Browsing by Department "Microbial Pathogenesis And Immunology"

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A Novel Technology for Engineering Binders to Membrane Proteins
Microbial Pathogenesis And Immunology; TAMHSC; National Institutes of Health
Antibodies have been coined the ‘magic bullets’ against many human diseases. However, there remains significant challenges in the engineering of antibodies targeting multi-pass membrane proteins, which encompass a large number of therapeutic targets such as cell surface receptors and the ion channel proteins. The difficulty in engineering binders to membrane proteins stems from the limitation of the current in vitro selection/panning technologies, such as phage display, which require highly purified target protein. Unfortunately, membrane proteins are often refractory to purification due to their dependence on the cell membrane for proper folding and activity. Currently, there is no effective in vitro technology for the discovery/engineering of binders to multi-pass membrane proteins. The overall goal of this study is to develop a novel technology – SMURF (Simple proxiMity coUpled mRNA display) – for engineering protein binders to protein targets on the cell surface, thus bypassing the need to purify the target protein. SMURF combines mRNA display with the proximity-assisted-DNA-assembly phenomenon and, unlike conventional panning in which all binders to a solid support are enriched, SMURF fosters the enrichment of binders only to a desired target protein on the cell surface. In Aim 1, we will demonstrate the SMURF principle using oligonucleotides and optimize the primer sequences. Aim 2 will establish the SMURF enrichment of a model protein in a mixture of non-target proteins in solution. Finally, in Aim 3, a model protein will be displayed on the mammalian cell surface and a library of binders will be screened to demonstrate and quantify the whole-cell SMURF enrichment efficiency. The successful completion of this study will establish a novel technology for facile discovery/engineering of binders to whole-cell-displayed membrane proteins and should greatly expand the repertoire of drug targets amenable to therapeutic intervention.
A Novel Technology for Engineering Binders to Membrane Proteins
Microbial Pathogenesis And Immunology; TAMHSC; https://hdl.handle.net/20.500.14641/217; National Institutes of Health
Antibodies have been coined the ‘magic bullets’ against many human diseases. However, there remains significant challenges in the engineering of antibodies targeting multi-pass membrane proteins, which encompass a large number of therapeutic targets such as cell surface receptors and the ion channel proteins. The difficulty in engineering binders to membrane proteins stems from the limitation of the current in vitro selection/panning technologies, such as phage display, which require highly purified target protein. Unfortunately, membrane proteins are often refractory to purification due to their dependence on the cell membrane for proper folding and activity. Currently, there is no effective in vitro technology for the discovery/engineering of binders to multi-pass membrane proteins. The overall goal of this study is to develop a novel technology – SMURF (Simple proxiMity coUpled mRNA display) – for engineering protein binders to protein targets on the cell surface, thus bypassing the need to purify the target protein. SMURF combines mRNA display with the proximity-assisted-DNA-assembly phenomenon and, unlike conventional panning in which all binders to a solid support are enriched, SMURF fosters the enrichment of binders only to a desired target protein on the cell surface. In Aim 1, we will demonstrate the SMURF principle using oligonucleotides and optimize the primer sequences. Aim 2 will establish the SMURF enrichment of a model protein in a mixture of non-target proteins in solution. Finally, in Aim 3, a model protein will be displayed on the mammalian cell surface and a library of binders will be screened to demonstrate and quantify the whole-cell SMURF enrichment efficiency. The successful completion of this study will establish a novel technology for facile discovery/engineering of binders to whole-cell-displayed membrane proteins and should greatly expand the repertoire of drug targets amenable to therapeutic intervention.
