Pilot Project 1 

Title: Interferon-Mediated Impairment in Macrophage Antibacterial Activity during SARS-CoV-2 and Klebsiella pneumoniae Co-Infection
Pilot Project Leader: Sunil More, Ph.D, The Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University

Project Summary: 

Respiratory viruses such as SARS-CoV-2 are known to predispose patients to secondary bacterial infections. This further complicates the disease pathogenesis and clinical management of the cases. Approximately 40% of COVID-19 patients have detectable bacterial coinfection and Klebsiella pneumoniae (Kp) has been identified as a secondary infection in SARS-CoV-2 patients. Despite the clinical significance of coinfections, comprehensive studies evaluating their impact on pathogenesis in COVID-19 patients are limited. To address this gap, we have developed a mouse model of SARS-CoV-2 and Kp coinfection, demonstrating a lethal phenotype upon coinfection. Surprisingly, despite an increase in macrophages, there is significant bacterial propagation in the lungs. We have also observed that interferon-treated macrophages failed to clear Kp. Thus, we hypothesize that type I interferons secreted upon SARS-CoV-2 infection are imparting dysfunctional phenotype to the macrophages. Therefore, our project aims to investigate the impact of interferons on the antimicrobial function of macrophages in the context of SARS-CoV-2 infection. We will evaluate the antibacterial activity of SARS-CoV-2 infected or interferon-treated macrophages and explore the underlying mechanisms involved. 

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Pilot Project 2

Title: Investigating SARS-CoV-2 persistence in a mouse model of Long COVID
Pilot Project Leader: Xufang Deng, Ph.D, The Department of Veterinary Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

Project Summary: 

Long COVID, or post-acute COVID-19 syndrome (PACS), affects more than 65 million individuals globally, presenting a substantial health challenge with no currently validated and effective treatments.  Despite recovery from acute SARS-CoV-2 infection, Long COVID manifests as a multisystemic condition impacting various organs. While the virus primarily causes respiratory complications, it can persist in diverse body sites such as the intestines, heart, brain, and kidneys. It is hypothesized that the persisting reservoir of SARS-CoV-2 instigates prolonged systemic inflammation, contributing to the persistent symptoms in Long COVID patients. However, this clinical observation-based hypothesis lacks experimental validation, and the connection between SARS-CoV-2 persistence and prolonged inflammation remains elusive. This project aims to address these gaps by establishing an animal model to understand the in vivo dynamics of host inflammation and SARS-CoV-2 infection. Leveraging a natural SARS-CoV-2 infection mouse model and an inflammation biosensor mouse, specific objectives will be pursued. Aim 1 involves determining the spatiotemporal dynamics of host inflammation during and after SARS2-N501YMA30 infection, utilizing bioluminescence imaging and transcriptomic profiling. Unique characteristics of SARS-CoV-2-induced inflammation will be identified by comparing it with influenza A infection. In Aim 2, we aim to define viral persistence and explore the relationship between viral infection/persistence and organ inflammation through dual-color in vivo imaging. Our goal is to demonstrate the impact of viral persistence on prolonged inflammation and tissue injury following acute SARS-CoV-2 infection.

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Pilot Project 3

Title: Non-tuberculous Mycobacterial Infection in Cystic Fibrosis Ferret Model
Pilot Project Leader: Yong Cheng, Ph.D, The Department of Biochemistry and Molecular Biology, Division of Agricultural Sciences and Natural Resources, Oklahoma State University

Project Summary: 

Cystic fibrosis (CF) is an autosomal recessive disease in humans caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR). Microbial lung infections lead to the majority of morbidity and mortality in patients with CF. Non-tuberculous mycobacterial (NTM) infections have been found in approximately 13% of CF patients, and related incidence and prevalence is increasing. Mycobacterium avium complex (MAC) (M.avium and M.intracellulare) and Mycobacterium abscessus complex (MABSC) (M.abscessus, M.massiliense and M.bolletii), are two main groups of NTM identified in CF patients and account for 95% of NTM infections. Our preliminary data indicate that M.abscessus infection dysregulates macrophages in the airway of CF mice, and in vitro airway macrophage culture prepared from CF mice. Similar results were observed in vitro in CF ferret bone marrow-derived macrophage culture, and human monocyte-derived macrophage culture. In this pilot project, we intend to establish a CF ferret model with M.abscessus lung infection and further investigate the effect of M.abscessus lung infection on macrophage function and lung pathology in CF ferrets.

Pilot Project 1 

Title: Glycosylation and Structural Studies of SARS-CoV-2 Entry Membrane Protein TMPRSS2
Pilot Project Leader: Gabriel Cook, Ph.D., The Department of Chemistry, College of Arts and Sciences, Oklahoma State University

Project Summary: 

Transmembrane Protease Serine 2 (TMPRSS2) plays an important role in viral entry into host cells for influenza and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV). SARS-CoV-2, the highly transmittable viral pathogen has infected over 760 million people, resulting in 6.9 million deaths worldwide. The spike glycoprotein of SARS-CoV-2 engages the host cell membrane glycoprotein TMPRSS2 gene for activation and entry into the cell. It is believed that the glycosylation state of both of these proteins in important for the interaction that leads to the entry of the virus. 

Utilizing recumbinantly expressed full-length TMPRSS2, we will use Nuclear Magnetic Resonance (NMR) Spectroscoph and Small-angle X-ray Scattering (SAXS) Spectroscoph to determin the structure and the dynamics of the membrane protein in a membrane mimicking environment. As we have shown previously, NMR is a powerful technique for determining the high-resolution structure and dynamics of membrane proteins. 

In addition, we will use the methods of in vitro glycosylation developed in our lab to examine the changes that occur as a result of this post-translational modification as glycosylation has been shown to be extremely important for the function of glycoproteins. 

Employing these methods, we will determine the structure and dynamics of this biologically important glycoprotein and will explore possible exposed cleavage regions and binding sites to provide a map for therapeutic design. Future detailed and specific study of TMPRSS2 are required to provide more information on the structure and function of its various domains which would be useful in determining the role of TMPRSS2 in viral entry and replication and thus beneficial for the diagnosis and therapy for these infections. 

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Pilot Project 2

Title: Interferon-Mediated Impairment in Macrophage Antibacterial Activity during SARS-CoV-2 and Klebsiella pneumoniae Co-Infection
Pilot Project Leader: Sunil More, Ph.D, The Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University

Project Summary: 

Respiratory viruses such as SARS-CoV-2 are known to predispose patients to secondary bacterial infections. This further complicates the disease pathogenesis and clinical management of the cases. Approximately 40% of COVID-19 patients have detectable bacterial coinfection and Klebsiella pneumoniae (Kp) has been identified as a secondary infection in SARS-CoV-2 patients. Despite the clinical significance of coinfections, comprehensive studies evaluating their impact on pathogenesis in COVID-19 patients are limited. To address this gap, we have developed a mouse model of SARS-CoV-2 and Kp coinfection, demonstrating a lethal phenotype upon coinfection. Surprisingly, despite an increase in macrophages, there is significant bacterial propagation in the lungs. We have also observed that interferon-treated macrophages failed to clear Kp. Thus, we hypothesize that type I interferons secreted upon SARS-CoV-2 infection are imparting dysfunctional phenotype to the macrophages. Therefore, our project aims to investigate the impact of interferons on the antimicrobial function of macrophages in the context of SARS-CoV-2 infection. We will evaluate the antibacterial activity of SARS-CoV-2 infected or interferon-treated macrophages and explore the underlying mechanisms involved. 

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Pilot Project 3

Title: siRNA-mediated gene silencing to identify functional NETosis pathways in a feline model for COVID-19
Pilot Project Leader: Jennifer Margaret Rudd, Ph.D, The Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University

Project Summary: 

The emergence of SARS-CoV-2 resulted in a pandemic with severe consequences for human and animal health as well as the global economy. In line with other acute respiratory infections, a significant driver of disease severity is the hyperresponsive and dysfunctional innate immune response triggering cytokine storm and collateral host tissue damage. Numerous innate responders play a role in this excessive inflammatory response; notably, neutrophilia, and the release of neutrophil extracellular traps (NETs), are significantly correlated with worsened clinical outcomes in COVID-19. The delicate balance between beneficial inflammation and neutrophil-derived host tissue damage emphasizes a crucial area of investigation for therapeutics to avoid severe illness. As SARS-CoV-2 viral variants drift toward global endemicity, it remains critical that effective therapies continue to be developed. While the pathobiology of the virus has been studied since emergence, bioinformatics tools designed explicitly for SARS-CoV-2 have only been developed recently. In addition, the use of small interfering RNA (siRNA) is emerging as a promising therapeutic approach to acute COVID-19. Previous studies in our lab developed and validated translational feline SARS-CoV-2 model capable of inducing clinical signs, lesions, and gene expression analogous to acute COVID-19 in hospitalized patients. In this study, we propose an in vitro feline neutrophil model as compared to a human-derived neutrophil in vitro model to delineate functional kinetics of neutrophils and NETS during SARS-CoV-2 infection and employ promising bioinformatics tools and siRNA to understand the gene regulatory mechanisms impacting NETs and neutrophil function during acute COVID-19. 

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Pilot Project 4

Title: DRS to Identify Epitranscriptomic Changes in Response to Influenza and Smoking
Pilot Project Leader: Susan Schroeder, Ph.D, The Department of Chemistry & Biochemistry, Microbiology & Plant Biology, College of Arts and Sciences, University of Oklahoma

Project Summary: 

Changes in ribonucleic acid (RNA) provide the genetic instructions for a host cell to respond to a virus infection. Direct RNA Sequencing (DRS) nanopore technology can provide novel insights into the RNA of both the host cell and the virus. Direct RNA nanopore sequencing provides single-molecule, long-read sequencing data and direct identification of modified nucleotides.  The changes in modified nucleotides can alter splicing and the way that the RNA folds and interacts with proteins and thus finetune host gene regulation in response to viruses. In collaboration with Dr. Jordan Metcalf at OUHSC and the Oklahoma Center for Respiratory Infectious Disease (OCRID), we have collected data on RNA from human lung cells infected with influenza and identified significant changes in adenosine methylation in both mRNA and noncoding RNA.  Based on our previous collaborative research and the known immunosuppressive effects of smoking, we hypothesize that the effects of smoking further alter the host epitranscriptomic re-sponse to influenza infection. We predict that the changes in RNA will be different with exposure to both influenza and cigarette smoke extract than either stressor alone. Long-read single cell sequencing with nanopore technology will provide complementary information to correlate the splicing changes with the levels of viral RNA in an individual cell.   Overall, this project will provide a single-molecule, single-cell RNA profile of the effects of smoking on influenza immune response.  This funding will support our col-laborative efforts to submit NIH RO1 proposals. 

