Microbiology and Immunology

Permanent URI for this collectionhttp://hdl.handle.net/10342/527

Browse

Now showing 1 - 20 of 269

Embargo
Molecular insights into the regulation of complement protease C1s by the multi-specific SERPIN C1 esterase inhibitor
(East Carolina University, July 2024) Garrigues, Ryan J.
Functioning as a sentinel of the innate immune response, the complement system is an evolutionarily ancient proteolytic cascade that is involved in the recognition, opsonization, and lysis of foreign pathogens and apoptotic cells. Initiation of this system is paramount to the potency of the immune response but must be carefully controlled by negative regulators to mitigate aberrant activation. One such regulator, known as C1 esterase inhibitor (C1-INH), is the only canonical complement regulator that directly inhibits the initiating proteases of the classical pathway of complement C1r and C1s. C1-INH is part of a superfamily of serine protease inhibitors (SERPIN), which covalently react with target proteases through a substrate-like reactive center loop (RCL). In addition to its regulatory role in complement, C1-INH is multi-specific targeting a dozen proteases of the coagulation, contact, and fibrinolytic systems. Dysregulation of C1-INH is most apparent in congenital loss-of-function mutations of its encoding gene SERPING1 which causes a chronic debilitating disease known as hereditary angioedema (HAE). However, the molecular basis for C1-INH recognition of any of its cognate proteases including C1s has yet to be elucidated. Towards this end we aimed to characterize the SERPIN domain of C1-INH using a bacterial expression system and determine the molecular basis for C1s interactions. Utilizing structure-guided site-directed mutagenesis coupled to a novel surface plasmon based C1s binding assay we show that C1-INH relies on an extensive binding surface outside of the RCL for full affinity. The implications of this structure to the fields of SERPIN biology are discussed.
Embargo
STRUCTURE-FUNCTION ANALYSIS OF A FAMILY OF BORRELIAL C1r INHIBITORS FROM LYME DISEASE AND RELAPSING FEVER SPIROCHETES
(East Carolina University, 2023-06-01) Booth, Charles E., Jr
The complement system is an ancient part of the innate immune system consisting of fluid-phase proteins that contribute to inflammation, homeostasis, and pathogen clearance. A group of vector-borne spirochetal pathogens are known as the Borrelia spp., are vector-borne bacterial pathogens transmitted the hard- or soft-bodied ticks. The most prominently studied spirochete pathogen in the U.S. is Borreliella burgdorferi, the causative agent of Lyme disease. Lyme disease is a debilitating sickness with an estimated 476,000 cases per year. Within the U.S., other borrelial pathogens are known to cause the diseases relapsing fever and Borrelia miyamotoi disease with the etiological agents being B. hermsii and B. turicatae, and B. miyamotoi respectively. The Lyme spirochete B. burgdorferi was previously found to express the lipoprotein BBK32 in the mammalian host. BBK32 was first discovered to interact with host fibronectin for adhesion and extravasation from the vasculature system. Later BBK32 was found to bind and inhibit the protease, C1r, of the classical pathway of complement for immune evasion purposes. Thus, BBK32 was indicated to be a multifunctional bacterial protein by interacting with multiple host proteins. The etiologic agents of both relapsing fever and B. miyamotoi disease were discovered to encode BBK32 orthologs denoted as the fibronectin binding proteins (Fbps). These orthologs are broken into three separate families termed FbpA, FbpB, and FbpC. Previous work indicated that FbpC from B. hermsii and B. recurrentis interacts with fibronectin and complement regulators, but neither FbpA nor FbpB fibronectin interactions have been characterized. More importantly, no work has investigated the C1r inhibitory potential for either Fbp protein. Herein we characterize the fibronectin interactions for FbpA and FbpB of B. miyamotoi and implement a structure-function approach to determine if the Fbps are capable of interacting with C1r. Utilizing biochemical and biophysical assays we determined that the Fbps demonstrate a differentially bind fibronectin, and C1r active states. Moreover, structural data of the complement inhibitory domains for BBK32 and the Fbps indicate that there are structural differences within the key C1r interacting domains. Molecular dynamic simulations of the complement inhibitory domains of BBK32 and its orthologs indicate that these proteins showcase differences in protein dynamics that may contribute to their C1r inhibition. Overall, our data supports that this family of borrelial C1r inhibitors exhibit similar, but non-overlapping functions. This work directly increases our understanding or host-pathogen interactions and immune evasion along with guiding the synthesis of specific small molecule inhibitors for complement-mediated pathologies.
