influence over biologically important processes and can modulate cell survival and human health. In fact, mutations, hyper-stability, and dissociation of GQs are implicated in neurodegenerative disease, mental retardation, premature-aging conditions, and various cancers. As such, GQs are novel drug targets. GQ-targeting therapeutics are developed to influence the folding and genetic interactions of GQs that are implicated in diseased states. To do so requires a greater understanding of GQ structure and dynamics and molecular dynamics (MD) simulations are well suited to provide these fundamental insights. Previous MD simulations of GQs have provided limited information due to inaccuracies in their models, namely the nonpolarizable nature of their force fields (FFs). The cutting-edge Drude polarizable FF models electronic degrees of freedom, allowing charge distribution to change in response to its environment. This is an important component for modeling ion-ion and ion-DNA interactions and can influence the overall stability of GQ structures. The work herein employs the Drude polarizable FF to 1) describe the role of electronic structure on the dynamics and folded stability of GQs, 2) determine the impact of ion interaction on GQ stability, and 3) characterize the role of G-hairpin motifs in GQ intermediates. Such fundamental investigations will help clarify GQs role in healthy and diseased states and transform our understanding of noncanonical DNA, improving human health, therapeutic design, and fundamental science.
From Structure, to Function, to Pathogenesis: Understanding the Immunological Consequences of The Unique Peptidoglycan of Borrelia burgdorferi
Davis, Marisela Martinez (Virginia Tech, 2020-05-21)
The bacterial pathogen responsible for Lyme disease ¬— Borrelia burgdorferi— is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. Like all Gram-negative bacteria the structural portion of the cell envelope known as peptidoglycan (PG) is sandwiched between the inner and outer membranes. Unlike virtually all bacteria, this PG layer is unique in B. burgdorferi in that the amino acid structure differs from most Gram-negative and Gram-positive bacteria by the addition of an Ornithine residue to the third amino acid location in the crosslinking structure. This unique motif is hypothesized to be responsible for the unusual clinical manifestations seen in Lyme disease, specifically Lyme arthritis, the most common late stage symptom of the disease in the United States. Peptidoglycan is only one component of the cell envelope in B. burgdorferi though; other portions of the cell envelope remain understudied specifically when viewed through the lens of the immune response they may elicit in addition to that of PG. The combined immunological effect of the unique bacterial antigen found in B. burgdorferi PG, as well as other potentially associated proteins contained within the cell wall, are explored here. These studies further our understanding of the B. burgdorferi cell envelope and provide critical information that underlies the elusive pathogenesis of Lyme disease.
Functional Characterization of Serine Hydrolases Mediating Lipid Metabolism and Protein Depalmitoylation in Asexual Stage Plasmodium Falciparum
Liu, Jiapeng (Virginia Tech, 2023-06-05)
Malaria is an infectious disease caused by Plasmodium parasites and transferred by Anopheles mosquitos. Due to Artemisinin resistance, new druggable targets identification and new drug development are urgently needed. Serine hydrolases (SHs) are one of the largest classes of enzymes having important roles in life processes. The deadliest malaria parasite, P. falciparum, encodes more than 50 SHs including proteases, lipases, esterase and others, while only several of them have been characterized. The study of uncharacterized SHs will shed light on future drug development to treat malaria. In this study, we applied chemical biology and genetic approaches to identify SHs important for the pathogenic asexual stage growth of P. falciparum parasites. We mainly focused on a depalmitoylase essential for merozoite invasion and lysophospholipases (LPLs) essential for acquiring fatty acids (FAs) from the host. Identifying essential metabolic enzymes will benefit the treatment to malaria. We focused on metabolic SHs and identified two SHs were refractory to