COVID-19: RAPID: Large-scale functional analysis of Ab repertoires elicited by SARS-CoV-2
Microbial Pathogenesis And Immunology; TAMU; https://hdl.handle.net/20.500.14641/470; National Science Foundation
The award to Texas A&M University will support research on a novel lab-on-a-chip system, to determine the identity, functionality, and clonality of antibodies (Abs) elicited by SARS-CoV-2, the causative agent of COVID-19. One of the most important functions of Abs during viral infection is neutralization, the process whereby Ab binding to viral or host targets prevents viral entry into host cells, thereby thwarting disease. Conventional approaches for determining the functions of pathogen-specific neutralizing Ab (nAb) repertoire are time- and labor-intensive, and thus have only been used to investigate small portions of Ab repertoires. This award will support research to develop a system that enables fast, direct, functional assays that measure viral neutralization activities of Ab-producing cells. In addition to the strong scientific impact, this award will support cross-disciplinary training postdoctoral, graduate student, and undergraduate student trainees, including women and scientists from historically under-represented groups. Results from the studies will be disseminated rapidly and in peer-reviewed journals and at scientific meetings. Until recently, the large-scale analysis of antibody repertoires was cost-prohibitive, owing to their massive sizes. However, over the past decade, novel sequencing approaches, including Ig-seq, have provided unprecedented insight into Ab gene repertoires. However, despite these dramatic advances, our understanding of the functions of Ab repertoires remains incomplete. Support from this award will advance a droplet microfluidics platform termed PRESCIENT, that enables fast, single-cell (digital) resolution, direct, functional assays that measure viral neutralization activities of Ab-producing B-cells, thereby providing a system for rapidly characterizing a large repertoire of nAbs against viral pathogens. The overall objectives are to deliver: A fully integrated microfluidic platform with dual fluorescence detection capability and system level throughput of at least 10 assays/sec achieved; demonstration that sera from inoculated mice react with recombinant SARS-CoV-2 spike protein antigen; and characterization (at single cell resolution) the nAb (functional) repertoire against pseudotyped SARS-CoV-2-GFP. This RAPID award will thus deliver the first global functional characterization of Abs that neutralize pseudotyped SARS-CoV-2, a timely response to the ongoing global pandemic caused by SARS-CoV-2 and an important complement to the ongoing computer simulation studies conducted by other labs. This RAPID award is made by the Division of Biological Infrastructure (DBI) using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
Elucidating the Roles of CMPK2 in Mitochondrial Homeostasis and Antiviral Immunity
Microbial Pathogenesis And Immunology; TAMHSC; https://hdl.handle.net/20.500.14641/248; DHHS-NIH-National Heart, Lung, and Blood Institute
PROJECT SUMMARY: Cytidine/uridine monophosphate kinase 2 (CMPK2) is an interferon-regulated enzyme that was originally reported to catalyze the ATP-dependent phosphorylation of dCMP and dUMP to diphosphate forms in vitro. Due to a putative mitochondrial targeting sequence, CMPK2 was postulated to function in the mitochondrial deoxyribonucleotide salvage pathway necessary for the synthesis and maintenance of mitochondrial DNA (mtDNA). However, more recent data have revealed that CMPK2 prefers ribonucleotide diphosphate substrates in vitro and functions to restrict HIV and other RNA viruses in cell based assays. Beyond these seemingly contrasting findings, no other studies have addressed the cellular localization and tissue expression patterns of CMPK2 or utilized genetic knockouts to determine true biological activity. Therefore, the overall objective of this proposal is to close these knowledge gaps and mechanistically advance understanding of CMPK2 in mitochondrial function, tissue homeostasis, and antiviral innate immunity using a diverse toolkit of cell and animal models. The central hypothesis is that by maintaining mitochondrial homeostasis, CMPK2 boosts cell-intrinsic innate immunity and limits runaway inflammation triggered by mitochondrial stressors and viral infection. In support of this hypothesis, ongoing studies have revealed that CMPK2 localizes strongly to mitochondria and that ectopic overexpression of CMPK2 is sufficient to protect cells from RNA virus infection. Moreover, after systemic challenge with innate immune agonists, CMPK2 is markedly upregulated in the lungs and liver, and CMPK2 knockout mice exhibit elevated expression of proinflammatory cytokines and type I interferon (IFN-I) responses after Toll-like receptor (TLR) stimulation. To gain additional insight into how CMPK2 functions in mitochondria and antiviral immunity, two related, but independent aims are proposed. Aim 1 will elucidate the molecular mechanisms by which CMPK2 maintains mitochondrial homeostasis at rest and during stress. Here, CMPK2 knockout cells, novel lines reconstituted with mutant CMPK2 vectors lacking nucleotide kinase activity or mitochondrial targeting, and whole body CMPK2 knockout mice will be utilized. Aim 2 will determine that the mitochondrial activity of CMPK2 restricts coronavirus replication and maintains mitochondrial function during infection. Here, an intranasal mouse hepatitis virus challenge protocol that models acute respiratory distress syndrome and closely mirrors coronavirus pneumonia in humans will be employed. This research will fundamentally advance our understanding of how CMPK2 functions in mitochondrial homeostasis and antiviral innate immunity at both the cellular and organismal levels. Moreover, it may have a positive impact on public health by revealing novel CMPK2-centered strategies to maintain mitochondrial homeostasis, boost antiviral immunity, and limit damaging inflammation during coronavirus infection.
