Pilot Projects

 

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.