knock out. We studied a likely essential SH named PfABHD17A, which is a human depalmitoylase homolog. PfABHD17A is localized on the rhoptry, an organelle essential for invasion. We expressed the recombinant PfABHD17A, conducted inhibitor screen and discovered that human depalmitoylase inhibitor ML211 inhibits PfABHD17A in vitro. ML211 inhibits merozoite invasion but not egress, which together with the localization of PfABHD17A on the rhoptries, suggested that PfABHD17A is essential in merozoite invasion. We also purified PfABHD17A and verified that PfABHD17A may exhibit depalmitoylase activity in vitro. LPLs are important for asexual stage parasites acquiring FAs from the host. The P. falciparum genome includes 17 putative LPLs while LPLs responsible for hydrolyzing FA from lysophosphatidylcholine (LPC) in the asexual stage are currently unknown. Using a chemical biology approach, we identified serine hydrolase inhibitor AKU-010 inhibits LPC hydrolysis effectively. Using activity-based protein profiling (ABPP) and genetic approaches, we identified that AKU-010 inhibits a series of SHs including Exported Lipases (XLs), Exported Lipases Homolog (XLH) and Plasmodium falciparum prodrug activation and resistance esterase (PfPARE). We generated a series of knockout parasite lines on the AKU-010 targets and identified that red blood cell (RBC)-localized XL2 and cytosolic XLH4 contribute to most LPC hydrolysis activity in the asexual stage. XLs and XLHs are important for parasites using LPC for growth and contribute to detoxification from accumulated LPC. XL2 and XL4 together are essential for parasite growth under high LPC concentration medium, such as human serum. XL/XLH-deficient parasites could still acquire FA from LPC, which is mainly contributed by parasite membrane- localized PfPARE. PfPARE has little impact on parasite growth and LPC metabolism with the existence of XLs and XLHs but is important after the loss of XLs and XLHs. Parasites deficient in PfPARE, XLs and XLHs have little ability to release FA from LPC and cannot use LPC as FAs source for growth. In summary, we identified metabolic SHs mediating protein depalmitoylation and lipid metabolism and in asexual stage Plasmodium falciparum, which may benefit future drug development to treat malaria.
The identification and characterization of unique FemX homologue in B. burgdorferi, and insights into the peptidoglycan biosynthesis pathway
Kushelman, Mara Rebecca (Virginia Tech, 2022-07-01)
Borrelia burgdorferi — the causative agent of Lyme borreliosis — accounts for ~500,000 infections in the United States per year. Relative to other bacteria, B. burgdorferi is highly unusual in many regards. For instance, the synthesis and composition of B. burgdorferi cell wall is extremely unique and plays a critical role in Lyme pathogenesis. The cell wall is made up of peptidoglycan (PG) - a mesh-like structure, composed of long rigid glycan strands of repeating sugars GlcNAc and MurNAc, and flexible peptide stems, interlinked by amino acid cross-bridges. PG is an essential component for survival of the bacterial cell, protecting it from the osmotic stress and environmental threats, as well as defining the shape of the bacterium and aiding in the motility. One unique feature of the B. burgdorferi PG is the chemical composition of stem peptide, which involves the atypical cross-link between Ornithine and Glycine. We identified gene bb0586 as a femX homologue in borrelial genome and hypothesize that it encodes a glycyl transferase enzyme responsible for synthesis of glycine cross-bridges, that hold together glycan strands in the peptidoglycan cell wall. Here, we predicted the structure of FemXBb, identified and characterized the substrate-binding site, and proposed a novel mechanism for substrate recognition and recruitment, involving previously uncharacterized elements of the structure. We have also determined the ability of recombinant FemXBb to add Glycine bridges to mDAP in E. coli and investigated the effect that femX knock-out can have on the B. burgdorferi. In addition, we have investigated the steps of PG biosynthesis in B. burgdorferi. The results of our research suggest the existence of a highly unusual mechanism of PG synthesis in Lyme disease spirochete, which has a potential to be used for development of targeted antibacterial therapies.
