Pilot Projects

 

Comparative transcriptomic analysis of IAV infection in porcine organoids

Dr. Laura Miller, College of Veterinary Medicine

There has been a re/emergence of zoonotic pathogens driven by multiple and complex factors, with livestock animals frequently in the interface of the disease transmission between wild animals and humans. These constant interactions have significantly increased the chances of potential spillover of viruses from wild animals to domestic animals and/or to humans, as evidenced by recent influenza and coronavirus outbreaks. Mammalian innate and adaptive immune responses are complex, interconnected and crucial for host defense against infectious disease. However, in some situations, some of these responses may lead to deleterious consequences. This highlights the need for alternative and adjunctive therapeutic options that target host-responses. Organoids can be used to document species-specific differences and are useful to assess the pandemic threat of animal influenza viruses. We have established an ex vivo three-dimensional (3D) organotypic air-liquid interface primary porcine respiratory epithelial cell culture system (ALI-PRECs) recreating a cell culture environment morphologically and functionally more representative of the epithelial lining of the swine trachea than traditional culture systems. ALI-PRECs are proposed for ex vivo comparison of influenza A virus infection. Specific Aim 1 will be to harness the ALI-PRECs for comparative influenza A virus studies. Intensive molecular and cellular characterization will be performed in parallel to virus infection tests. Specific Aim 2 will be to perform genomic analysis of ALI-PRECs following human-, swine-, avian-lineage influenza A virus infection(from Specific Aim 1) to evaluate the virus-host relationship. Transcriptomic annotation will allow clustering of shared expression patterns. Moreover, genome-wide profiling will delineate the epigenetic regulation of both viral and host factors, thus sequentially determining productive infection and clinical disease (pathogenesis) outcome. Understanding pathogenesis will help in the development of novel therapeutic options that minimize immunopathology without impairing beneficial host defenses.

 

Target Genes of Global regulators SinRI in Clostridioides difficile

Dr. Revathi Govind , College of Arts & Sciences

Clostridioides difficile is an important nosocomial pathogen that has been classified as an “urgent threat” by CDC.Antibiotic use is the primary risk factor for the development of C. difficile associated disease because it disrupts healthy protective gut flora and enables C. difficile to colonize the colon. C. difficile damage host tissue by secreting toxins and disseminates by forming spores. Biofilm formation further aids in successful colonization and persistence of C. difficile in the host gut. We discovered that a new set of global regulators (SinR and SinI) to regulate toxin production, sporulation, and biofilm formation in C. difficile. In this grant proposal, we hypothesize that Sin regulators controls the regulatory networks of all three pathogenic traits by controlling the regulators in these pathways. Main objective of this proposal is to identify the genes that are directly under the control of Sin regulators.

 

The Role of Conserved ORF3a Protein Domains in SARS-CoV-2 Replication and Pathogenesis

Dr. Edward Stephens, Professor, Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center

