Kim, G.L., Hooven, T.A., Norambuena, J., Li, B., Boyd, J.M., Yang, J.H. and Parker, D., 2021. Growth and Stress Tolerance Comprise Independent Metabolic Strategies Critical for Staphylococcus aureus Infection. Mbio, 12(3), pp.e00814-21
This excellent paper from Dane Parker’s lab is timely. Two of its authors, Dane Parker and Jason Yang, will soon speak at our Fall Virtual Symposium. The study used a transposon insertion sequencing approach (which I had fun learning about) to define genetic requirements for survival of Staphylococcus aureus in the murine respiratory tract. Mice respiratory tracts were infected with a S. aureus transposon library for 24 hrs, the mice were then sacrificed, lungs removed, homogenized and surviving bacteria were outgrown in LB for a short time. Transposon junctions were then PCR amplified and prepared for Illumina sequencing to identify genes important for survival in the airway. If few to no insertion reads were then found for a locus, it was likely to be essential for survival. If several to many reads were found for a locus, it was very likely to be dispensable. A high proportion of the essential genes turned out to be central metabolic (such as pyruvate carboxylase), factors connected with growth and survival against host killing instead of being immunomodulatory in nature. For example, protein A which famously binds immunoglobulin and S. aureus’ alpha-toxin deployment has been shown to be an immunomodulatory strategy. Both these genes were found in the sequence data indicating they were possibly dispensable unless cross complimented by neighboring sloughing/secreting S. aureus with intact genes. They then characterized and validated many of the presumptive essential genes using a truly remarkable number of phenotypic assays: a macrophage survival assay, hemolytic assay, protein A expression, temperature sensitivity, in vivo (mouse) growth attenuation, in vitro complex vs. minimal growth media, biofilm formation (with & w.o. the host metabolite itaconate), arachidonic acid sensitivity, and respiration (O2 consumption rates, extracellular acidification rates). The authors found many genes involved in growth processes were critical including those in the TCA cycle, ATP production, and nucleotide metabolism supporting the ability of S. aureus to grow in the respiratory tract. Additionally, genes involved in stress tolerance-resistance to host defenses were also found. The study is a major contribution to understanding pathogenesis of S. aureus pneumonia: over 100 genes were defined as having a role in pulmonary infection, many of the factors were extensively tested and validated, and many of the genes identified had no previous known function. The study is a treasure trove of data that greatly contributes to understanding a very important pathogen. First author Gyu-Lee Kim is now a senior researcher at Korea Disease Control and Prevention Agency.