Open Access
Regulation of Human T-cell Leukemia Virus Type 1 Antisense Promoter by Myocyte Enhancer Factor-2C in the Context of Adult T-cell Leukemia and Lymphoma
(2022-05-26) Rushing, Amanda W.; Lemasson, Isabelle; et al
Open Access
Cellular miR-150-5p May Have a Crucial Role to Play in the Biology of SARS-CoV-2 Infection by Regulating nsp10 Gene
(2022) Akula, Shaw M.; Bolin, Paul; Cook, Paul P.
Open Access
Lipoproteome Screening of the Lyme Disease Agent Identifies Inhibitors of Antibody-Mediated Complement Killing
(2022) Garrigues, Ryan J.; Garcia, Brandon L.; et al
Open Access
FliL Ring Enhances the Function of Periplasmic Flagella
(2022-03-07) Xu, Hui; Motaleb, Md A.; et al
Open Access
Outer Surface Lipoproteins from the Lyme Sisease Spirochete Exploit the Molecular Switch Mechanism of the Complement Protease C1s
(2022-09-29) Garrigues, Ryan J.; Thomas, Sheila; Garcia, Brandon L.; Leong, John M.
Open Access
TLR-4 agonism induces CD25+ MHCIIhigh dendritic cells in association with tolerogenic antigen recognition
(East Carolina University, 2022-12-06) Bastian, Alexander
Autoimmune disease is a result of the breakdown in immunological self-tolerance leading to destruction of self-tissues mediated by the aberrant immune attack. In Multiple Sclerosis (MS), CD4+ T cells mediate destruction of myelin in the central nervous system (CNS) leading to debilitating symptoms in afflicted individuals. MS is the leading cause of non-injury disability in young adults, impacting nearly 1 million people in the United States alone. As is the case for many autoimmune diseases, there is no cure for MS, highlighting the urgent need for new therapeutic platforms. A therapeutic approach to reestablish self-tolerance is likely to be more effective and have less side effects than current treatments, highlighting the importance for developing such an approach. At the center of this approach lies CD4+ FOXP3+regulatory T cells (Tregs), which are a subset of T cells that suppress the immune system and play an integral role in controlling autoimmunity. Therapies aimed at increasing Tregs, especially in a disease targeted manner, have the potential to reestablish self-tolerance and cure autoimmunity.All T cells, including Tregs, must recognize antigen on antigen presenting cells (APCs) to perform effector functions. Therefore, targeting an APC niche that supports the development of Tregs is an effective approach for development of autoimmune therapeutics. Dendritic cells (DCs) are a class of APCs known to support Treg development and function. In this study, we show that agonism of Toll-like receptor 4 (TLR-4) with lipopolysaccharide (LPS) or monophosphoryl Lipid A (MPLA) on DCs leads to a CD25⁺ MHCIIhigh DC phenotype. We show that the expression of CD25 is unique to DCs and that it binds IL-2 from the environment without detectable downstream signaling. Importantly, we show that this bound IL-2 can be utilized by responder T cells, highlighting a potential function of CD25 on DCs. We then combined TLR-4 agonism with our lab's DC-targeting tolerogenic vaccine, GMCSF-MOG, which we have previously shown leads to high levels of Tregs and amelioration of experimental autoimmune encephalomyelitis (EAE) in mice. When GMCSF-MOG is combined with MPLA, Treg levels are increased and extend out to 78 days post injection. The enhanced and extended Treg levels observed when MPLA is included in the vaccine likely play a role in accelerating the GMCSF-MOG-mediated amelioration of EAE and preventing observed EAE relapse. At the crux of DC-mediated tolerance induction is the efficiency of the antigen recognition event. Lower affinity TCR ligation supports tolerance through lower induction of costimulatory molecules (CD40L) and increased induction of inhibitory molecules (PD-1). Furthermore, inhibiting the CD40L/CD40 axis increases Treg induction. Overall, we show that TLR-4 agonism leads to CD25⁺ MHCIIhigh DCs and functions as a tolerogenic adjuvant when combined with the DC targeting GMSCF-MOG vaccine through support of low-efficiency Treg-favorable antigen recognition events.
