Cloning of aaa and isdB <em>Staphylococcus aureus</em> Antigens as Candidates for Bovine Vaccines

Staphylococcus aureus is a gram-positive bacterium that is classified as a high-burden antibiotic-resistant pathogen. In humans, S. aureus is commonly found on the skin where it is mainly harmless, but can enter through open wounds, causing pneumonia as well as infections in the bloodstream, heart, and joints (Juhász-Kaszanyitzky, 2007). S. aureus is also a major contributor to disease in cattle populations. Antibiotic-resistant strains of S. aureus have been found to cause bovine mastitis in cows, resulting in reduced milk production and an annual loss of about $2 billion to the dairy industry in the United States alone (Park, 2021). Although vaccines for S. aureus are already in development, improving existing options and creating new vaccines are vital to preventing infections in cattle. Reducing the use of antibiotics through vaccination can also help mitigate bacterial resistance. S. aureus expresses many virulence factors that enable it to colonize the udder. These include Aaa (autolysin/adhesin) and IsdB (iron-regulated surface determinant B). Aaa is an adhesin protein that allows S. aureus to attach to and infect host cells, contributing to immune evasion (Hirschhausen, 2012). Due to its invasive activity, Aaa is a strong vaccine candidate and could serve as the antigen bound component of a well characterized and experimentally safe adjuvant, cholera toxin B (CTB). This goal is to trigger an organism's immune response against Aaa with the idea of reducing the binding activity of S. aureus to host cells. Another important protein, IsdB, facilitates iron transport from the host cell to S. aureus through a variety of heme transport systems (Torres, 2006). This is another antigen that can potentially be used in vaccines, aiming to deprive S. aureus of the essential nutrients it needs to replicate. We have separately cloned the aaa and isdB genes into E. coli successfully for the construction of fusions to CTB (CTA2/B chimeras). These plasmids have been sequenced to confirm the correct insertion of the gene (pAAS001 and pAAS002). The next steps will be to express and purify these fusions using a D-galactose resin. Overall, our research aims to identify new S. aureus antigens that can be successfully cloned and purified as fusions with CTB, for use in developing new vaccine formulations. As a result, this will reduce S. aureus infections in cows and decrease the biological and economic threats to the dairy industry. References: Hirschhausen N, Schlesier T, Peters G, Heilmann C (2012) Characterization of the Modular Design of the Autolysin/Adhesin Aaa from Staphylococcus aureus. PLOS ONE 7(6): e40353. https://doi.org/10.1371/journal.pone.0040353 Juhász-Kaszanyitzky, E., Jánosi, S., Somogyi, P., Dán, A., van der Graaf-van Bloois, L., van Duijkeren, E., & Wagenaar, J. A. (2007). MRSA transmission between cows and humans. Emerging infectious diseases, 13(4), 630–632. https://doi.org/10.3201/eid1304.060833 Park, S., & Ronholm, J. (2021). Staphylococcus aureus in Agriculture: Lessons in Evolution from a Multispecies Pathogen. Clinical microbiology reviews, 34(2), e00182-20. https://doi.org/10.1128/CMR.00182-20 Torres, V. J., Pishchany, G., Humayun, M., Schneewind, O., & Skaar, E. P. (2006). Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization. Journal of bacteriology, 188(24), 8421–8429. https://doi.org/10.1128/JB.01335-06