Inositol Pyrophosphate Phosphatases as Key Enzymes to Understand and Manipulate Phosphate Sensing in Plants
Freed, Catherine P. (Virginia Tech, 2022-01-28)
Phosphorus (P) is one of the three major macronutrients that plants need to grow and survive. When P is scarce, plants utilize a network of characterized responses known as the Phosphate Starvation Response (PSR) to remobilize internal stores of P as well as external P from soil. Emerging evidence shows the PSR is regulated by a specialized group of secondary messenger molecules, inositol pyrophosphates (PP-InsP). PP-InsPs and their precursors, inositol phosphates (InsPs), are important for plant abiotic stress responses, hormone signaling, and other stress responses. While PP-InsPs are critical for plant survival, much about the roles of PP-InsPs and how they are regulated remains to be understood. Further, the enzymes responsible for the synthesis of PP-InsPs in plants have been recently discovered; however, not much is known about the enzymes that degrade PP-InsPs in plants. The goal of the work presented herein is to understand critical aspects of the PP-InsP signaling in plants and leverage this information into a P phytoremediation strategy. To achieve this, I have investigated a group of PP-InsP phosphatases and assessed long-term impacts of depleting PP-InsPs in two plant species, Arabidopsis thaliana (Arabidopsis) and Thlaspi arvense (Pennycress). Exploring the impact of plant PP-InsP phosphatases has allowed me to explore critical aspects of PP-InsP sensing that show great promise for informing P remediation strategies.
The interplay between pathogenic bacteria and bacteriophage Chi: New directions in motility and phage-host interactions in Enterobacterales
Esteves, Nathaniel Carlos (Virginia Tech, 2024-04-15)
The bacterial flagellum is a rotary motor that propels motile bacteria through their surroundings via swimming motility, or on surfaces via swarming motility. The flagellum is a key virulence factor for motile pathogenic bacteria. Viruses that infect bacteria via this appendage are known as flagellotropic or flagellum-dependent bacteriophages. Much like other phages, flagellotropic phages are of interest for clinical applications as antibacterial agents, particularly against multidrug resistant (MDR) bacteria. Bacteriophage χ is a flagellotropic phage that infects multiple species of motile pathogens. In the projects described below, we characterized several aspects of the complex interactions between χ and two of its hosts: Salmonella enterica and Serratia marcescens. In Chapter I, we describe in detail the existing knowledge on flagellum-dependent bacteriophages, pathogenic bacteria, and the flagellar motility system. We also expand significantly on flagellotropic phage χ. In Chapter II, we describe our discovery of S. enterica cellular components other than motility that are crucial for bacteriophage χ infection, making the key discovery that the AcrABZ-TolC multi-drug efflux system is required for infection to proceed. We additionally found that the host molecular chaperone trigger factor is important for the χ phage lifecycle. In Chapter III, we outline our characterization of the initial binding interaction between χ and the flagellum, determining that of flagellin's seven domains, C-terminal domain D2 is the most important for χ adsorption. In Chapter IV, we expand on this by discussing our work that determined that the χ tail fiber protein is encoded by the gene CHI_31, purification of this recombinantly-expressed protein, and demonstration of its direct interaction with the flagellar filament. Lastly, in Chapter V, our findings indicate that S. marcescens is able to detect χ infection and lysis in the surroundings and alter gene expression, resulting in an increase in the production of the red pigment prodigiosin. Overall, our hypothetical model for χ infection is as follows: χ binds to the flagellum of its host using its single tail fiber, composed of monomers of the CHI_31 gene product gp31. This tail fiber interacts with CTD2 of flagellin, and the rotation of the flagellum brings the phage to the cell surface, where it interacts with AcrABZ-TolC to inject its genetic material into the host cytoplasm. At some point during the process of production of phage particles and subsequent cell lysis, the host molecular chaperone trigger factor likely assists with proper folding of χ proteins. After cell lysis, cells in the surroundings are capable of detecting lysis and responding accordingly, at least in the case of S. marcescens. This research is clinically relevant for a number of reasons. Phage therapy, the use of bacteriophages as antibacterial agents, requires knowledge of phage infection pathways for optimal implementation. The fact that the flagellum and a complex mediating MDR are both essential for χ infection leads to particular interest in χ for this application. Knowledge of the host-determining factors between χ and Salmonella may lead to the ability to alter the χ phage genome to target specific pathogenic Salmonella or Escherichia coli strains while avoiding disruption of beneficial bacterial communities.