Open Access
A Wzx-Wzy-like Polysaccharide Biosynthetic Pathway Contributes to Virulence in Brucella abortus 2308 through an Unknown Mechanism
(East Carolina University, 2022-08-05) Hopersberger, Dariel Anne
Brucella abortus is an intracellular pathogen that causes spontaneous abortion in cattle and undulant fever in humans. As a member of the a-proteobacteria, B. abortus is closely related to the bacterial species Caulobacter crescentus and Agrobacterium tumefaciens, which have been shown to produce a holdfast and a unipolar polysaccharide (UPP), respectively, at one pole of the bacterial cell. Although little is currently known about exopolysaccharide production and function in Brucella species, recent evidence suggests that these bacteria are capable of producing exopolysaccharides, and with the exception of a flippase gene, B. abortus 2308 carries the genes for a complete UPP biosynthetic pathway. In A. tumefaciens, UPP can be detected by crystal violet, Congo red, or lectin staining; however, UPP production was not detected in B. abortus 2308 using these assays. UPP production can also be increased in A. tumefaciens by overexpressing the diguanylate cyclase PleD, which regulates intracellular cyclic di-GMP levels, but we were unable to demonstrate that UPP is regulated by cyclic di-GMP in B. abortus 2308. Nonetheless, upp homologs in B. abortus 2308 are able to restore biofilm production in A. tumefaciens. In A. tumefaciens, knockout mutants of the outer membrane transporter uppC and the polyisoprenylphosphate hexose-1-phosphate transferase uppE result in strong biofilm phenotypes, wherein the ability of A. tumefaciens to produce a biofilm is nearly abrogated. B. abortus 2308 uppC and uppE homologs restore biofilm production in the respective Agrobacterium upp mutants, indicating that uppC and uppE from B. abortus 2308 produce functional proteins. A uppCE mutant in B. abortus 2308 also exhibits significant attenuation in mice and an altered intracellular replication profile in cultured murine macrophages. Accordingly, uppCE appears to be required for virulence in B. abortus 2308, although by a currently unknown mechanism. Additional studies will be needed to determine whether B. abortus 2308 produces an authentic UPP as well as define the mechanisms by which the putative upp genes contribute to virulence.
Open Access
A Novel Formamidase is Required for Riboflavin Biosynthesis in Invasive Bacteria
(2002) Baumgartner, John; Pitzer, Josh E.; Roop, R. Martin II; Yurge, Svetlana N.
Open Access
Acinetobacter baumannii Regulates Its Stress Responses via the BfmRS Two-Component Regulatory System
(2022-02-15) Palethorpe, Samantha; Farrow III, John M.; Wells, Greg; Pesci, Everett; Milton, Morgan E.; Cavanagh, John; Actis, Luis
Open Access
Analysis of Six tonB Gene Homologs in Bacteroides Fragilis Revealed That tonB3 is Essential for Survival in Experimental Intestinal Colonization and Intra-Abdominal Infection
(2022-01-25) Parker, Anita C.; Seals, Nathaniel L.; Rocha, Edson R.; Baccanale, Cecile L.
Open Access
gC1qR/C1qBP/HABP-1: Structural Analysis of the Trimeric Core Region, Interactions With a Novel Panel of Monoclonal Antibodies, and Their Influence on Binding to FXII
(2022-07-05) Garcia, Brandon L.; Zhang, Ying
Open Access
Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions
(2022-05-27) Booth, Charles E. Jr; Garcia, Brandon L.
Open Access
Pathobiology and Therapeutic Relevance of GSK-3 in Chronic Hematological Malignancies
(2022-05-31) McCubrey, James A.; Martelli, Alberto M.; Paganelli, Francesca; Evangelisti, Camilla; Chiarini, Francesca
Open Access
Effects of the Mutant TP53 Reactivator APR-246 on Therapeutic Sensitivity of Pancreatic Cancer Cells in the Presence and Absence of WT-TP53
(2022-02-24) Abrams, Stephen L.; Akula, Shaw M.; Steelman, Linda S.; McCubrey, James A.
Open Access
APR-246—The Mutant TP53 Reactivator—Increases the Effectiveness of Berberine and Modified Berberines to Inhibit the Proliferation of Pancreatic Cancer Cells
(2022-02-08) McCubrey, James Andrew; Abrams, Stephen L.; Steelman, Linda S.
Open Access
Wild Type and Gain of Function Mutant TP53 can Regulate the Sensitivity of Pancreatic Cancer Cells to Chemotherapeutic Drugs, EGFR/Ras/Raf/MEK, and PI3K/mTORC1/GSK-3 Pathway Inhibitors, Nutraceuticals and Alter Metabolic Properties
(2022-04-27) McCubrey, James A.; Meher, Akshaya K.; Akula, Shaw M.; Abrams, Stephen L.; Steelman, Linda S.; LaHair, Michelle M.; Franklin, Richard A.; Martelli, Alberto M.; Ratti, Stefano; Cocco, Lucio; Barbaro, Fulvio; Duda, Przemysław; Gizak, Agnieszka
Open Access
A Structural Basis for Inhibition of the Complement Initiator Protease C1r by Lyme Disease Spirochetes
(2021-07-04) Garrigues, Ryan J.; Garcia, Brandon L.; Pierce, Alexandra D. Powell; Hammel, Michal; Skare, Jon T.
Open Access
A Gain and Dynamic Range Independent Index to Quantify Spillover Spread to Aid Panel Design in Flow Cytometry
(2021) Ratlif, Michelle L.; Bhowmick, Debajit; van Diepen, Frank; Pfauth, Anita; Tissier, Renaud