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Pilot Project 5

Title: Identification of Antimicrobial Peptides against S. aureus produced by D. pigrum in the Nose
Pilot Project Leader: Reed Stubbendieck, Ph.D, The Department of Microbiology & Molecular Genetics, College of Arts and Sciences, Oklahoma State University

Project Summary: 

Pilot Project 1 

Title: Determining the Cellular Mechanisms Whereby Skeletal Muscle (Fiber Type) Prevents Influenza-Induced Mortality in Sarcopenic Obesity
Pilot Project Leader: Joshua Butcher, Ph.D., The Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

Project Summary: 

Simultaneous or separate presentation of obesity and influenza is known to significantly increase morbidity and mortality in aging patients. Although the mechanisms are unclear, new evidence shows that skeletal muscle (fiber type) likely plays a key role in the progression of both diseases. Augmented muscle mass (a by-product of exercise) protects against obesity-derived cardiovascular dysfunction, as well as reducing the onset, symptoms, recovery time, and mortality of influenza. What remains unknown is whether influenza infection targets a specific fiber type to drive overall morbidity and myopathy? Further, could preseveration of a specific fiber type be targeted to protect against influenza severity in aged lean or obese patients? These questions will be interrogated in the following pilot study using mouse models of obesity (the db/db) in combination with myostatin deletion and PGC1α-overexpression. Importantly, this project will examine the dysfunction (both the diaphragm and gastrocnemius) in the skeletal muscle system that accompanies obesity, aging, and influenza, and the how the combination drives overall pathology. Taken together, the following project should yield clinically relevant targets for combating obesity and influenza-derived disease progression.

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Pilot Project 2

Title: Genetic Loci Associated with SARS-CoV-2 Rapid Adaptation
Pilot Project Leader: Xufang Deng, Ph.D, The Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

Project Summary: 

New SARS-CoV-2 variants are constantly emerging and sicken individuals who have insufficient immunity against the infection. These variants carry many genetic mutations and can escape the neutralization by antibodies elicited by vaccines or prior infection. Some variants outcompeted other variants and became globally prevalent strains, as exemplified by the Omicron variant. Identifying what genetic loci/mutations are associated with SARS-CoV-2 rapid adaptation and investigating its underlying mechanisms is crucial for discovering viral Achilles’ heel and developing novel antiviral strategies. To this end, this project is focused on the Omicron variant that exhibits exceptional transmissibility and characteristic changes in tissuetropism and interferon antagonism. Omicron carries over fifty genetic mutations that are located at the spike gene and the genes encoding immune modulators. Therefore, we hypothesize that the Omicron mutations cause the alteration of tissue tropism and IFN antagonism by 1) altering the spike-mediated viral entry and 2) reshaping the cellular antiviral immune environment. The goal of this project is to identify the key mutations that are responsible for the characteristic phenotype of Omicron and determine to what extent these mutations contribute to SARS-CoV-2 adaptation. To test this hypothesis and achieve these
goals, we take an integrated reverse genetics approach combining human respiratory cell models to pinpoint key genetic loci and faithfully evaluate their biological effects during authentic viral infection. This project will not only provide insights into SARS-CoV-2 tissue tropism but also expand the knowledge of SARS-CoV-2 evolution and pathogenesis.

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Pilot Project 3

Title: Modeling Transmission Aerobiology of SARS-CoV-2 Aerosols in Human and Mouse Lungs
Pilot Project Leader: Yu Feng, Ph.D, The Department of Chemical Engineering, The College of Engineering, Architecture, and Technology, Oklahoma State University

Project Summary: 

Transmission-blocking interventions are a critical modality in the control of SARS-CoV-2, which needs a thorough understanding of the infection risks associated with different exposure conditions. However, fundamental understanding of the connections remains deficient among the SARS-CoV-2 laden aerosol airborne transmission, pulmonary transport, and infection, associated with different emission activities such as cough, sneeze, and talk. Existing in vitro and animal studies were not appropriately designed, since the virus input (i.e., "seeding") to the animals or tissues are not precisely correlated to the real exposure conditions to airborne SARS-CoV-2 laden aerosols. This is due to the lack of experimental measurement capabilities and appropriate predictions of the physical, chemical and rheological properties of the SARS-CoV-2 laden aerosol droplets. To fill the gap, the proposed research will refine and calibrate the established multiscale CFPD-HCD model to predict the chain of events associated with SARS-CoV-2 laden aerosol droplets transmission in human and mouse, including (1) emission in exhalation clouds that propel SARS-CoV-2 laden droplets, (2) the rapid evolution of the droplets subject to different environmental factors (moisture, temperature, etc.), (3) inhalation and rehydration in pulmonary routes, and (4) the host cell dynamics after their deposition in the lung. Upon completion, the numerical research will provide insights into SARS-CoV-2 airborne transmission that cannot be provided using in vitro or in vivo measurements to reduce significantly the burden of gathering evidence to develop new and more accurate in vitro and animal models amenable to experimental studies of controlling SARS-CoV-2 aerosol transmission. 

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Pilot Project 4

Title: Interaction of human CTRP6 with SARS-CoV-2 NSP14 protein 
Pilot Project Leader: Xia Lei, Ph.D, The Department of Biochemistry and Molecular Biology, Division of Agricultural Sciences and Natural Resources, Oklahoma State University

Project Summary: 

SARS-CoV-2, the causative agent of Coronavirus Disease 2019 (COVID-19), has infected more than 423M people worldwide with more than 5.8M deaths, which represents the greatest threat to global public health and economies. It is critical to understand viral pathogenesis at the molecular level for developing effective antiviral therapy. Non-structural protein NSP14 is a highly conserved enzyme necessary for viral replication via shutting down host protein synthesis. NSP14 forms a stable complex with another non-structural protein Nsp10 and exhibits exoribonuclease (ExoN) and N7-methyltransferase activities (N7-MTase), which is required for its translation inhibition activity. Protein interaction screens using the yeast two-hybrid (Y2H) system has identified human CTRP6 is a binding partner of NSP14. Although CTRP6 plays critical roles in obesity, diabetes, autoimmune diseases and tumorigenesis, its exact cellular functions have not been clearly elucidated yet. We propose to characterize the nature of the interaction of CTRP6 and NSP14 in the following two aspects: Aim 1, Determine the interaction of human CTRP6 and NSP14. Co-immunoprecipitation, co­localization, and thermal stability assay will be used to confirm the binding of these two proteins and binding domain of CTRP6 will be identified. Aim 2, Determine the role of human CTRP6 during SARS-CoV-2 infection. The effect of CTRP6 on the activity of NSP14 including ExoN, N7-MTase, translation inhibition and inhibition of IFN-stimulated genes induction will be examined. This work will produce more detailed molecular-level insight into the mechanism of NSP14 protein during viral infection. Interaction of CTRP6 and NSP14 protein may open novel avenues for therapeutic interventions. 

Pilot Project 1 

Title: Drug Targeting of Oxysterol-Binding Protein (OSBP) for Respiratory Viral Infections
Pilot Project Leader: Anthony Burgett Ph.D., Department of Pharmaceutical Sciences, College of Pharmacy, The University of Oklahoma Health Science Center

Project Summary: 

Drug Targeting of Oxysterol-Binding Protein (OSBP) for Respiratory Viral Infections Summary: The current COVID-19 crisis starkly illustrates the need to develop new modalities for the therapeutic treatment of pathogenic RNA respiratory viruses, including against novel viruses that have yet to emerge. Recently, human oxysterol-binding protein (OSBP) was identified as a critical mediator in the replication of a broad spectrum of (+) ssRNA viruses, including the Enterovirus genus human pathogens (i.e., poliovirus, enteroviruses, rhinoviruses, coxsackieviruses), hepatitis, Zika, and Dengue Fever viruses. Additionally, our preliminary results also indicate that OSBP is required for the replication of coronaviruses. These discoveries present the opportunity for a paradigm shift in antiviral drug development: potentially drug target a human host protein, OSBP, required for viral proliferation, as opposed to targeting individual viral proteins.

We have a published collaborative project focused on the role of OSBP in viral proliferation and antiviral drug development through targeting OSBP. We published two papers in 2019 on the antiviral activity and cellular biology of OSBP-targeting compounds, including the natural product compound OSW-1. This proposed research project will develop and test novel antiviral compounds for drug development as novel prophylactic broad-spectrum antiviral therapeutics. The potential new OSBP-targeting compounds will provide chemical probes to study the role of OSBP in viral proliferation, and the new OSBP-targeting compounds will constitute a possible route to the pharmacological intervention of RNA respiratory viral infections, including against new viral pathogens yet to emerge.

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Pilot Project 2

Title: Molecular Components of the two heme uptake pathways of mycobacterium tuberculosis
Pilot Project Leader: Avishek Mitra Ph.D., Department of Microbiology and Molecular Genetics, College of Arts and Sciences, Oklahoma State University

Project Summary: 

Mycobacterium tuberculosis (Mtb) has a strict requirement for iron to colonize the human host. The necrotic centers of granulomas (infected macrophages) contain high concentration of heme(Hm)- and hemoglobin(Hb)-sequestering proteins, possibly to limit access of Mtb to heme iron. This is extremely relevent because: 1) Hm and Hb store more than 75% of the host iron and 2) macrophages recycle senescent erythrocytes for Hb production in new erythrocytes and 3) Mtb resides and replicates within macrophages. While heme is a major iron source for Mtb in the human host, we have no knowledge of the relevance of Hm acquisition in Mtb virulence because we lack basic understanding of Mtb heme uptake mechanisms. Our studies show that Mtb has at least two Hm acquisition pathways. The outer membrane PPE36 and PPE62 proteins and the inner membrane Dpp transporter constitute one pathway. We also discovered that Mtb has an entirely unknown pathway where Hm acquisition is mediated by albumin. Despite our discoveries, the following gaps remain in our knowledge: 1) how are heme utilizing PPE proteins exported to the outer membrane for Hm acquisition, 2) how Mtb captures heme from host hemoproteins (e.g. hemoglobin), and 3) what factors are required for albumin-Hm acquisition. By answering these gaps in knowledge, we will pave the way to understand the biological relevance of heme utilization in Mtb disease progression. This will facilitate development of chemotherapeutic approaches against Mtb by inhibiting the uptake of heme, which is the major iron source in the human host during infection.