Investigating proteins that influence membrane-associated germination processes in Bacillus subtilis spores
Flores, Matthew Jose (Virginia Tech, 2023-06-30)
Many endospore-forming bacteria cause diseases such as anthrax and food poisoning. Spores however also contribute to various agricultural and industrial processes. Spores possess extreme resistance properties, notably to chemical, et and dry heat, desiccation, and UV damage. For pathogenic spore formers, this poses an issue as spores are resistant to most decontamination methods currently in use. This work focuses on characterizing proteins thought to contribute to spore stability and efficient spore germination. Understanding how spores can remain stable for long periods of dormancy and against various insults and rapidly initiate germination could allow for the development of techniques that induce germination early and rapidly, promoting inexpensive decontamination. Physiological studies found that a family of spore-associated lipoproteins is needed for efficient spore germination and influences membrane fluidity in dormant spores. All the members of the lipoprotein family serve the same function, as each can fulfill the role of another. In vivo cross-linking was used to characterize protein-protein interactions found on the inner spore membrane. Glutaraldehyde crosslinking revealed that the four lipoproteins appear to interact. Bacterial two-hybrid analysis on individual protein domains further suggests the lipoproteins seem to interact through their predicted ring-building motif within their otherwise uncharacterized domains. Additionally, the absence of the spore lytic enzyme SleB seems to alter the crosslinking pattern of the lipoproteins, suggesting either it's interacting or helping facilitate lipoprotein interactions. Fluorescence microscopy reveals an unequal spatial distribution of the lipoproteins on the spore membrane, which seems to be supported by preliminary super-resolution microscopy studies. Further work aiming to characterize the entire inner spore membrane interactome is currently being conducted. The presented research used many methods and built many collaborations with the goal of providing insight to spore dormancy and efficient spore germination with an additional goal of understanding inner spore membrane protein behavior and how it leads to the highly resistant properties native to bacterial endospores.
Investigating the enzyme activity of a Clostridioides difficile amidase complex
Kohler, Brian Jacob (Virginia Tech, 2023-07-24)
Clostridioides difficile is a highly antibiotic resistant and infectious endospore-forming bacterium, responsible for an estimated 450,000 cases per year. The formation of an endospore is necessary for the survival of the bacterium while in-transit between hosts and while passing through the toxic environment of the host's stomach. Essential to the endospore's resistance is a thick layer of highly modified peptidoglycan called the cortex. While the endospore cortex is forming, the enzymes CwlD and PdaA convert N-acetylmuramic acid (NAM) into muramic--lactam (MAL). MAL serves as a recognition element for germination-specific lytic enzymes that degrade cortex peptidoglycan layers during germination. Without the MAL residues the endospore cannot complete germination. Unique to the Peptostreptococcaceae family, which includes C. difficile, is the lipoprotein GerS, which is required for the function of CwlD. The interaction between these two proteins is poorly understood. In this work, attempts to complement a Bacillus subtilis cwlD mutant using C. difficile gerS and/or cwlD were unsuccessful. No MAL residues were produced, and spores produced were incapable of completing germination. In vitro assays of CwlD activity on purified peptidoglycan revealed binding and activity of C. difficile CwlD, which were significantly increased when in complex with GerS. The ability of C. difficile CwlD+GerS to function in vitro but not to complement in B. subtilis suggests that in vivo activity is blocked by some factor in this heterologous system. Such a factor might be the in vivo ionic environment or a failure to properly localize within the forespore in B. subtilis cells. Furthering the understanding of C. difficile's germination machinery will potentially provide new targets for therapies.