Coronaviruses infect numerous animal species including pigs, dogs, cats, cattle, horses, llamas, camels, civets, bats, and pangolins. In the last two decades, three highly pathogenic coronaviruses (MERS-CoV, SARS-CoV, and SARS-CoV-2) have evolved to cross the species barrier to cause morbidity and mortality in humans. As an example, SARS-CoV-2, the causative agent of COVID-19, caused a pandemic with over 6 million deaths worldwide and over 1 million deaths in the United States alone (as of April 3, 2023). With the concurrent global expansion of human and domestic animal populations, it is likely that novel coronaviruses will emerge because of cross-species transmission among humans and domestic and wild animals. The immunity generated from current vaccines is transitory and requires periodic boosters. Concomitant with the monitoring of species for new variants or distinct new coronavirus species, additional studies on the pathogenesis of SARS-CoV-2 is required to develop better vaccines in the future that generate long-term immunity. Using SARS-CoV-2 as the best-studied member of the β-coronaviruses, which is highly related to SARS-CoV, we propose to generate recombinant viruses with select mutations within the ORF3a protein of SARS-CoV-2. The ORF3a protein causes ER stress, activates the NLRP3 inflammasome, induces apoptosis, causes incomplete autophagy, and may facilitate in virus release through the endosome/lysosome pathway of virion assembly and release. Orf3a also has a PDZ-binding domain (PBM), which is known to interact with host cell proteins through its PDZ domain. The Orf3a is transported to the cell plasma membrane and co-localizes with the LAMP-1 marker for lysosomes. Proteins containing small linear motifs such as NPXY, YxxΦ, and dileucine sorting signals can be targeted to different compartments of the cell including the plasma membrane, recycling endosomes, late endosomes, lysosomes, and the trans-Golgi network (TGN). While the Orf3a has no dileucine or NPXY signals, it has three potential tyrosine sorting motifs in its cytoplasmic domain. Additionally, ORF3a has a PBM at its C-terminus. We removed the tyrosine-based sorting signals (Orf3a-∆YxxФ) and showed they were not significantly transported to the cell surface or associated with lysosomes. In Aim 1, we will analyze Orf3a-∆YxxФ and ORF-mutPBM proteins for the functions discussed above. In Aim 2, we propose to construct viruses expressing ORF3a-∆YxxΦ, ORF3a-mutPBM, Orf3a-[∆YxxФ/mut-PBM] and analyze these viruses for replication in cell lines (VeroE6, A549) or primary human small airway epithelial cells to determine if these viruses have reduced/decreased pathogenicity in cell cultures. We will also analyze these viruses for the functions described above. Finally, we propose to analyze the pathogenicity of these viruses in the K18 ACE-2 mouse model. Overall, the results of these studies will further our knowledge of the Orf3a transport and the PBM in the SARS-CoV-2 replication cycle and pathogenicity, may aid in the development of more effective vaccines with long-term immunity against this disease, and provide important preliminary data used to secure NIH funding.

Role of ICP0/CIN85 interaction during HSV-1 infection

Dr. Maria Kalamvoki, Associate Professor, Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center

Following productive, lytic infection in mucosal epithelial cells located at the portal of entry in the body, herpes simplex virus-1 (HSV-1) establishes a lifelong, silent infection in sensory neurons. The virus is occasionally reactivated due to weakened immune response or stress causing diseases that range in severity from benign cold sores to encephalitis. HSV-1 contributes to exacerbation of neurodegenerative diseases such as Alzheimer’s and facilitates infection by other pathogens such as HIV-1.
To infect and persist in the host, HSV-1 has evolved strategies to counteract host antiviral responses. The immediate early protein of the virus Infected Cells Protein No 0 (ICP0) plays a fundamental role in this process. ICP0 is a non-essential protein for the virus in cell cultures, particularly at a high multiplicity of infection, however ICP0 is essential in vivo to promote successful onset of lytic infection and productive reactivation of viral genomes from latency. Following its expression, ICP0 localizes in the nucleus where it activates viral gene transcription by blocking repressors of the viral genome, by inducing viral chromatin remodeling, and by inhibiting antiviral responses. The onset of virus replication triggers translocation of ICP0 to the cytoplasm. The functions of ICP0 out of the nucleus have not been studied. We have discovered that ICP0 in the cytoplasm interacts with the adaptor protein CIN85, a binding partner of the Cbl E3 ligase, which has a major role in cell surface receptor internalization, endocytic processing and protein sorting. The virus via ICP0 appears to subjugate this endocytic machinery to remove surface receptors, which could initiate antiviral signals and affect the ability of the virus to spread. Known manifestations of the ICP0/CIN85 interaction include the removal of the epidermal growth factor receptor (EGFR) and of the viral entry receptor Nectin-1 from the surface of the infected cells to facilitate virus spread. The internalized receptors are targeted either for degradation and/or exocytosis. We also discovered that the ICP0/CIN85 interaction was critical for exocytosis of numerous hostile factors, including innate immunity components and autophagy related factors. We hypothesize that ICP0 via its association with CIN85 subjugates an endocytosis pathway and diverts internalized cargo for degradation and/or exocytosis. This appears to be a novel mechanism by which the virus suppresses antiviral responses. We have designed two Aims to test this hypothesis. In Aim 1, we will determine the importance of ICP0/CIN85 interaction for HSV-1 infection in vitro. In Aim 2, we will determine the importance of ICP0/CIN85 interaction for HSV-1 infection in vivo. These studies are expected to unravel the importance of ICP0/CIN85 interaction and endocytosis of surface molecules during HSV-1 infection and their implication in immunoevasion.