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Pilot Project 3

Title: Pathogenesis of SARS-CoV-2 and Klebsiella pneumoniae coinfection
Pilot Project Leader: Sunil More Ph.D., Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

Project Summary: 

Sever acute respiratory syndrome cronavirus 2 (SARS-CoV-2) infections are associated with coinfections by other bacterial, fungal, and viral pthogens in COVID-19 patients. There are multiple pathologic changes in the respiratory system including damage to the upper and lower respiratory tract epithelium, reduced surfactant production, and pulmonary edema. These changes reduce the mucociliary clearance which in turn allows easy attachment and colonization of bateria and other microbes. The coinfections determine therapeutic measures and clincal outcomes in COVID-19 patients. Our project aims to understand how these coinfections paticularly by Klebsiella pneumoniae affects immune response, develop a mouse model and identify biomarkers that can be utilized for therapeutic intervention. 

Pilot Project 1 

Title: A Screening Platform for Pan-Coronavirus Assembly Modulators
Pilot Project Leader: Christina Bourne Ph.D., Department of Chemistry and Biochemistry, College of Arts and Sciences, The University of Oklahoma - Norman

Project Summary: 

The current SARS Coronavirus pandemic has brought a long-standing need to develop anti-viral therapeutics to the forefront of attention. Immediate efforts are focusing on immune modulation and vaccine development based on the spike, or S, glycoprotein. However, even with vaccination, some people will fail to be protected and require a viable therapeutic option. Other therapeutic strategies are focused on inhibition of viral enzymes, which imparts selective pressures that could drive mutations. To address issues with long term immunity and provide additional (non-enzymatic) therapeutic options, the current proposal draws from established successful ideas from
other anti-viral strategies to develop a new platform for a novel beta- coronavirus therapeutic target: viral assembly modulation.

This proposal seeks to take advantage of the PI’s expertise in biochemistry and previous experience in a similar anti-viral approach. The objective is to build screening platforms focused on identifying modulators of the membrane (M) and N protein self- and cross- interactions that are essential for CoV assembly. These proteins display conservation in interactions among beta-Coronaviruses, making this target likely applicable across outbreaks. This validated platform will provide a means to assess readily available commercial compound libraries for protein-protein interaction modulators. It is expected that identified compounds can be progressed through medicinal chemistry approaches to optimize cell-based activity.

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Pilot Project 2

Title: Augmented Muscle Mass as a Buffer Against Influenza
Pilot Project Leader: Joshua Butcher Ph.D., Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

Project Summary: 

Simultaneous presentation of obesity and influenza is known to significantly increase morbidity and mortality in patients. Although the mechanisms are unclear, elevated oxidant stress likely plays a key role in the progression of both diseases. Augmented muscle mass (a by-product of exercise) protects against obesity-derived cardiovascular dysfunction, as well reducing the onset, symptoms, recovery time, and mortality of influenza. What remains unknown is whether augmented muscle mass generated by inhibition of myostatin (an exercise mimetic) can protect against influenza severity in lean or obese mice. If so, will the mechanism of protection be via the ability of augmented muscle mass to reduce oxidant stress by a specific oxidant enzyme (NOX1)? These questions will be interrogated in the following pilot study using mouse models of obesity (the db/db) in combination with myostatin deletion and NOX1 deletion. Importantly, this project will examine both the vascular and skeletal muscle (the diaphragm) dysfunction in the respiratory system that accompanies obesity, influenza, and the how the combination drives overall pathology. Taken together, the following project should yield clinically relevant targets for combating obesity and influenza-derived disease progression.

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Pilot Project 3

Title: Xanthophylls in RIG-I-MAVS modulated antiviral innate immunity
Pilot Project Leader: Dingbo (Daniel) Lin Ph.D., Department of Nutritional Sciences, College of Education and Human Sciences, Oklahoma State University

Project Summary: 

Deficiency of the innate immunity has emerged as a key contributor to the increased susceptibility to respiratory infection diseases, such as influenza. Elderly and other vulnerable populations are at increased risks of respiratory infection and have significantly decreased levels of circulatory xanthophylls. However, there is a knowledge gap in understanding the significance of xanthophylls in host immune responses and influenza. Recent evidence supports the premise that RIG-I-MAVS and STING modulate innate immunity and play a pivotal role in protection from influenza in humans. Xanthophylls are oxygenized carotenoids solely metabolized by β-carotene oxygenase 2
(BCO2) in the inner membrane of mitochondria. Previous work from our group suggest that xanthophylls, such as zeaxanthin, stimulated MAVS oligomerization and potentiated STING protein expression and IFN-β1 transcription in Raw264.7 macrophage cells. Accumulation of xanthophylls distinctly elevated mitochondrial cardiolipin levels and increased the survival rates in BCO2 knockout mice exposed to influenza A virus (IAV). Here, we hypothesize that xanthophylls enhance the resistance to IAV through STING and RIG-I-MAVS signaling pathways. We will determine xanthophylls increase cardiolipin externalization to the outer membranes of mitochondria, which in turn enhances MAVS oligomerization and RIG-I-MAVS signaling. We will determine xanthophylls triggers STING signaling which will restrict flu virus replication. We will also examine that xanthophylls boost the innate immunity using macrophage cells collected after feeding of xanthophylls in the elderly. The results are expected to have a significant impact on public health by providing mechanistic investigation why some populations are more vulnerable to influenza and associated comorbidities.

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Pilot Project 4

Title: A comprehensive approach to analyze corona viral protein evolution towards novel drug discovery strategies
Pilot Project Leader: Rakhi Rajan Ph.D., Department of Chemistry and Biochemistry, College of Arts and Sciences, The University of Oklahoma - Norman

Project Summary: 

With its high virulence, the Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) has caused thousands of deaths and severely disrupted our economy and daily life. Our preliminary phylogenetic analysis has provided clues on its high virulence: it showed that some SARS-CoV-2 proteins (especially the Spike protein) have evolved substantially since SARS CoV (2003 outbreak strain) to gain more rapid entry into the host cell, whereas other proteins (such as the 3CL protease) have been largely conserved to maintain viral biogenesis. In this project, we will focus on the highly evolved spike protein, with the goal of opening up new treatment and prevention options against current and future SARS-CoV-2 strains. Our study will be extended to conserved proteins in a future R01 project. Our synergistic approach combining bioinformatics, biochemistry, and computational methods is unique in three ways. First, it fills a critical knowledge gap in SARS-CoV-2 research. A detailed evolutionary pathway of the spike protein (and thus a deeper understanding of its enhanced transmission/virulence) will emerge from our systematic assessment of the effect of each key mutation of the spike protein on its binding to receptors on human/animal host cells. Second, it will broaden our therapeutic options against SARS-CoV-2. Through identifying multiple binding pockets on the spike protein, we can develop lead compounds targeting each of these pockets via in silico and in vitro screening. Finally, by developing a recurrent neural network model, we can help predict future SARS-CoV-2 viral evolution and thus facilitate the development of proactive treatment options.

Pilot Project 1 

Title: Investigation & Potential Drug Targeting of Oxysterol-Binding Protein (OSBP) in Viral Respiratory Disease
Pilot Project Leader: Earl L. Blewett, Ph.D., Department of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences

Project Summary: 

Viral pathogenic respiratory infections annually sicken millions and kill thousands, with limited to no direct therapeutic interventions currently available. In 2015, oxysterol-binding protein (OSBP) was identified as a critical mediator in the replication of a broad spectrum of Enterovirus genus human pathogens. Recently, even more RNA viruses including hepatitis C, Zika and  engue Fever viruses have also been shown to require OSBP for viral proliferation. OSBP is postulated to have a role in membrane formation of the  viral replication organelles (RO) which form at the ER-Golgi interface. These  discoveries present the opportunity for a paradigm shift in anti-viral drug  development: potentially drug targeting a human host protein, OSBP, that is  required for viral proliferation, as opposed to targeting viral proteins. Our  published collaborative project has explored biological effects and antiviral properties of OSBP small molecule inhibitors, including a prophylactic antiviral activity of the OSW-1 compound. In this proposal, we will define the role of OSBP and the antiviral activity of the panel of OSBP-targeting compounds on a series of virus pathogens, including human rhinovirus (HRV), human coronavirus (HCoV), Enterovirus A71 (EV-A71), EV-D68, Dengue virus (DENV), cowpox (CPXV), and herpes simplexvirus-1 (HSV-1). Our hypothesis is that any virus that requires the formation of a replication organelle or  membrane-wrapped virus factory will be susceptible to OSBP small molecule  inhibition. We will test the hypothesis by determining the sensitivity of a panel of viruses to OSBP small molecule inhibitors. This project will provide a foundation for future federal funding applications and therapeutic development programs.