Investigating the Role of Trimeric Autotransporter Adhesins in Fusobacterium nucleatum Pathogenesis
Yoo, Christopher Charles (Virginia Tech, 2019-07-09)
Fusobacterium nucleatum is a Gram-negative bacterium that serves as a bridging organism in polymicrobial biofilms within the oral cavity. Although the bacterium is abundant in healthy gingival tissue, recent studies have found that F. nucleatum is associated with a wide-spectrum of human diseases which include periodontal disease, preterm birth, endocarditis, colorectal cancer, and pancreatic cancer. Previous studies of F. nucleatum virulence have uncovered two surface adhesins, Fap2 and FadA, that interact with the surface of human cells; however, the study of new virulence factors was previously limited as there was no gene deletion system available to functionally analyze F. nucleatum proteins. Interestingly, F. nucleatum has a diverse landscape of structurally unique surface adhesins called Type 5c secreted trimeric autotransporter adhesins (TAAs), which are a family of proteins that are historically known for their contributions to bacterial pathogenesis. This dissertation encompasses the use of recombinant protein expression systems and newly developed gene deletion technology to provide a foundational understanding of the contribution of Type 5c secreted proteins in F. nucleatum pathogenesis. Our results show that the presence of TAAs on the surface of F. nucleatum contribute to the bacterium's ability to bind and invade human cells, establishing the need to characterize other F. nucleatum surface proteins. Additionally, our studies analyzed the proinflammatory landscape induced by F. nucleatum through the identification of specific cytokines that are being secreted during in vitro infections of human cells. Cytokine signaling is a critical aspect of the host cell immune response as it promotes the recruitment of immune cells to the site of infection for efficient clearance of bacterial pathogens. While it has been well established that F. nucleatum modulates the secretion of IL-8, our studies identified that the bacterium also promotes the secretion of CXCL1, which is an important signaling protein that promotes tumor metastases. Overall, the work provided in this dissertation has delivered the initial characterization of TAAs in F. nucleatum virulence, a framework for future studies of Type 5c secreted proteins in Fusobacterium pathogenesis, and the role of Fap2 and FadA in promoting pro-inflammatory and pro-metastatic signaling from colorectal cancer cells.
A large screen identifies beta-lactam antibiotics which can be repurposed to target the syphilis agent
Hayes, Kathryn A.; Dressler, Jules M.; Norris, Steven J.; Edmondson, Diane G.; Jutras, Brandon L. (Springer Nature, 2023)
Syphilis, caused by the spirochete Treponema pallidum subsp. pallidum (hereafter called T. pallidum), is re-emerging as a worldwide sexually transmitted infection. A single intramuscular dose of benzathine penicillin G is the preferred syphilis treatment option. Both supply shortage concerns and the potential for acquired antibiotic resistance further the need to broaden the repertoire of syphilis therapeutics. We reasoned that other β-lactams may be equally or more effective at targeting the disease-causing agent, Treponema pallidum, but have yet to be discovered due to a previous lack of a continuous in vitro culture system. Recent technical advances with respect to in vitro T. pallidum propagation allowed us to conduct a high-throughput screen of almost 100 β-lactams. Using several molecular and cellular approaches that we developed or adapted, we identified and confirmed the efficacy of several β-lactams that were similar to or outperformed the current standard, benzathine penicillin G. These options are either currently used to treat bacterial infections or are synthetic derivatives of naturally occurring compounds. Our studies not only identified additional potential therapeutics in the resolution of syphilis, but provide techniques to study the complex biology of T. pallidum— a spirochete that has plagued human health for centuries.