Elucidating the Roles of CMPK2 in Mitochondrial Homeostasis and Antiviral Immunity
Microbial Pathogenesis And Immunology; TAMHSC; https://hdl.handle.net/20.500.14641/248; DHHS-NIH-National Heart, Lung, and Blood Institute
PROJECT SUMMARY Cytidine/uridine monophosphate kinase 2 (CMPK2) is an interferon-regulated enzyme that was originally reported to catalyze the ATP-dependent phosphorylation of dCMP and dUMP to diphosphate forms in vitro. Due to a putative mitochondrial targeting sequence, CMPK2 was postulated to function in the mitochondrial deoxyribonucleotide salvage pathway necessary for the synthesis and maintenance of mitochondrial DNA (mtDNA). However, more recent data have revealed that CMPK2 prefers ribonucleotide diphosphate substrates in vitro and functions to restrict HIV and other RNA viruses in cell based assays. Beyond these seemingly contrasting findings, no other studies have addressed the cellular localization and tissue expression patterns of CMPK2 or utilized genetic knockouts to determine true biological activity. Therefore, the overall objective of this proposal is to close these knowledge gaps and mechanistically advance understanding of CMPK2 in mitochondrial function, tissue homeostasis, and antiviral innate immunity using a diverse toolkit of cell and animal models. The central hypothesis is that by maintaining mitochondrial homeostasis, CMPK2 boosts cell-intrinsic innate immunity and limits runaway inflammation triggered by mitochondrial stressors and viral infection. In support of this hypothesis, ongoing studies have revealed that CMPK2 localizes strongly to mitochondria and that ectopic overexpression of CMPK2 is sufficient to protect cells from RNA virus infection. Moreover, after systemic challenge with innate immune agonists, CMPK2 is markedly upregulated in the lungs and liver, and CMPK2 knockout mice exhibit elevated expression of proinflammatory cytokines and type I interferon (IFN-I) responses after Toll-like receptor (TLR) stimulation. To gain additional insight into how CMPK2 functions in mitochondria and antiviral immunity, two related, but independent aims are proposed. Aim 1 will elucidate the molecular mechanisms by which CMPK2 maintains mitochondrial homeostasis at rest and during stress. Here, CMPK2 knockout cells, novel lines reconstituted with mutant CMPK2 vectors lacking nucleotide kinase activity or mitochondrial targeting, and whole body CMPK2 knockout mice will be utilized. Aim 2 will determine that the mitochondrial activity of CMPK2 restricts coronavirus replication and maintains mitochondrial function during infection. Here, an intranasal mouse hepatitis virus challenge protocol that models acute respiratory distress syndrome and closely mirrors coronavirus pneumonia in humans will be employed. This research will fundamentally advance our understanding of how CMPK2 functions in mitochondrial homeostasis and antiviral innate immunity at both the cellular and organismal levels. Moreover, it may have a positive impact on public health by revealing novel CMPK2-centered strategies to maintain mitochondrial homeostasis, boost antiviral immunity, and limit damaging inflammation during coronavirus infection.
