Browsing by Author "Schubot, Florian David"

Now showing 1 - 6 of 6
  • Thumbnail Image
    Impact of mutations in non-structural proteins on SARS-CoV-2 replication
    Datsomor, Eugenia Afi (Virginia Tech, 2024-06-14)
    The late 2019 marked the onset of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that led to the unprecedented COVID-19 pandemic, with profound global health and socioeconomic impacts. This thesis offers a thorough examination of the molecular biology, evolution, and disease-causing mechanisms of SARS-CoV-2, as well as recent advancements in understanding the structural and functional implications of mutations in viral proteins. The prevailing belief is that SARS-CoV-2 originated from a zoonotic transmission involving bats as the natural reservoir hosts, with an unknown intermediate host facilitating transmission to humans. Genomic sequencing and phylogenetic analysis have identified similarities between SARS-CoV-2 and bat coronaviruses, particularly RaTG13, indicating a potential bat origin. However, the exact circumstances and intermediate hosts of the spillover event remain under investigation. In its structure, SARS-CoV-2 is an enveloped virus with a positive-sense single-stranded RNA genome. This genome encodes both structural and non-structural proteins crucial for viral replication and the development of the disease. The spike (S) protein facilitates viral entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. Meanwhile, non-structural proteins are involved in viral RNA synthesis, immune evasion, and the assembly of virions. Alterations in the genetic makeup of the SARS-CoV-2 genome, notably within the spike protein, can impact transmission efficiency, viral load, and immune evasion. Notable mutations such as D614G, N501Y, and E484K have been associated with increased transmissibility and reduced neutralization by antibodies. Understanding the effects of these mutations on viral fitness and pathogenicity is crucial for informing public health interventions and vaccine development efforts. The impacts of Non-structural proteins (NSPs) on viral replication and transmission are however understudied. In this study, we focused on mutations in the several NSPs including NSP1, 2, 3, 13,14, and 15 of the early Omicron (BA.1) and XBB 1.5 variants and investigated their impact on structure and the functional implications using bioinformatics tools and protein structure prediction methods. Our analysis focused on potential alterations in NSP1's structure and hence its ability to suppress host gene expression and modulate immune responses, shedding light on the mechanisms by which SARS-CoV-2 evolves to evade host defenses. Overall, this thesis gives insights into the emergence, structure, replication cycle, evolution, and pathogenesis of SARS-CoV-2, highlighting the importance of ongoing research efforts in understanding and combatting this global health threat and provides a detailed structural analysis of mutations in NSPs.
  • Thumbnail Image
    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.
  • Thumbnail Image
    The little engine that could: Characterization of noncanonical components in the speed-variable flagellar motor of the symbiotic soil bacterium Sinorhizobium meliloti
    Sobe, Richard Charles (Virginia Tech, 2022-06-07)
    The bacterial flagellum is a fascinating corkscrew-shaped macromolecular rotary machine used primarily to propel bacterial cells through their environment via the conversion of chemical potential energy into rotational power and thrust. Flagella are the principal targets of complex chemotaxis systems, which allow microbes to navigate their habitats to locate favorable conditions and avoid harmful ones by continuous sampling of environmental compounds and cues. Flagella serve as surface and temperature sensors, mediators of host cell adherence by bacterial pathogens and symbionts alike, and important virulence factors for disease-causing microbes. They play several essential roles in accelerating the foundational stages of biofilm formation, during which bacteria build highly intricate microbial communities with increased resistance to predation and environmental assaults. Flagellum-mediated chemotaxis has broad and impactful implications in fields of bioremediation, targeted drug delivery, bacterial-mediated cancer therapy and diagnostics, and cross-kingdom horizontal gene transfer. While the core structural and functional components of flagella have been well characterized in the closely related enteric bacteria, Escherichia coli and Salmonella typhimurium, major departures from this paradigm have been identified in other diverse species that merit further investigation. Many bacteria employ additional reinforcement modules to surround and stabilize their more powerful flagellar motors and provide increased contact points in the inner membrane, the peptidoglycan sacculus, and, in Gram-negative bacteria, the outer membrane. Additionally, the soil-dwelling bacterium Sinorhizobium meliloti exhibits marked distinctions in the regulation, structure, and function of its navigation systems. S. meliloti is a nitrogen-fixing symbiont of the agronomically valuable leguminous plant, Medicago sativa Lucerne, and uses its coupled chemotaxis and flagellar motility systems to search for host plant roots to colonize. Following root colonization, the bacterium converts to a nitrogen-fixing factory for the plant and the combined influences of this symbiosis can quadruple the yields of the host. This dissertation is aimed at delivering a thorough representative overview of the processes facilitating bacterial