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Pilot Project 2

Title: Functional characterization of a lysophospholipase that influences P. aeruginosa biofilm formation
Pilot Project Leader: Matthew Cabeen, Ph.D., Department of Microbiology and Molecular Genetics, College of Arts and Sciences, Oklahoma State University

Project Summary: 

Many bacterial species form biofilms, in which cells are protected by a self-produced extracellular matrix. Infectious biofilms are resistant to host immune responses and to antibiotic therapies. The opportunistic human pathogen Pseudomonas aeruginosa is notorious for forming biofilms in burn wounds, diabetic ulcers, and in the lungs of cystic fibrosis patients. Effective biofilm prevention strategies require new molecular “control points” where biofilm signaling can potentially be interdicted. We recently discovered one such control point: a phosphotransfer system (PTS) in P. aeruginosa that suppresses biofilm formation when a key regulatory protein is unphosphorylated by reducing cellular levels of cyclic-di-GMP (cdG), a signal that promotes biofilm formation. PTS-mediated biofilm suppression requires PA14_04030, a protein with homology to lysophospholipase D proteins, which cleave phosphodiester bonds in phospholipids and in some cases in nucleotides. Here, we aim to learn how 04030 modulates biofilm signaling, testing the hypothesis that 04030 acts as a cdG phosphodiesterase. We will rigorously test the impact of 04030 on biofilm formation and cdG levels. We will assess the importance of its conserved phospholipase D-like active site with respect to biofilm control. We will test the in vitro degradation activity of 04030 towards cdG. We will also examine how the PTS influences 04030 production or activity. Our experiments have potential to establish lysophospholipases as an unappreciated class of bacterial signaling enzymes, and we will use our data from this pilot project to support an NIH R01 proposal in 2020 to further characterize the roles of phospholipases in bacterial biology and pathogenesis.

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Pilot Project 3

Title: Sex differences in human group 2 innate lymphocytesin response to influenza virus 
Pilot Project Leader: Susan E. Kovats, Ph.D., Arthritis & Clinical Immunology Research Program, Oklahoma Medical Research Foundation

Project Summary: 

     Humans show significant sex differences in the incidence and severity of respiratory diseases including asthma and virus infection, with women often experiencing stronger immune responses and morbidity. Sex disparities including sex hormones regulate the type 2 immune mechanisms that predispose to asthma. However, we lack information about sex differences in type 2 inflammation that is important for promoting immune response resolution upon respiratory virus infection. Lung resident type 2 innate lymphocytes (ILC2s) regulate type 2 responses in humans and mice. In contrast, some ILC2s exhibit functional plasticity and acquire the capacity to promote type 1 inflammation in influenza infection.
     We and others have identified sex differences in the numbers and phenotype of lung ILC2s in homeostasis. Our preliminary data show that the phenotype and function of lung resident ILC2s in females and males significantly differ in response to type 1 inflammation in murine influenza virus infection. ILC2s in females are preferentially functionally suppressed, and this is associated with their increased IFNGR expression and signaling involving STAT1. Herein, we will study human ILC2s, as this will increase the impact of our findings. We will test the hypothesis that human female ILC2s show greater IFNGR signaling, leading to increased suppression of ILC2 phenotypes in type 1 environments. Experiments will assess human blood ILC2 phenotypes and functional responses upon stimulation by type 1 cytokines and influenza virus. These studies will increase mechanistic understanding of sex differences in ILC2 plasticity during respiratory virus infection.

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Pilot Project 4

Title: Chemical cartography: a novel approach to study respiratory infections
Pilot Project Leader: Laura-Isobel McCall, Ph.D., Department of Chemistry and Biochemistry, College of Arts and Sciences, The University of Oklahoma 

Project Summary: 

Influenza virus (IFV) is a leading cause of respiratory illness and death worldwide, with over 5 million cases and 600,000 deaths annually in non- pandemic years. Although vaccination can prevent influenza, it may not be effective in immunocompromised populations, and is hampered by viral variability. Recently, there has been significant interest in developing host-directed therapies against influenza. This will however require improved understanding of IFV pathogenesis and tissue mechanisms of damage vs repair. The overall goal of this pilot project is to generate proof-of-concept that a new approach combining spatially-resolved tissue metabolite analysis by liquid chromatography-tandem mass spectrometry, with 3D modeling and big data analytics (“chemical cartography”), can generate such insights. In aim 1, we will use chemical cartography to demonstrate that standard serum and bronchoalveolar fluid analyses do not fully capture the complexity and spatiality of metabolomic changes occurring in the infected lung. In aim 2, we will differentiate between metabolic pathways that are modulated throughout the infected lung vs pathways only affected at the site of highest viral load, using chemical cartography. Overall, these results will establish methods to perform chemical cartography in the context of respiratory infections and demonstrate the strength of chemical cartography to study these diseases. Results will also identify candidate pathways for pharmacological modulation to treat IFV. This pilot project will serve as the foundation for future grant applications to investigate their potential for IFV treatment, and to use chemical cartography to identify local lung metabolic pathways differing between mild and severe IFV infection.

Pilot Project 1 

Title: Interactions of murine pulmonary macrophage subsets withCryptococcus neoformans
Pilot Project Leader: Karen Wozniak, Ph.D., Department of Microbiology and Molecular Genetics, College of Arts and Sciences, Oklahoma State University

 

Project Summary: 

Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that causes pneumonia and disseminates from the lung to the brain via intracellular transport in macrophages to cause meningitis. This infection affects approximately 225,000 individuals with AIDS each year, resulting in over 181,000 annual deaths. Understanding the interaction of C. neoformans with macrophage subsets in pulmonary tissues is critical for elimination of the pathogen and ultimately prevention of dissemination to the brain. The long term goal of our laboratory is to understand the mechanisms of cryptococcal pathogen control by cells of the innate immune system. Many studies have shown that cryptococcal virulence factors enable C. neoformans to survive and replicate within host macrophages, but macrophage-Cryptococcus interactions can result in either killing or intracellular cryptococcal growth, suggesting that differential interactions may be macrophage mediated. The pulmonary macrophage subsets involved and signaling pathway(s) governing this difference in non-activated macrophages are unknown. Preliminary data in our laboratory have shown that human PBMC-derived macrophage subsets have different interactions with C. neoformans. We hypothesize that subsets of pulmonary murine macrophages interact differently with C. neoformans, resulting in either intracellular growth or fungicidal activity by macrophages. The goals of this proposal are to identify subsets of macrophages with differential anti-cryptococcal activity and to examine gene expression and signal transduction pathways of each subset following interaction with C. neoformans in order to design immunotherapies to target macrophage intracellular growth of C. neoformans.

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Pilot Project 2 

Title: OHet72: a potential new drug in the armamentarium against TB and MDR-TB
Pilot Project Leader: Lucila Garcia-Contreras, Ph.D. Department of Pharmaceutical Sciences, College of Pharmacy, The University of Oklahoma Health Science Center

 

Project Summary: 

The global control of tuberculosis (TB) is threatened by an increased number of multi-drug resistant TB (MDR-TB) cases and the low rates of cure achieved with current treatments.  Thus, potentially useful new drugs and alternative drug delivery strategies are urgently needed. Recently, we discovered that SHetA2, a novel anticancer drug also has significant activity against Mycobacterium tuberculosis, (MTB). However, SHetA2 has an oral bioavailability of 1-10%. As an alternative administration strategy, we developed two inhalable dry powder formulations of SHetA2, a microparticle (MP) or a nanocrystal-microparticle (NCMPs) formulations, with optimized physicochemical properties for maximum delivery to the alveolar region of the lung, the main place of MTB residence. The NC-MPs powder had a faster dissolution rate in simulated lung fluid, but pharmacokinetic studies in mice revealed that the amount of powder to be inhaled for therapy would be larger than that of other inhalable products. Our early studies found that OHet72 was more potent against MTB than SHetA2 (MIC 10-fold smaller). We hypothesize that an inhalable powder of OHet72 would be a better candidate for TB therapy because it would require smaller doses administered 2-3 times per week. Thus, we propose to use the pilot project funds to (1) Assess the disposition of OHet72 after pulmonary administration and compare it to that of SHetA2; and (3) Probe the possible mechanism by which OHet72 kills MTB. The results of this project will be used in an R01 application to evaluate the efficacy of this drug in the TB-guinea pig model.

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Pilot Project 3

Title: Effect of airway mucosal cooling and hyperosmolarity on innate immune function
Pilot Project Leader: Michael Davis, Ph.D., Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

 

Project Summary: 

Respiratory viral infections exhibit seasonal fluctuations, with the greatest prevalence observed during winter months in the northern hemisphere.  Virus biology and human behavioral patterns are believed to contribute to the seasonal fluctuations, but whether variation in host defense plays a role has received minimal investigation.  Breathing cold air is known to produce alterations in airway biology, including mucosal injury, bronchoconstriction, and airway inflammation, and preliminary data suggests prolonged alteration in airway cytokine expression following cold air exposure.  In this project, we will use a novel animal model (horses) to evaluate the effects of breathing cold air on airway innate immune function, with exercise used to amplify the magnitude of the resulting airway cooling and desiccation.  Airway lavage obtained after exercise will be analyzed ex vivo for reduced production of key cytokines and resistance to replication of live equine influenza virus.  Human and equine airway epithelial cell cultures will be exposed to cooling and desiccation similar to that measured during in vivo challenges of humans to assess the direct effects of cold air on mucosal function and the similarities of these effects between humans and horses.  The equine cell cultures will also be challenged with live equine influenza while co-cultured with post-exercise lavage cells and supernatant to estimate the resistance of the airways to viral infection.  These studies will establish the foundation for in vivo live virus exposure studies in horses following cold air exposure to directly assess the effect of ambient air temperature on airway innate immune function.

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Pilot Project 4

Title: Viral RNA structures, function, and energetics
Pilot Project Leader: Susan J. Schroeder, Ph.D., Department of Chemistry and Biochemistry, College of Arts and Sciences, The University of Oklahoma - Norman

 

Project Summary: 

Human Endogenous Retroviral (HERV) RNA are part of immune response signaling pathways, but the structural and functional roles of HERV RNA are not fully known.  HERV RNA is expressed abundantly in patients with cancer and virus infections. HERV-K is expressed in epithelial cells in lungs and over-expressed in several forms of lung cancer.  HERV-W is highly overexpressed in response to influenza A virus infection.  New StructureSeq methods can probe the dynamic changes in host and viral RNA.   The main hypothesis is that HERV RNA is an RNA shapeshifter that changes shape when interacting with different protein partners.  We will generate in vivo, genome-wide chemical probing data for HERV RNA in cells to generate structural models of HERV RNA and test RNA structure prediction methods.   HERV RNA structural motifs have the potential to be an effective target for RNA aptamer or small molecule inhibitors.  We will study the structures of HERV RNA loops and compare the structures to known target sites for antibiotics. The transcriptome-wide probing data will reveal the changes in RNA expression and folding during influenza infections and apoptosis and thus provide new insights into the RNA biology of cell stress responses. This project will be pursued in collaboration with the Molecular Biology Core at Oklahoma State University.  In the long-term, this project will use the newly funded 800 MHz NMR spectrometer at the statewide NMR facility, thus capitalizing on the molecular and structural biology resources in the state of Oklahoma.