Innate Immune Signaling and Type I Interferon Responses as Novel Modifiers of Mitochondrial Disease Pathology
Microbial Pathogenesis And Immunology; TAMHSC; https://hdl.handle.net/20.500.14641/248; DOD-Army-Medical Research and Materiel Command
Fiscal Year 2016 Peer Reviewed Medical Research Program Topic Area: Mitochondrial Disease Mitochondrial diseases are a group of disorders caused by the malfunction of cellular organelles called mitochondria. Because mitochondria are responsible for generating the energy that powers vital cellular processes, mitochondrial malfunction can result in extensive disease throughout the body, including the nervous, musculoskeletal, digestive, and reproductive systems. Much of the research on mitochondrial disease has focused on the role of mitochondria in energy generation. However, recent studies have shown that mitochondria are key regulators of the immune system and orchestrate many aspects of inflammation during viral or bacterial illness, as well as in non-infectious diseases. In fact, abnormal inflammatory responses have been implicated in a number of diverse pathologies, some of which are present in the multi-organ disease of patients afflicted with mitochondrial syndromes. It is therefore possible that mitochondrial dysfunction aberrantly engages the immune system, resulting in inflammatory responses that exacerbate the pathology of mitochondrial disorders. To test this hypothesis, we will use a mouse model of mitochondrial disease (called POLG-mutator mice) that mirrors pathology seen in human patients with mitochondrial disease. We will use an array of techniques to characterize inflammatory responses through the progression of disease in these mice. Next, we will use POLG-mutator mice deficient in key immune signaling pathways to determine whether the absence of these pathways attenuates mitochondrial dysfunction and disease, thus demonstrating their importance in driving it. Based on preliminary studies, we predict that inhibition of the immune system will slow or alleviate pathology in this mouse model of mitochondrial disease. This proposal is innovative in several ways. First, it will examine the novel, unexplored paradigm that inflammatory mechanisms exacerbate multi-organ pathology in mitochondrial disorders and will provide a robust foundation for future research that focuses on immune pathology of mitochondrial diseases in other experimental and clinical settings. Second, there are presently no cures for many mitochondrial disorders, and few treatments are available to slow the progression of these diseases. This research may lay the foundation for studies exploring the therapeutic targeting of inflammatory pathways as a means to attenuate multi-system pathology of mitochondrial diseases.
Internal Toxin Neutralizer for Treating STEC-infection
Microbial Pathogenesis And Immunology; TAMHSC; DHHS-NIH-National Institute of Allergy and Infectious Diseases
Abstract The Shiga toxin-producing E. coli (STEC) is the most common cause of bloody diarrhea and afflicts an estimated 73,000 people in the US annually, causing significant morbidity. The most recent and largest STEC outbreak occurred in Germany in 2011, affecting >3,800 people, including 54 deaths. Currently there is no effective treatment for STEC infection. The pathology of STEC infection derives from two exotoxins – Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) – that are secreted by STEC in the gut. Although antibiotic treatment can reduce the load of STEC, it also augments Shiga toxin release, leading to increased risk of developing the more serious hemolytic uremic syndrome (HUS) and kidney failure (up to 25%). Consequently, the CDC recommends that antibiotics not be used in STEC patients and that only supportive therapy (e.g. oral and i.v. fluid, pain control) be used. Although anti-toxin antibodies have been identified, the inability of antibodies to cross the cell membrane renders them powerless against toxins already absorbed by the host cells, limiting their clinical application. We hypothesize that a cytosol-accessible anti-toxin should be able to neutralize both extracellular and intracellular Shiga toxin, leading to a much-prolonged therapeutic window and better therapeutic efficacy. The overall goal of this study is to engineer a panel of intracellular toxin neutralizers (ITNs) against Shiga toxin 2 (Stx2). As a scaffold for the proposed ITN, we will use a designed ankyrin repeat protein (DARPin). DARPins represent a versatile class of binding proteins that have been engineered to bind diverse targets with up to picomolar affinity and possess low immunogenicity. In this project, we will first isolate DARPins that bind and neutralize Stx2 (Aim 1). Concurrently, we will screen a panel of cell-penetrating peptides (CPPs) for their ability to transport ITNs into cells (Aim 2). In Aim 3, we will assemble anti-Stx2 ITNs using the best anti-Stx2 DARPin and CPP and evaluate the therapeutic potential of these anti-Stx2 ITNs in vitro and in vivo. The approach of using ITN to combat toxins in circulation offers a new paradigm for the treatment of both STEC and non-STEC bacterial infections.