flagellum-mediated chemotaxis and motility. Chapter 1 describes the interplay between chemotaxis and flagellar motility pathways as well as the structure, function, and regulation of these systems in several model bacteria. Particular emphasis is placed on the comparison of flagellar systems from the soil-dwelling legume symbiont, Sinorhizobium meliloti with other model systems, and a brief introduction is provided for its primary counterpart, the agronomically valuable legume, Medicago sativa, more commonly referred to as alfalfa. Chapter 2 embodies the first report of a flagellar system to require two copies of a protein known as FliL for its function. FliL is found in all bacterial flagellar systems reported to date but is only essential for some to drive motility. The more conserved copy of the protein has retained the title of FliL and several experiments to assay the proficiency of flagellar motor function revealed that in the absence of FliL swimming is essentially abolished as is the presence of flagella on the cell body. Flagellar motor activity and swimming proficiency of mutants lacking the FliL-paralog MotF was nearly as abysmal as those without FliL but flagellation was essentially normal indicating distinct roles for the two proteins. FliL is implicated in initial stator recruitment to the motor while MotF was found to serve as a power or speed modulator. A model to accommodate and explain the roles of these proteins in the flagellar motor of S. meliloti is provided. Chapter 3 links a never-before characterized flagellar protein, currently named Orf23, to a role in promoting maximum swimming velocity and perhaps stator alignment with the rotor in a peptidoglycan-dependent manner. The loss of LdtR, a transcriptional regulator of peptidoglycan-modification genes, caused defects in swimming motility that are restored only by removal of Orf23 or by replacing a nonpolar glycine with a polar serine in the periphery of stator units. Bioinformatics analyses, immunoblotting, and membrane topology reporter assays revealed that Orf23 is likely embedded in the inner membrane and that the remainder of the protein extends into the periplasm. Building on findings from Chapter 2, Orf23 is anticipated to influence stator positioning through interactions with MotF, FliL, and/or stator units directly. The chapter is concluded with the description of future experiments aimed to more thoroughly characterize Orf23. Altogether, this work increases the depth and breadth of knowledge regarding the composition and function of the speed-variable bacterial flagellar motor. We have identified several components required for stator incorporation and function, as well as an accessory component that improves stator performance. A wise society will draw inspiration from these fascinating and powerful machines to inform new technologies to achieve modern goals including targeted drug delivery, bioremediation, and perhaps one day our own exploration.
  • Thumbnail Image
    Phosphatidylinositol 3-phosphate binding properties and autoinhibition mechanism of Phafin2
    Tang, Tuoxian (Virginia Tech, 2021-05-26)
    Phafin2 is a member of the Phafin protein family. Phafins are modular with an N-terminal PH (Pleckstrin Homology) domain followed by a central FYVE (Fab1, YOTB, Vac1, and EEA1) domain. Both the Phafin2 PH and FYVE domains bind phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide mainly found in endosomal and lysosomal membranes. Phafin2 acts as a PtdIns(3)P effector for endosomal cargo trafficking, macropinocytosis, apoptosis, and autophagy. The PtdIns(3)P binding activity is critical to the localization of Phafin2 on a specific membrane and, subsequently, helps the recruitment of other binding partners to the same membrane surface. However, there are no studies on the structural basis of PtdIns(3)P binding, the PtdIns(3)P-binding properties of each domain, and the apparent redundancy of two PtdIns(3)P binding domains in Phafin proteins. In the present dissertation, different biochemical and biophysical techniques were utilized to investigate the structural features of Phafin2 and its lipid interactions. This dissertation shows that Phafin2 is a moderately elongated monomer with a predicted α/β structure and ~40% random coil content. Phafin2 binds lipid bilayer-embedded PtdIns(3)P with high affinity; its PH and FYVE domains display distinct PtdIns(3)P-binding properties. Unlike the PH domain, the Phafin2 FYVE domain binds both membrane-embedded PtdIns(3)P and water-soluble dibutanoyl PtdIns(3)P with similar affinity. An intramolecular autoinhibition mechanism is found in Phafin2, in which a conserved C-terminal aspartic acid-rich (polyD) motif inhibits the binding of Phafin2 PH domain to PtdIns(3)P. The polyD motif specifically interacts with the Phafin2 PH domain. Using negative-stain Transmission Electron Microscopy, Phafin2 was found to cause membrane tubulation in a PtdIns(3)P-dependent manner. In conclusion, this study provides the structural and functional basis of Phafin2 lipid interactions and evidence of an intramolecular autoinhibition mechanism for PtdIns(3)P binding to the Phafin2 PH domain, which is mediated by the C-terminal polyD. The distinct PtdIns(3)P binding properties of the Phafin2 PH and FYVE domains may indicate that these two domains have different functions. Considering that the Phafin2 PH domain's PtdIns(3)P binding is intramolecularly regulated, cells may employ a unique mechanism to release the Phafin2 PH domain from the conserved C-terminal motif and control the functions of Phafin2 in PtdIns(3)P- and PH domain-dependent signaling pathways.