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Pilot Project 5 

Title: A precise scale-up method from mice to men on the infection of influenza a virus 
Pilot Project Leader: Yu Feng, Ph.D., Department of Chemical Engineering, College of Engineering, Architecture, and Technology, Oklahoma State University

 

Project Summary: 

Effective prediction of the transport, deposition, and within-host dynamics of influenza A virus (IAV) requires extrapolation of data obtained from animal studies to humans. As the respiratory tracts of rodents and humans are anatomically very different and will significantly influence the deposition patterns of inhaled aerosols, there is a critical need to study airflow and virus-laden droplet deposition patterns, as well as the immune responses in airways of these animals and compare them to human. However, there are no translational success from animal studies to human because of the failure of the development and employment of a precise scale-up method, considering inter-species anatomical difference of respiratory tracts. Thus, it is necessary to develop a precise scale-up strategy to realize precise inter-species extrapolations. The primary goal of this research is to validate and use a Computational Fluid-Particle Dynamics (CFPD) plus Hose Cell Dynamics (HCD) model to perform an inters-pecies comparison of inhaled micron IAV-laden droplets in mouse and human airways under realistic breathing and exposure conditions. Virus deposition patterns will be predicted, as well as the after-deposition dynamics in the respiratory tracts. The CFPD-HCD model will be validated and optimized with infected mice studies in animal and immunopathology cores at OCRID. We will analyze the inter-species and inter-subject geometric, kinematic and dynamic differences, and generate novel mice-to-men correlations. Upon completion, the precise scale-up method will significantly increase the extrapolation accuracy from animal studies and greatly reduce the burden of gathering evidence from clinical studies with human.

Pilot Project 1 

Title: The role of glucose homeostasis during respiratory infections.
Pilot Project Leader: Veronique A. Lacombe, DVM, Ph. D., Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

 

Project Summary: 

While diabetes, defined by a persistent hyperglycemic state, has reached epidemic levels, respiratory infections (e.g., influenza) have long held a high spot in the list of worldwide causes of death. Importantly, the incidence of hyperglycemia is a major and independent risk factor for the development and worsening severity of pulmonary infection. Although the lung is a major organ to utilize glucose, the role and the regulation of glucose homeostasis in the lung have received little attention. Glucose uptake from the bloodstream, the rate-limiting in glucose utilization, is tightly regulated by a family of specialized proteins, called the glucose transporters (GLUTs). Because every cell expresses these GLUTs, they are recognized as major regulators of whole-body glucose metabolism and thus are key pharmacological targets. However, little is known about the regulation of glucose transport in the respiratory system, particularly during a hyperglycemic state. Therefore, we hypothesize that GLUT activity in the diabetic lung modulates airway surface liquid glucose concentration and viral proliferation. The specific aims of this project are to test the hypotheses that: 1) diabetes will alter GLUT activity in the lung through an Akt/AS160 dependent pathway; 2) rescuing GLUT activity will improve airway surface liquid glucose concentration and thus decrease viral proliferation in the lung and the severity of influenza infection of diabetic animals. We will use a comprehensive, integrated approach at multiple system levels using state-of-the-art techniques. Insights gained from this study could lead to the identification of novel metabolic therapeutic targets for patients affected by diabetes and concurrent respiratory infections, a crucial outcome of this award.

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Pilot Project 2 

Title: Evasion of Host RNA Decay Machinery by the NS1 Protein of 2009 Pandemic Flu
Pilot Project Leader: Shitao Li, Ph.D., Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

 

Project Summary: 

Influenza A virus (IAV) is a highly transmissible respiratory pathogen and presents a continued threat to global health, with considerable economic and social impact. IAV comprises a plethora of strains with different virulence determinants that contribute to influenza pathogenesis. Several determinants in non-structural protein 1 (NS1) of high pathogenic IAV strains have been found to subvert host defense and increase virulence. However, the NS1 of 2009 pandemic IAV lacks all these virulence determinants. To discover the new virulence determinant, we recently systematically analyzed NS1 protein complexes and found that the host factor, zinc finger C3H1-type containing protein (ZFC3H1) specifically interacted with NS1 of 2009 pandemic IAV. Our pilot experiments revealed that ZFC3H1 facilitates RNA exosome to degrade viral RNA, thereby limiting influenza replication. Based on our preliminary data, we hypothesize that the pandemic flu NS1 possesses a new virulence determinant that inhibits ZFC3H1-mediated RNA degradation and improves viral RNA stability. Aim 1 will identify the new NS1 virulence determinant of pandemic flu and determine its role in IAV pathogenesis. Aim 2 will define how ZFC3H1 restricts IAV replication via destabilizing viral RNA. Aim 3 will determine the mechanisms by which the new NS1 determinant antagonizes ZFC3H1. Overall, this proposal will uncover a new NS1 virulence determinant of 2009 pandemic flu and reveal how the determinant perturbs host RNA decay machinery by engagement with ZFC3H1. The outcomes of our study will not only help develop effective therapeutics, but is also crucial for prediction of future potential epidemics and pandemics.

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Pilot Project 3

Title: Phage-like Chromosomal Islands and Virulence in Pneumococci
Pilot Project Leader: William Michael McShan, Ph.D., Department of Pharmaceutical Sciences, College of Pharmacy, The University of Oklahoma Health Science Center

 

Project Summary: 

Streptococcus pneumoniae is a major cause of human respiratory disease worldwide. 30% of severe cases are caused by strains that are fully resistant to one or more clinically relevant antibiotics. Previously, the PI has shown that phage-like chromosomal islands (CI) in group A streptococci cause the cell to 1) adopt a mutator phenotype through disruption of DNA mismatch repair, which promotes antibiotic resistance, and 2) alter global transcription, up-regulating virulence genes that can enhance pathogenecity, including biofilm formation and antibiotic resistance. We have constructed strains of S. pneumoniae TIGR4 that differ by the presence or absence of a novel Cl in S. pneumoniae (SpnCI) has been identified that has the potential to inactivate a required gene for nucleotide excision repair. Preliminary studies show that such element alter global transcription patterns, including genes that contribute to survival and/or biofilm formation. The hypothesis of this application is that SpnCI also confers a mutator phenotype and alters global transcriptional patterns that may increase virulence or promote survival. To test this hypothesis, the following specific aims will be performed: 1) analyze impact SpnCI has in the acute immune response in an infection model, and 2) determine the SpnCI-associated impact upon biofilm formation and virulence. The PI is highly qualified to perform these studies, having pioneered the discovery and characterization of these streptococcal Cl. The proposed studies will have a positive impact, fundamental advancing our understanding of the biology of this pathogen and may lead to new antimicrobial strategies and improved patient care.

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Pilot Project 4

Title: Identiying the function of novel Ca-binding protein mediating Ca regulation of P. aeruginosa virulence 
Pilot Project Leader: Marianna Patrauchan, Ph.D.,  Department of Microbiology and Molecular Genetics, College of Arts and Sciences, Oklahoma State University

 

Project Summary: 

Calcium ion (Ca2+) is perhaps the most versatile intracellular messenger in eukaryotic cells, regulating directly or indirectly all the vital cellular processes. However the role of Ca2+ in bacteria still remains elusive. Ca2+ plays role in regulating host hyperinflamatory responses to bacterial infection and accumulates in pulmonary fluids of cystic fibrosis patients and in mitral annulus of endocarditis patients with progressive atherosclerosis and calcified stenosis. This increase in host Ca2+ may serve as a trigger that turns on virulence of invading pathogens. In support, our preliminary studies showed that elevated Ca2+ positively regulates biofilm formation, and several virulence factors in P. aeruginosa, an opportunistic human pathogen and a leading cause of severe chronic infections in CF patients. However the molecular mechanisms of such regulation are not known. Our previous OCRID pilot funding enabled us to study one of the proteins regulated by Ca2+ and mediating Ca2+-regulated virulence. The protein was designated CarP. Based on the obtained results, the PI organized a team of researchers and applied for NIH RO1 funding. The application was reviewed positively and we were encouraged to resubmit a strengthen application. To strengthen the premises for the application, here we propose to 1) characterize CarP predicted phytase activity and its relationship with Ca2+; 2) identify the pathways regulated by CarP in response to Ca2+; and 3) determine the role of CarP in homeostasis of periplasmic Ca2+. This will provide the insight into the mechanism of CarP function and deepen the foundation for further in-depth studies.

Pilot Project 1

Title:Plakophilin 2 Controls Polymerase Assembly of Influenza A Virus
Pilot Project Leader: Shitao Li, Ph.D, Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

 

Project Summary: 

Influenza A virus (IAV) is a highly transmissible respiratory pathogen and a major cause of morbidity and mortality around the world. The emerging new viral strains are often refractory to conventional treatments. Thus, we determine to identify therapeutic targets that are at the influenza PB1-host interface because host factors controlling viral polymerase activity will be potential candidates. By proteomics approach, we identified plakophilin 2 (PKP2) as a common interactor with PB1 proteins of influenza strains. PKP2 is known as a scaffold protein at cell junctions. PKP2 also exhibits nuclear localization that is regulated by phosphorylation. Nonetheless, the role of PKP2 in viral infection is unknown. Pilot experiments found that PKP2 disrupted IAV polymerase assembly in nucleus and subsequently restricted viral replication. We hypothesize that PKP2 is a host inhibitor of IAV polymerase complex and propose the following two aims. Aim1 will establish the role of PKP2 in IAV polymerase assembly and viral infection. We hypothesize that PKP2 competes with PB2 for binding to PB1 and disintegrates the polymerase complex. We will define the domain in PKP2 essential for inhibiting PB1-PB2 interaction and the requirements for viral restriction. Aim 2 will determine the mechanisms regulating PKP2 restriction of IAV polymerase assembly. We hypothesize that phosphorylation results in PKP2 cytosolic retention, thereby preventing PKP2 from entering the nucleus to inhibit viral replication. The kinase and phosphatase of PKP2 will be identified. Taken together, the proposed study defines a natural inhibitor of IAV polymerase and provides mechanistic insights for developing potential antivirals.