The Lyme disease spirochete's BpuR DNA/RNA-binding protein is differentially expressed during the mammal-tick infectious cycle, which affects translation of the SodA superoxide dismutase
Jutras, Brandon L.; Savage, Christina R.; Arnold, William K.; Lethbridge, Kathryn G.; Carroll, Dustin W.; Tilly, Kit; Bestor, Aaron; Zhu, Haining; Seshu, Janakiram; Zuckert, Wolfram R.; Stewart, Philip E.; Rosa, Patricia A.; Brissette, Catherine A.; Stevenson, Brian (2019-07)
When the Lyme disease spirochete, Borrelia burgdorferi, transfers from a feeding tick into a human or other vertebrate host, the bacterium produces vertebrate-specific proteins and represses factors needed for arthropod colonization. Previous studies determined that the B. burgdorferi BpuR protein binds to its own mRNA and autoregulates its translation, and also serves as co-repressor of erp transcription. Here, we demonstrate that B. burgdorferi controls transcription of bpuR, expressing high levels of bpuR during tick colonization but significantly less during mammalian infection. The master regulator of chromosomal replication, DnaA, was found to bind specifically to a DNA sequence that overlaps the bpuR promoter. Cultured B. burgdorferi that were genetically manipulated to produce elevated levels of BpuR exhibited altered levels of several proteins, although BpuR did not impact mRNA levels. Among these was the SodA superoxide dismutase, which is essential for mammalian infection. BpuR bound to sodA mRNA in live B. burgdorferi, and a specific BpuR-binding site was mapped 5 ' of the sodA open reading frame. Recognition of posttranscriptional regulation of protein levels by BpuR adds another layer to our understanding of the B. burgdorferi regulome, and provides further evidence that bacterial protein levels do not always correlate directly with mRNA levels.
The Lyme disease spirochete’s BpuR DNA- / RNA-binding protein is differentially expressed during the mammal-tick infectious cycle, and affects translation of the SodA superoxide dismutase
Jutras, Brandon L.; Arnold, William; Savage, Christina R.; Tilly, Kit; Beastor, Aaron; Rosa, Patti; Brissette, Catherine A.; Stevenson, Brian (Wiley, 2019-09)
Peptidoglycan in osteoarthritis synovial tissue is associated with joint inflammation
Holub, Meaghan N.; Wahhab, Amanda; Rouse, Joseph R.; Danner, Rebecca; Hackner, Lauren G.; Duris, Christine B.; McClune, Mecaila E.; Dressler, Jules M.; Strle, Klemen; Jutras, Brandon L.; Edelstein, Adam I.; Lochhead, Robert B. (2024-03-27)
Objectives: Peptidoglycan (PG) is an arthritogenic bacterial cell wall component whose role in human osteoarthritis is poorly understood. The purpose of this study was to determine if PG is present in synovial tissue of osteoarthritis patients at the time of primary total knee arthroplasty (TKA), and if its presence is associated with inflammation and patient reported outcomes. Methods: Intraoperative synovial tissue and synovial fluid samples were obtained from 56 patients undergoing primary TKA, none of whom had history of infection. PG in synovial tissue was detected by immunohistochemistry (IHC) and immunofluorescence microscopy (IFM). Synovial tissue inflammation and fibrosis were assessed by histopathology and synovial fluid cytokine quantification. Primary human fibroblasts isolated from arthritis synovial tissue were stimulated with PG to determine inflammatory cytokine response. Results: A total of 33/56 (59%) of primary TKA synovial tissue samples were positive for PG by IHC, and PG staining colocalized with markers of synovial macrophages and fibroblasts by IFM. Synovial tissue inflammation and elevated IL-6 in synovial fluid positively correlated with PG positivity. Primary human fibroblasts stimulated with PG secreted high levels of IL-6, consistent with ex vivo findings. Interestingly, we observed a significant inverse correlation between PG and age at time of TKA, indicating younger age at time of TKA was associated with higher PG levels. Conclusion: Peptidoglycan is commonly found in synovial tissue from patients undergoing TKA. Our data indicate that PG may play an important role in inflammatory synovitis, particularly in patients who undergo TKA at a relatively younger age.

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