Illuminating Dark Antibiotics: A Novel Synthesis of Streptothricin F and First Total Syntheses of BD-12 and Albothricin”

Dr. Shyam Sathyamoorthi, Assistant Professor, Department of Medicinal Chemistry, Kansas University School of Pharmacy

Since the commercialization of penicillin in 1928, antibiotics have been hailed as “magic bullets”. In recent years, however, the number of bacterial strains resistant to clinically used antibiotics has sharply increased. The lack of viable first-line treatments for these bacterial infections has forced clinicians to consider second-line antibiotic options such as polymyxins and aminoglycosides, traditionally avoided because of significant toxicity. Thus, the development of antibiotics with new mechanisms of action for the control of pernicious bacterial infections is of vital importance. The synthesis of natural products and simplified derivatives has been a particularly successful strategy for the enrichment of the anti-bacterial armamentarium. The specific aims of this proposal are: 1. Completion of The Shortest Total Synthesis of Streptothricin F to date and First Syntheses of BD-12 and Albothricin. A key step in the syntheses will be an aza-Wacker cyclization recently developed in our laboratory. 2. Synthesis and Biological Evaluation of Streptothricin F Analogues. We aim to synthesize a collection of simplified, structurally modified analogues that retain the antibacterial activity of the parent compounds but that have more “drug-likeness” and less cytotoxicity. Our plan for these analogues will be guided by function/diversity-oriented synthesis principles and in silico structure-based design. The latter will be executed by Dr. David Johnson (COBRE Computational Chemical Biology Core Laboratory, University of Kanas). Antibacterial activity will be evaluated in collaboration with the COBRE Infectious Disease Assay Development Core (University of Kansas). If analogues show promising antibiotic activity, their murine toxicity will be assessed in collaboration with the CEZID Animal Model/Pathology Core (Kansas State University). The rationale for this proposed research is that its success would allow for access to a diverse collection of antibacterial compounds with modes of action that are likely mechanistically distinct from FDA-approved antibiotics. The expected outcome of this research is the completion of several important steps towards the timely development of new, broad-spectrum, tolerable antibiotics and probe compounds for microbiological studies. The successful execution of the research proposed herein is expected to have a significant positive impact by aiding the global effort to reduce human morbidity and mortality resulting from pernicious bacterial infections.

Impact of the Borrelia burgdorferi adenylate cyclase cyaB at the host-pathogen interface

Vanessa Marie Ante, Assistant Professor, Division of Biology

Lyme disease is the most common vector-borne illness in the United States with rapidly increasing incidence. The symptoms of Lyme disease range from mild flu-like illness with fatigue and nausea to debilitating headaches and arthritis that can persist for years. The longstanding and severe morbidity of Lyme disease makes it a significant public health concern, especially considering a human vaccine is currently not available. Lyme disease is caused by the spirochete Borrelia burgdorferi, transmitted to humans through the bite of an infected Ixodes scapularis tick. Our long-term research goal is to characterize the mechanisms utilized by B. burgdorferi that support pathogenesis in the mammalian host to potentially identify novel targets for therapeutic intervention. We have previously established a role for cyaB, an adenylate cyclase responsible for producing the important second messenger cAMP, in B. burgdorferi pathogenesis. We found that B. burgdorferi cyaB modulates gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host. The objective of this proposal is to investigate the influence of cyaB at the B. burgdorferi-host interface to continue defining this regulatory pathway. Based on
preliminary and published data, we hypothesize that direct adherence to host epithelial cells induces cyaB expression, impacting B. burgdorferi and the host. To test this hypothesis, in Aim 1 we will characterize the role of B. burgdorferi cyaB during host cell adherence through investigating the ability of a cyaB mutant B.
burgdorferi to adhere to host cells. Additionally, we will examine cyaB expression levels in a non-adherent strain of B. burgdorferi following exposure to host cells. In Aim 2 we will identify transcriptional changes impacted by B. burgdorferi cyaB during host cell exposure by examining both B. burgdorferi and host transcriptomes following co-cultivation. The results from this proposal will provide significant insight into the mechanisms of Borrelia-host interaction and the dynamic genetic regulation taking place at the hostpathogen interface. Moreover, the data generated may provide a deeper understanding of the environmental sensing and second messenger signaling being initiated upon exposure to the host.