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Pilot Project 2

Title: Computational Modeling of Tuberculosis Granuloma Activation
Pilot Project Leader: Ashlee N. Ford Versypt, Ph.D., School of Chemical Engineering, College of Engineering, Architechture, and Technology, Oklahoma State University

 

Project Summary: 

Tuberculosis (TB) is one of the most common infectious diseases and deadliest diseases worldwide. It isestimated that one-third of the world's population is infected with TB, and 1.5 million TB-related deaths were reported in 2014. TB is spread by aerosol droplets containing Mycobacterium tuberculosis (Mtb). The Mtb bacteria enter through the respiratory system and are attacked by the immune system in the lungs, primarily by aveolar macrophages. The bacteria are clustered and contained by the macrophages into cellular aggregates called granulomas. These granulomas can hold the bacteria dormant for long periods of time, even decades, in a condition called latent TB. However, the bacteria persist and can be activated when the granulomas are compromised by other immune response events in a host, such as cancer, HIV, or aging. The activation and subsequent spread of bacteria leads to active TB disease. It is difficult to study the activation process in humans because those with latent TB are asymptomatic and are often undiagnosed. Current animal models all have limitations. Computational and mathematical models can be useful tools for inexpensively conducting short- and long-term in silico experiments with multiple, interacting factors and can aid in generating and testing hypotheses. Several previous computational and mathematical models have been developed to describe the infection or granuloma formation stages of TB. No computational approach has been proposed considering the dynamics of matrix metalloproteinase 1 (MMP-1) regulation and the impact on TB activation. MMP-1 dysregulation has been recently implicated in TB activation through experimental studies, but the mechanism is not well understood. Animal and human studies currently cannot probe the dynamics of activation, so a computational approach is proposed to fill this gap. The overall objective of the study is to predict TB cavity formation (a hallmark of activation) in response to the dynamics of MMP-1 dysregulation. Mathematical and computational tools will be developed and used to test the hypothesis that the dynamics of MMP-1 regulation play a key role in the transition from latent TB to active TB.

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Pilot Project 3

Title: Regulation of glucose transportation in the healthy and diabetic lung: novel targets
Pilot Project Leader: Veronique A. Lacombe, DVM, Ph.D., Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

 

Project Summary: 

While diabetes, defined by a persistent hyperglycemic state, has reached epidemic levels, respiratory infections have long held a high spot in the list of worldwide causes of death. Importantly, the incidence of hyperglycemia is a major and independent risk factor for the development and worsening severity of pulmonary infection. Although the lung is a major organ to utilize glucose, the role and the regulation of glucose uptake in the healthy and diabetic lung have received little attention. Glucose uptake from the bloodstream is tightly regulated by a family of specialized proteins, called the glucose transporters (GLUTs). Because every cell expresses these GLUTs, they are recognized as major regulators of whole body metabolism and thus key pharmacological targets. However, little is known about GLUTs of the respiratory system, particularly during a hyperglycemic state. Therefore, we hypothesize that GLUT activity in the diabetic lung modulates airway surface liquid glucose concentration and bacterial proliferation. The specific aims of this project are to test the hypothesis that: 1) diabetes alters the gene and protein expression of GLUTs (1, 2, 8, 10) in the lung; 2) metformin treatment rescues GLUT activity and airway surface liquid glucose concentration and thus prevents bacterial proliferation in the lung of type 2 diabetic animals. We will use a comprehensive, integrated approach at multiple system levels using state-of-the-art techniques. Insights gained from this study could lead to the identification of novel metabolic therapeutic targets for patients affected by diabetes and concurrent respiratory infections, a crucial outcome of this award.

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Pilot Project 4

Title: The role of cytomegalovirus in immunosenescene and influenza susceptibility
Pilot Project Leader: Dianne McFarlane, DVM, Ph.D., Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

 

Project Summary: 

This project brings together the expertise of laboratories at Oklahoma State University and the resources of the Oklahoma’s Health Sciences Center to study the impact of chronic, latent viral infection on immunosenescence and influenza susceptibility in the elderly. Cytomegalovirus (CMV) is a common respiratory herpesvirus that establishes lifelong infections in humans and other primates. CMV infection has been implicated in age- related immune dysfunction, known as immunosenescence, due to repeatedly exposing the immune system to viral antigens. Immunosenescence leads to an impairment in the elderly's ability to respond to immune challenges especially novel challenges. As a consequence, the elderly are at higher risk of developing infectious diseases. Notably, the elderly show increased susceptibility to influenza during seasonal flu outbreaks and therefore influenza vaccine is recommended for all persons aged 65 years and older. However, the elderly also show impaired vaccination response, making it difficult to provide effective preventative measures. The mechanisms by which age and CMV impair immune function, specifically immunity to influenza, are not well understood. Therefore, we will test the impact of CMV on the age-related decline in immune function. This project will lead to a better understanding of the role that chronic pathogens, such as CMV, play on immunosenescence and influenza susceptibility by helping to clarify the cause and effect relationship. It will also help determine suitable interventions that may improve immune health and function in the elderly.

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Pilot Project 5

Title: Phage-like Chromosomal Islands and Virulence in Pneumococci
Pilot Project Leader: William M. McShan, Ph.D., Department of Pharmaceutical Sciences, College of Pharmacy, The University of Oklahoma Health Science Center

 

Project Summary: 

StreptocoCCUS pneumoniae is a major cause of human respiratory disease worldwide. 30% of severe cases are caused by strains that are fully resistant to one or more clinically relevant antibiotics. Previously, the PI has shown that phage-like chromosomal islands (CI) in group A streptococci cause the cell to 1) adopt a mutator phenotype through disruption of DNA mismatch repair, which promotes antibiotic resistance, and 2) alter global transcription, up-regulating virulence genes that can enhance pathogenecity. An uncharacterized, novel CI in S. pneumoniae has been identified that has the potential to inactivate a required gene for nucleotide excision repair. The overall hypothesis of this application is that this pneumococcal CI, named SpnCI, also confers a mutator phenotype and alters global transcriptional patterns that may increase virulence or promote survival. To test this hypothesis, the following specific aims will be performed: 1) create an isogenic pair of S. pneumoniae strains that differ only by the presence or absence of phage-like chromosomal island SpnCI, and 2) perform global transcriptional analysis of these isogenic strains to observe SpnCI associated changes that could promote virulence or survival. This study will provide the first evidence of the biological impact a phage-like CI may have in S. pneumoniae. The PI is highly qualified to perform these studies, having pioneered the discovery and characterization of these streptococcal CI. The proposed studies will have a positive impact, fundamental advancing our understanding of the biology of this pathogen and may lead to new antimicrobial strategies and patient care.

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Pilot Project 6

Title: Ribosome Analysis of Pseudomonas Biofilms from Cystic Fibrosis Patient Strains
Pilot Project Leader: Kevin S. Wilson, Ph.D., Department of Biochemistry & Molecular Biology, Ferguson College of Agriculture, Oklahoma State University

 

Project Summary: 

Cystic fibrosis patients succumb to chronic airway infections, frequently caused by biofilms, predominantly composed of Pseudomonas aeruginosa. Such biofilms are almost impossible to eradicate once they are established in the lungs. Patients conventionally receive long-term therapies of antibiotics. One of the most common treatments is tobramycin, an antibiotic that inhibits ribosomes and translation in planktonic bacteria, effectively killing these solitary cells. Biofilms cells, encased within protective extracellular matrices, can survive tobramycin treatments not because the antibiotic fails to penetrate the matrices but because of their physiological dormancy. We hypothesize, by analogy to our recently published on dormant E. coli persister cells, that biofilm cells survive tobramycin by remodeling their ribosomes and globally suppressing translation. In this CoBRE application, we propose two Aims designed to test the predictions of our hypothesis. In Aim 1, we will assess translational activities by comparing the proteomes of biofilms and planktonic cells of P. aeruginosa. In Aim 2, we will profile ribosomes from biofilms treated with tobramycin. Our preliminary data support the feasibility of our proposed experiments and provide strong evidence of exciting data to come. In addition to globally suppressing protein synthesis in biofilms, we anticipate finding specific proteins synthesized at higher levels than in planktonic cells. In ribosomes from biofilms, we anticipate finding regulatory proteins that suppress ribosome activities. These aims will set the stage for future studies of translational regulation in P. aeruginosa biofilms. These studies will provide important insights into how biofilms can survive tobramycin and other antibiotics.

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Pilot Project 7

Title: Multi-scale Dosimetry Modeling of Influenza Virus-Laden Droplets through the Pulmonary Route
Pilot Project Leader: Yu Feng, Ph.D., Department of Chemical Engineering, College of Engineering, Architecture, and Technology, Oklahoma State University

 

Project Summary: 

The goal we were focusing on is to enable the first known effect to quantitatively simulate virus-laden droplets/aerosols transport and deposition in the entire human respiratory tracts, and their following translocation into systemic regions, based on natural laws of physics. The three research activities performed in this period paved the way to achieve the goal. Our preliminary CFPD simulations indicated that under the indoor exposure, the virus-laden droplets would mostly deposit in the nasal cavity and upper airways. The local deposition data did not show high deposition in lower lung airways. The mentioned simulation results provide initial evidence that the transport of virus into the deeper lung airways may be due to the resuspension of the virus after they deposit in the upper airway and reproduce themselves. Therefore, it is important to consider the reproduction and release dynamics in the CFPD model.