Internal Toxin Neutralizer for Treating STEC-infection
Microbial Pathogenesis And Immunology; TAMHSC; https://hdl.handle.net/20.500.14641/217; DHHS-NIH-National Institute of Allergy and Infectious Diseases
The Shiga toxin-producing E. coli (STEC) is the most common cause of bloody diarrhea and afflicts an estimated 73,000 people in the US annually, causing significant morbidity. The most recent and largest STEC outbreak occurred in Germany in 2011, affecting >3,800 people, including 54 deaths. Currently there is no effective treatment for STEC infection. The pathology of STEC infection derives from two exotoxins – Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) – that are secreted by STEC in the gut. Although antibiotic treatment can reduce the load of STEC, it also augments Shiga toxin release, leading to increased risk of developing the more serious hemolytic uremic syndrome (HUS) and kidney failure (up to 25%). Consequently, the CDC recommends that antibiotics not be used in STEC patients and that only supportive therapy (e.g. oral and i.v. fluid, pain control) be used. Although anti-toxin antibodies have been identified, the inability of antibodies to cross the cell membrane renders them powerless against toxins already absorbed by the host cells, limiting their clinical application. We hypothesize that a cytosol-accessible anti-toxin should be able to neutralize both extracellular and intracellular Shiga toxin, leading to a much-prolonged therapeutic window and better therapeutic efficacy. The overall goal of this study is to engineer a panel of intracellular toxin neutralizers (ITNs) against Shiga toxin 2 (Stx2). As a scaffold for the proposed ITN, we will use a designed ankyrin repeat protein (DARPin). DARPins represent a versatile class of binding proteins that have been engineered to bind diverse targets with up to picomolar affinity and possess low immunogenicity. In this project, we will first isolate DARPins that bind and neutralize Stx2 (Aim 1). Concurrently, we will screen a panel of cell-penetrating peptides (CPPs) for their ability to transport ITNs into cells (Aim 2). In Aim 3, we will assemble anti-Stx2 ITNs using the best anti-Stx2 DARPin and CPP and evaluate the therapeutic potential of these anti-Stx2 ITNs in vitro and in vivo. The approach of using ITN to combat toxins in circulation offers a new paradigm for the treatment of both STEC and non-STEC bacterial infections.
TRIM Proteins Polarize DNA Sensing Outcomes During the Innate Immune Response to Mycobacterium Tuberculosis
Microbial Pathogenesis And Immunology; TAMHSC; https://hdl.handle.net/20.500.14641/513; National Institutes of Health
Project Summary Mycobacterium tuberculosis (Mtb) is an incredibly successful human pathogen that currently infects one-third of the world's population and kills 1.5 million people every year. While interaction of Mtb bacilli and macrophages activates numerous antimicrobial pathways, this bacterium has evolved an exquisite array of adaptations to counteract such responses in order to establish a niche and promote infection. As such, when Mtb is internalized into macrophages, innate immune sensing of bacterial DNA in the host cell cytosol triggers both anti-bacterial and pro-bacterial responses: selective autophagy destroys a population of bacilli and restricts Mtb growth, while activation of the antiviral type I interferon response promotes bacterial infection and pathogenesis. An innate immune kinase called TBK1 is central to both of these processes; however, the mechanism by which this kinase comprises both selective autophagy and type I interferon signaling complexes is unknown. Our new work has uncovered an important role for the tripartite motif protein TRIM14 in regulating the kinase TBK1 and eliciting the type I IFN response during Mtb infection. We hypothesize that TRIM14 is a key modulator of DNA sensing during Mtb infection and that post-translational modification of TRIM14 influences the shuttling of TBK1 away from selective autophagy to promote type I IFN production. Using biochemical, proteomic and microscopy-based approaches we will (1) determine the mechanism by which TRIM14 influences DNA sensing outcomes during Mtb infection (2) elucidate the role of post-translational modifications in regulating TRIM14 and (3) determine the role of TRIM30a in negatively regulating type I IFN production and controlling Mtb pathogenesis. Because these two DNA sensing pathways lead to such strikingly different disease outcomes, there is an obvious opportunity to develop therapeutics that target molecules like TRIMs, in hopes of activating selective autophagy while inhibiting the type I interferon response during Mtb infection.