  • Thumbnail Image
    Structural Studies of the Bacterial Histidine Kinases RetS and GacS, Key Components of the Multikinase Network that Controls the Switch Between a Motile Invasive Lifestyle and a Sessile Biofilm Lifestyle in Pseudomonas aeruginosa
    Ryan, Kylie Meghan (Virginia Tech, 2021-11-15)
    Signal transduction networks enable organisms to respond to environmental stimuli. Bacteria utilize two-component systems (TCSs) and phosphorelays as their primary means of signal transduction. Histidine kinase (HK) and response regulator (RR) proteins comprise these TCSs and phosphorelays. Previously, signal transduction within TCSs and phosphorelays was thought to only occur through a linear series of phosphotransfers between HKs and RRs. Recently multikinase networks have been shown to be involved in TCS and phosphorelay signal transmission. A multikinase network that includes the HKs RetS and GacS controls the switch between the motile invasive lifestyle and the sessile biofilm lifestyle of the opportunistic human pathogen Pseudomonas aeruginosa. GacS promotes the sessile biofilm lifestyle, while RetS promotes the motile invasive lifestyle via the inhibition of GacS. This inhibition occurs through three distinct mechanisms. Two of the mechanisms are dephosphorylating mechanisms and the third mechanism is a direct interaction between RetS and GacS which results in the inhibition of GacS autophosphorylation. This study examines the direct binding interaction between RetS and GacS using structural biology. We observed a heterodimeric RetS-GacS complex in which the canonical homodimerization interface was replaced with a heterodimeric interface. Heterodimerization between bacterial HKs is currently a novel observation, but it is likely that other HKs heterodimerize. The RetS-GacS direct interaction can serve as a model for HK-HK binding in multikinase networks.
  • Thumbnail Image
    The Type IV Pilus Assembly ATPase PilB as a Regulator of Biofilm Formation and an Antivirulence Target
    Dye, Keane (Virginia Tech, 2022-06-02)
    Bacterial type IV pili (T4P) are filamentous surface appendages with a variety of functions including motility, surface attachment, and biofilm formation. In many species of bacteria a clear understanding of how the functions of T4P in lifestyle switching are regulated remains to be elucidated. Here, we focus on understanding the regulation of the T4P assembly ATPase PilB. We examined its interactions with the secondary messenger cyclic-di-GMP (cdG). Specifically we investigated how cdG binding regulates PilB functions not only as the assembly ATPase, but also as an EPS signaling molecule in Myxococcus xanthus biofilm regulation. Chapter 2 focuses on the development of a microplate-based biofilm assay for M. xanthus. This new assay allows for the analysis of the M. xanthus submerged biofilms under vegetative conditions in a high throughput format which has been absent in the published literature. M. xanthus biofilm formation tightly correlates with EPS production, suggesting that the assay can be used as a convenient method of examining EPS production. Chapter 3 examines the regulation of M. xanthus PilB (MxPilB) by cdG binding in vivo. We carried out a mutational analysis of the MshEN cdG binding domain in MxPilB. Mutations were created that either diverge with or converge from the MshEN consensus sequence. These two classes of MxPilB variants are expected to either decrease or increase cdG binding affinity, respectively. We examined the motility, EPS production, and piliation phenotypes of these mutants. Our results were consistent with a model where the function of MxPilB is altered in response to cdG binding, and suggesting that PilB responds to different thresholds of cdG concentration. In Chapter 4, we examine the ligand binding to the N-terminal cdG binding domain and C-terminal ATPase domain of Chloracidobacterium thermophilum PilB (CtPilB) in vitro. Our results confirm that these two domains bind to their respective ligands specifically, and demonstrate these domains communicate with each other in response to ligand binding. The results from all of the studies help us to establish a model where cdG binding fine tunes the functions of PilB to regulate the switch of bacteria between the motile and planktonic states. In addition to their roles in motility and biofilm formation, T4P are key virulence factors in many significant human pathogens. Antivirulence chemotherapeutics are considered to be a promising alternative to antibiotics, as they target disease processes rather than bacterial viability. Because PilB is essential for T4P biogenesis, we sought to identify PilB inhibitors for the development of antivirulence therapies. In Chapter 5, we describe the development of the first high throughput screen (HTS), for PilB inhibitors. This assay is uses the reduction of the binding of a fluorescent ATP analog to CtPilB in vitro, leading to the discovery of the plant flavonoid quercetin as a PilB inhibitor. Using M. xanthus as a model a bacterium, quercetin was found to inhibit T4P-dependent motility and T4P assembly in vivo. Builds on this initial success with CtPilB, Chapter 6 describes the development and implementation of a second HTS based on the inhibition of CtPilB as an ATPase. Screening a large chemical library led to the identification of benserazide and levodopa as CtPilB inhibitors. We show that both compounds inhibit T4P assembly in M. xanthus without any detrimental effects on bacterial growth. Furthermore we demonstrate that both levodopa and benserazide inhibit T4P-mediated motility in Acinetobacter nosocomialis, a human pathogen, providing the first evidence that the compounds identified with CtPilB can be effective against a pathogenic bacterium. Both of these studies validate the effectiveness not only of our HTSs, with of CtPilB as a model protein for the identification of PilB inhibitors.