Pilot Project 1

Title: Exploration of ClpP Activation to Treat Respiratory Infections in Cystic Fibrosis
Pilot Project Leader: Adam Duerfeldt, Ph.D., Department of Chemistry and Biochemistry, College of Arts and Sciences, The University of Oklahoma

 

Project Summary: 

Caseinolytic protease P (ClpP) has been identified as a promising new antibacterial target.  Activation of ClpP with natural product acyldepsipeptides (ADEPs) leads to uncontrolled proteolysis and bacterial cell death. Since the ADEP mechanism of action operates via activation of a target rather than inhibition, ADEP efficacy is not contingent upon an actively growing phenotype. As such, it is no surprise that ADEP-mediated ClpP activation provokes bactericidal activity in actively growing populations, dormant persister cells, and tolerant biofilms. The ability to eradicate bacterial communities exhibiting growth state heterogeneity is an initiative with grand implications, especially as a treatment strategy for chronic infections in cystic fibrosis (CF) patients (e.g. Pseudomonas aeruginosa). ADEPs are the only natural product ClpP activators that have been identified to date and unfortunately, these lipopeptides suffer from poor bioavailability, limited solubility, effluxation and metabolic liabilities−all of which impede their clinical utility.
    The proposed studies represent a multidimensional approach to expand the ClpP activator arsenal and therefore the ability to fully investigate ClpP activation as a treatment strategy for opportunistic respiratory infections. The specific aims presented in this proposal are 1) to identify novel natural product ClpP modulators; and 2) to rationally design, synthesize, and evaluate small molecule ClpP activators. Completion of the proposed studies will afford a foundation for the continued development of broad spectrum antimicrobials.  Although the goal is to alleviate the threat of infectious pathogens that afflict CF patients, the implications of this research stretch far beyond this specific pathogen subset.

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Pilot Project 2

Title: Influenza-Host Protein Interactions Control Viral Infection and Pathogenesis
Pilot Project Leader: Shitao Li, Ph.D., Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

 

Project Summary: 

Influenza A virus (IAV) is a human respiratory pathogen that causes seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. Despite extensive studies of IAV, worldwide resistance to anti-influenza drugs has rapidly increased among circulating flu viruses. Thus, it is pressing to find new therapeutic targets and develop antiviral drugs based on these new targets. Viral infection induces dynamic alterations between virus and host proteins which ultimately modulates host immunity and leads to viral pathogenesis. Our studies investigate these IAV-host protein interactions and elucidate the novel underlying molecular mechanisms. In Aim1 we will systematically and comprehensively map the physical interaction network between host and influenza virus. We examine viral protein complexes from 6 IAV strains in 2 human cell lines and determine the changes in the proteome after influenza infection. Importantly, we biochemically validate the interactions between influenza and novel host factors. Aim 2 examines the mechanisms underlying host factors which regulate IAV infection and pathogenesis. We will define the role of host factors in four checkpoints of the influenza virus life cycle. To provide mechanistic insights of how the interactions affect IAV, we will also examine a novel flu interactor TRIM32 in detail.Preliminary data show that TRIM32 restricts IAV infection. The in vivo role of TRIM32 in host defense will be investigated using TRIM32 deficient mice. This study will provide a basis for defining new host factors involved in IAV infection, thereby providing potential drug targets for therapeutic interventions.

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Pilot Project 3

Title: β,β-carotene 9’,10’-oxygenase 2 (BCO2) in influenza virus pneumonia
Pilot Project Leader: Daniel Dingbo Lin, Ph.D., Department of Nutritional Sciences, College of Education and Human Sciences, Oklahoma State University

 

Project Summary: 

Host immune cell mitochondrial dysfunction in influenza pandemics is not well understood. Little is known about precisely how the mitochondrial function is linked to the host immune response in mammals. These retard the development of therapeutic strategies, especially during pandemics of a new influenza virus. β,β-carotene 9’,10’-oxygenase 2 (BCO2) is a mitochondrial inner membrane protein mediating inflammation and mitophagy. We reported that intact BCO2 is essential to mitochondrial integrity and stress response in chronic inflammation in obesity and diabetes. Impaired IL-18 signaling and mitochondria lstress occur in BCO2 knockout (BCO2-/-) mice. Thus, the long-term goal of this research is to dissect the role of BCO2 in mitochondrial integrity in influenza infection so that evidence-based approaches can be developed for treating influenza virus infection. For this project, the central hypothesis is that BCO2-mediating mitochondrial integrity responds to virus infection through activation of mitophagy, JNK and inflammasome. The goal of this project is to define the novel role of BCO2 in inflammasome activation and influenza pneumonia. Accordingly, the specific aims are: 1) to identify how BCO2 impacts mitochondrial integrity in mice infected with sublethal influenza virus, and 2) to determine the expression profiles of genes associated with BCO2 in host immune cells. We expect that BCO2-/- mice develop more severe phenotypes of influenza virus pneumonia due to disruption of mitochondrial integrity and subsequent inactivation of JNK and inflammasome. The outcome of the research will have a high therapeutic potential and advance our knowledge of the mitochondrial integrity in the innate immune response.

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Pilot Project 4

Title: Does PA0327 bind calcium and regulate Pseudomonas aeruginosa virulence?
Pilot Project Leader: Marianna Patrauchan, Ph.D., Department of Microbiology and Molecular Genetics, College of Arts and Sciences, Oklahoma State University

 

Project Summary: 

Since calcium (Ca2+) regulates many essential processes in eukaryotes, many human diseases are associated with abnormalities in cellular Ca2+ homeostasis. These include cystic fibrosis (CF) and pulmonary diseases that are commonly associated with bacterial infections. Therefore, it is possible that Ca2+ disbalance serves as an environmental trigger of virulence in opportunistic microbial pathogens. In support, the PI’s group has shown that Ca2+ enhances production of several virulence factors, infectivity, and antibiotic resistance in Pseudomonas aeruginosa, an opportunistic human pathogen and a leading cause of deadly chronic infections in CF patients. However, the molecular mechanisms of this regulation have not been clearly defined. Therefore there is a critical need for identification and characterization of proteins involved in Ca2+ regulatory and signaling pathways. We hypothesize that the earlier identified putative Ca2+-binding protein PA0327 plays role in Ca2+-induced virulence of P. aeruginosa. The goals of the proposed research are to confirm that PA0327 binds Ca2+ and characterize its role in Ca2+-induced virulence, antibiotic resistance, as well as it responsiveness to oxidative stress as a host factor. Understanding the mechanisms by which the virulence of P. aeruginosa is mediated by Ca2+ will not only enable experimental confirmation of the signaling role of Ca2+ in prokaryotes, it will also advance research on infectious diseases associated with Ca2+ imbalance, which will likely lead to the development of novel therapeutic approaches designed to prevent or treat respiratory infectious diseases. The proposed research will involve six trained students (two graduate and four undergraduate) ensuring it successful and timely completion. It will generate the data necessary to support the PI’s NIH R01 application in 2016 on the role of Ca2+ signalling in P. aeruginosa virulence.

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Pilot Project 5

Title: The role of angiogenic factors in the development if artheros clerosis during Chlamydia pneumoninae infection
Pilot Project Leader: Jennifer H. Shaw, Ph.D., Department of Integrative Biology, Oklahoma State University

 

Project Summary: 

Chlamydia pneumoniae is a respiratory pathogen that accelerates atherosclerotic cardiovascular disease (CVD). C. pneumoniae is an obligate intracellular bacterium that infects the respiratory tract and disseminates via circulating peripheral blood mononuclear cells to the vessel wall; persistence in the vasculature chemoattracts monocytes and induces endothelial oxidative stress and vascular smooth muscle cell (SMC) proliferation, which is integral to plaque formation. However, the mechanisms by which C. pneumoniae may regulate this atherogenic process are not fully understood. Another key factor in atherogenesis is the formation of microvessels (angiogenesis) within plaques, but little is known about the host angiogenic profile during C. pneumoniae infection. Placenta Growth Factor (PLGF), a cytokine associated with plaque progression, is a potential player in C. pneumoniae-driven atherogenesis due to its ability to chemoattract monocytes, induce SMC proliferation, and drive angiogenesis. Our long term objective is to identify angiogenic factors that mediate progression of atherosclerosis during C. pneumoniae infection and the mechanisms by which C. pneumoniae induces their expression. Our central hypothesis is that host expression of angiogenic factors, including PLGF, in response to C. pneumoniae infection will amplify atherosclerotic plaque development. We will test our hypothesis by measuring: i) the host angiogenic gene expression profile in C. pneumoniae-infected endothelial cells, ii) functional responses to C. pneumoniae-infected endothelial cells upon silencing expression of candidate angiogenic factors and, iii) the influence of candidate angiogenic cytokines on the progression of atherosclerotic plaques in C. pneumoniae-infected mice.

Pilot Project 1

Title: Develop Single Domain Antibodies for Blocking Interleukin 17 Receptor Signaling
Pilot Project Leader: Junpeng Deng, Ph.D., Department of Biochemistry & Molecular Biology, Agricultural Sciences and Natural Resources, Oklahoma State University

 

Project Summary: 

Respiratory syncytial virus (RSV) is the most important cause of serious lower respiratory tract infection in children. RSV is also dangerous for elderly patients, patients with chronic lung disease and asthma, and deadly for immunocompromised individuals. Interleukin 17 (IL17) plays a key role in mediating RSV associated airway hyperresponsiveness (AHR) and greatly exacerbates RSV induced proinflammatory response. It was shown blocking IL17 signaling resulted in significant inhibition of mucus production during RSV infection, and treatment of RSV-infected animals with anti-IL17 significantly reduced inflammation and decreased viral load.  The inhibition of IL17 pathway thus presents a novel therapeutic approach. Therefore better understanding the molecular mechanism of IL17 signaling is in critical need and also ties very well with the central theme of OCRID. A unique intracellular signaling domain termed ‘SEFIR’ was identified within all IL17 receptors (IL17Rs) and the key adaptor protein, nuclear factor κB (NF-κB) activator 1 (Act1).  A key step in IL17 signaling is the recruitment of Act1 to IL17Rs via SEFIR mediated protein-protein associations. Our recent data provided insights into the structure and mechanism by which SEFIR functions. In this OCRID pilot proposal, we aim for generating single domain antibodies specific for IL17 SEFIR domains that can be further developed into intracellular IL17R inhibitors in the future. The outcome from this pilot project will be significant and the obtained antibodies could be further developed in the future for blocking IL17 signaling for treatment of a number of inflammatory and infectious diseases including RSV infection.

