Category B. Infectious Diseases and Vaccine Discovery
B1. Characterization and Protective Efficacy Assessment of Potential Subunit Vaccine-S1S2 Against Salmonella enterica
Prashant Kumar1, Francisco J. Martinez Becerra1, Olivia Arizmendi1, Russell Middaugh2, William D. Picking1, Wendy D. Picking1
1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA;
2Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, KS, USA
Diarrhea caused by Salmonella enterica is an important public health problem. It is difficult to develop a broadly protective vaccine for S. enterica due to the presence of multiple serotypes and immunodominance of LPS. We have designed novel subunit vaccines, S1 (SipD-SipB) and S2 (SseB-SseC) fusion proteins, based on the highly conserved type-III secretion systems (T3SS) of Salmonella pathogenicity islands SPI-1 and SPI-2, respectively. The proteins were characterized using spectroscopic techniques to understand their structural and biophysical properties. Far-UV circular dichroism (CD) indicated both possess predominant alpha-helical secondary structure. Tertiary and quaternary structures were monitored using fluorescence and static light scattering (SLS) techniques, respectively. The resulting data sets from the spectroscopic techniques were collectively viewed using multi-index empirical phase diagrams and radar charts for assessment of protein structural integrity as a function of pH and temperature. S1 was found to be stable at lower temperature (below 25 ⁰C-30 ⁰C) while S2 was more thermostable (50 ⁰C - 55 ⁰C) over a wide pH range. We immunized mice with these proteins intramuscularly with Alum and MPL as adjuvants. Serum antibodies against the individual Tip proteins were measured. In addition, immunization with both fusion proteins yielded significant protection against Salmonella enterica serovar Typhimurium and Enteritidis. Since both the fusion proteins were tested for their broad range protection in different studies, their pre-formulation biophysical characterization is vital for further developing this protective Salmonella vaccine.
B2. Characterization of Novel Metallophore Biosynthetic Enzymes Conserved by the Bacterial Pathogens Pseudomonas aeruginosa and Staphylococcus aureus
Jeff McFarlane and Audrey L. Lamb
Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
Increasing antibiotic resistance among major pathogens has highlighted the need to more thoroughly understand fundamental mechanisms leading to virulence with the goal of identifying new targets for antimicrobial therapy. Pseudomonas aeruginosa and Staphylococcus aureus are commensal, opportunistic pathogens in human populations and are the leading cause of ventilator-associated pneumonia (P. aeruginosa) and bacteremia and endocarditis (S. aureus), each exhibiting high mortality rates. Critical to the pathogenesis of these species is their shared need to compete with the human host for essential metals such as iron. Human physiology has evolved elaborate means to sequester metals from potentially infectious bacteria, a defense known as nutritional immunity. In response, bacteria biosynthesize small molecules known as metallophores, such as pyoverdine in P. aeruginosa and staphyloferrin in S. aureus. These metallophores counteract nutritional immunity by scavenging metals required for growth. Recently, a novel metallophore biosynthetic pathway conserved between P. aeruginosa and S. aureus, has been identified. The enzymes in this pathway produce small molecule opine metallophores named pseudopine and staphylopine. Mutants with a gene deletion in this operon have been shown to significantly attenuate P. aeruginosa and S. aureus infections in mouse models.
We have recently determined the substrates required for the production of pseudopine and are completing a kinetic analysis of the nicotianamine synthase and opine dehydrogenase enzymes responsible for the production of both metallophores. This work will provide the necessary basic science knowledge to guide future drug design efforts directed at these novel antimicrobial targets.
B3. Infectious Disease Assay Development Core: High Throughput Screening Laboratory at the University of Kansas
High Throughput Screening Laboratory, University of Kansas, Lawrence, KS, USA
The University of Kansas High Throughput Screening Laboratory (KU-HTSL) is a fee-for-service, state-of-the-art facility dedicated to providing academia, not-for-profit institutions, biotech, and pharmaceutical industries with exceptional assay development and high throughput screening services at economical rates. The staff has experience in executing cell-based, biochemical, siRNA as well as high content screening campaigns against a plethora of target classes. Clients have the option of using our collection of 300,000 compounds and/or a client's own chemical library. Our chemical library is augmented with the KU Medicinal Chemistry department's legacy compound collection. KU-HTSL is innovative and flexible in providing superior service to the drug discovery research community, including assay development, screening, compound profiling and data mining. The integrated automation of the HTS system allows us to screen about 100,000 compounds in two days for endpoint assays, or one week for kinetic assays, at an affordable cost. KU-HTSL further leverages the strengths of the KU Core facilities and the KU Medical Center's Institute for Advancing Medical Innovations to support your new lead discovery research.