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Pilot Project 2

Title: Pseudomonas Aeruginosa Intra- Species Interactions
Pilot Project Leader: Erika Lutter, Ph.D., & Noha Youssef, Ph.D., Department for Microbiology and Molecular Genetics, College of Arts and Sciences, Oklahoma State University , 

 

Project Summary: 

Cystic fibrosis (CF) patients are prone to chronic lung infections with intermitted exacerbations and progressive pulmonary decline. CF lung infections consist of a polymicrobial microbiome which is dominated by Pseudomonas aeruginosa. Multiple strains of P. aeruginosa exist within the complex microbiome and are in a constant state of dynamic evolution leading to a high degree of phenotypic variability and specialization. However, while the importance and complexity of the diverse polymicrobial environment within the CF lung is currently being explored, very little is known about how bacteria, such as P. aeruginosa, interact during the course of infection and how this can mediate infection outcomes. Currently, most experiments focusing on understanding the pathogenic mechanisms of P. aeruginosa are done in pure cultures. In contrast, the work outlined herein aims to characterize intra-species interactions by assessing CF isolates in mixed cultures. We hypothesize that intraspecies interactions between different P. aeruginosa isolates can mediate gene expression and virulence factor production that affects the overall virulence of the Pseudomonas population. Specifically, we intend to accomplish this with two key aims: 1) Examine gene expression of P. aeruginosa CF isolates exposed to culture supernatant of another CF isolate and 2) Survey newly isolated P. aeruginosa CF isolates responses to control P. aeruginosa cell-free supernatant. The proposed research will produce insights into how intra-species interactions within the polymicrobial environment in the CF lung contributes to the chronic nature of P. aeruginosa infections and provide novel tools to assess mixed microbial infections.

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Pilot Project 3

Title: Nanotherapeutic Modulation of Autophagy for Treatment of Lung Pathogens
Pilot Project Leader: Ashish Ranjan, Ph.D., Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

 

Project Summary: 

Intracellular pathogens evade host defenses by subverting autophagy to establish chronic infection. Traditional antimicrobials treatment for such pathogen is hampered by their lack of cell specificity. We hypothesize that drug-loaded nanocarriers that are targeted at restoring or enhancing normal autophagy can significantly enhance intracellular clearance of the pathogen. To test the hypothesis, we will a) Prepare a library of nanocarrier formulations (liposomes and bacterial outer membrane vesicle-based minicells) containing a cell impermeable antimicrobial and autophagy inducing peptide, and b) Determine the mechanism, and ability to clear an intracellular bacteria (Psuedomonas aeruginosa) in vitro. For autophagy induction, we will use an m-TOR inhibitor (Rapamycin). We believe that a successful demonstration of autophagy restoration as a means to treat chronic infections can be applicable to a variety of diseases in veterinary and human patients.

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Pilot Project 4

Title: The Effect of “Avirulent” Rickettsial Infections on Rocky Mountain Spotted Fever Pathogenesis: Aerosol and Needle Inoculation
Pilot Project Leader: Edward Shaw, Ph.D., Department of Microbiology & Molecular Genetics, College of Arts and Sciences, Oklahoma State University

 

Project Summary: 

Rocky Mountain spotted fever (RMSF) is a tick-borne disease caused by the obligate intracellular bacteria Rickettsia rickettsii. RMSF is characterized by profound vascular pathogenesis affecting literally every organ system. While predominantly acquired from tick bites, it has long been suspected that exposure to aerosolized tick blood/materials could cause RMSF. Indeed, respiratory acquired cases have been reported in laboratory workers. While rickettsial infections like RMSF cause severe, potentially fatal disease, little is known about the stunning array of spotted fever group Rickettsia spp. (SFGR) that cycle between tick vectors and mammalian hosts. In addition, established and novel SFGR are increasingly recognized as disease agents. Indeed, infections with SFGR remain under recognized because some SFGR agents historically considered “avirulent” may actually be pathogens, but also because prior exposure to “avirulent” SFGR likely influences the transmission dynamics of virulent agents, limiting subsequent infections in mammals and ticks. Our long term goal is to determine whether SFGR (historically considered less or “avirulent” and often found cycling in ticks in nature) may cause disease via both nasal and needle inoculation and whether infection with these agents may reduce or mute the effects of a subsequent infection with R. rickettsii. Our central hypothesis is that “avirulent” SFG Rickettsia spp. cycling in nature elicits an immune response and influences subsequent transmission dynamics and disease. We will initiate testing of this hypothesis by determining the influence of “avirulent” Rickettsia spp. on subsequent infection with virulent R. rickettsii in a controlled G. pig infection model of disease.

Pilot Project 1

Title: Validation of bacterial condensins as drug targets
Pilot Project Leader: Valentin V. Rybenkov, Ph.D, Department of Chemistry and Biochemistry, College of Arts and Sciences, The University of Oklahoma - Norman

 

Project Summary: 

Bacterial pneumonia is the leading cause of childhood mortality in the world and affects more than 5 million people per year in the US. It poses a particularly severe risk for patients with impaired immunity and in intense care units, which is exacerbated by the spreading multidrug resistance among pathogens. This project seeks to develop bacterial condensins as a new drug target. Condensins play a central role in global chromatin packing, a fundamental cellular activity, and their inactivation dramatically affects cell viability and antibiotic susceptibility. They display low sequence conservation from bacteria to humans pointing to the possibility of designing highly selective inhibitors. This proposal intends to validate condensins as drug targets in pathogenic bacteria Yersinia pestis and Pseudomonas aeruginosa. During the course of the project, we will establish an animal model of lung infection, which would complement the applicant’s expertise in biophysical, biochemical and microbiological aspects of condensins. The generated data and resources will create a complete framework for target validation studies in bacterial lung infection and might ultimately lead to the design of a new family of antibacterials.

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Pilot Project 2

Title: The role of glutamate in the initiation and maintenance of pleurisy
Pilot Project Leader: Kenneth E. Miller, Ph.D., Anatomy & Cell Biology, College of Medicine, Oklahoma State University - Tulsa

 

Project Summary: 

Inflammation of the lung lining (pleura) is called pleurisy and this causes sharp chest pain during breathing and coughing. Pleurisy is an outcome of lung infection, e.g., pneumonia, or infectious disease, e.g., tuberculosis. Pain from inflammation is an unmet medical need and represents a major health burden affecting forty per cent of all U.S. adults. Sensory nerve fibers contact the pleural membranes, initiate and maintain inflammatory states, and transmit pleural sensory information to the central nervous system. Initiation and maintenance of pleurisy involves pleura nerve fibers. Release of peptides from nerve fibers assists in the initiation of pleurisy, but little is known about the role of glutamate release from pleural sensory fibers. This is major gap in our understanding about the pleura since all sensory nerve fibers contain and release glutamate. Glutamate release is a prominent feature of inflammation and peripheral sensitization in many, if not all, tissues. Our research platform is directed toward evaluating peripheral glutamate at the onset and during acute and chronic inflammation. We propose that pleurisy is initiated and maintained by way of glutamate release from pleural sensory nerve fibers.

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Pilot Project 3

Title: Azoreductase characterization of Pseudomonas aeruginosa strain FRD1, a cystic fibrosis isolate
Pilot Project Leader: Gilbert H. John, Ph.D., Microbiology & Molecular Genetics, College of Arts and Sciences, Oklahoma State University

 

Project Summary: 

Cystic fibrosis patients suffer from severe lung infection caused by Pseudomonas aeruginosa. There is a rise in P. aeruginosa antibiotic resistance thereby new and novel strategies are needed. This study seeks to introduce a new and novel strategy involving azorductase activity, a major enzyme present in numerous bacteria including P. aeruginosa. Azoreductase is known to have dual activity, in which the metabolism of azo dyes and neutralization of reactive oxygen species (ROH) through quinone reductase activity are present. Our hypothesis is quinone reductase enhances the survivability of the bacterium, thereby, increasing antibiotic resistance. We have preliminary data showing azoreductase activity P. aeruginosa strain FRD1, which is a cystic fibrosis isolate. An important finding is FRD1 displays different azo dye specificities, compared to other Pseudomonas strains, suggesting FRD1 may have different azorductase proteins. The current study will isolate and characterize the azoreductase genes from FRD1, show dual activity, and demonstrate improved survival of the bacterium based on a comparative study of wildtype and mutant azoreductase cultures. The results will support the goal of finding a new and novel mechanism associated with pathogenicity, antibiotic resistance, and treatment of P. aeruginosa lung infections.

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Pilot Project 4

Title: Photoreceptors as a novel class of virulence factors in opportunistic pathogens
Pilot Project Leader: Wouter  D. Hoff, Ph.D, & Marianna Patrauchan, Ph.D., Microbiology & Molecular Genetics, College of Arts and Sciences, Oklahoma State University

 

Project Summary: 

Genome sequencing has unexpectedly revealed that more than 25% of all bacteria, including dedicated and opportunistic pathogens, contain photoreceptors. Recently, evidence has been building that these photoreceptors (i) in many cases are likely involved in biofilm formation; and (ii) in some cases are involved in light-regulation of virulence. Here we examine the hypothesis that the recently identified bacteriophytochrome photoreceptor in Pseudomonas aeruginosa acts as a virulence factor and affects biofilm formation in this medically important opportunistic pathogen. Our aims are to determine the effect of light on: P. aeruginosa biofilm formation (aim 1); the expression of virulence factors in this organism (aim 2); and the degree of infectivity of P. aeruginosa in two different disease models (aim 3). In addition, we will use disruption mutants to determine the involvement of known virulence signaling pathways in the light regulation of pathogenicity of P. aeruginosa (aim 4) as observed in aims 1-3. We will utilize the availability of well-developed assays for biofilm formation, virulence factor expression, and infectivity for this organism. In addition, we will exploit the comprehensive P. aeruginosa gene disruption strain collection to rapidly test which signaling pathways are involved in the observed photoresponses. Anticipated results are that the bacteriophytochrome in P. aeroginosa affects both its biofilm formation and infectivity. The elucidation of the role that photoreceptors play in modulating the virulence of opportunistic pathogens promises to open novel avenues of treatment of infections, particularly through combined antibiotics treatment with a controlled regime of light exposure of patients.