Higgins, DarrenKim, Jenny2026-07-0720252025-09-042025Kim, Jenny. 2025. Uncovering Genes Involved in Staphylococcus aureus Survival and Persistence in Macrophages. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32240479https://dash.harvard.edu/handle/1/42743965Staphylococcus aureus (S. aureus) is a Gram-positive bacterial pathogen that causes serious infections in humans, colonizing the respiratory tract, skin, nose, soft tissue, and even bone. Though traditionally thought to be an extracellular pathogen, S. aureus is also capable of surviving intracellularly in both phagocytotic (macrophages, neutrophils) and non-phagocytotic cells (epithelial, endothelial cells). This intracellular survival is believed to protect S. aureus from the host immune system and cell impermeable antibiotics, resulting in difficult-to-treat recurring infections of the bone and heart such as osteomyelitis and endocarditis. At the same time, intramacrophage S. aureus can also adopt a state of antibiotic tolerance, protecting bacteria even from cell-permeable antibiotics. It is therefore critical to elucidate the mechanisms of S. aureus intracellular survival and persistence as it could provide insight into the treatment of persistent and recurrent S. aureus infections in the clinic. In this dissertation, we established a model of infection to characterize S. aureus survival in murine macrophages and conducted transposon-sequencing (Tnseq) screens in vitro and in vivo to identify genes involved in S. aureus intramacrophage survival. The most significant hit was MazF, a toxin in the toxin-antitoxin (TA) module mazEF. Through infection of bone marrow derived macrophages, we determined that deletion of the full toxin-antitoxin module reduced intramacrophage S. aureus survival by ~50%, which was restored by plasmid complementation of the module under the control of its native promoter. Because of the role of MazEF in other bacterial species in the control of temperate phages, we conducted additional studies probing the role of the mazEF TA module in regulating prophage activation as well as the function of prophages themselves in influencing bacterial survival in macrophages, revealing that deleting prophage phi 11 significantly improves survival in intramacrophage conditions. Altogether, this work uncovers two novel genes that significantly affect S. aureus survival in macrophages, providing critical information for the treatment of S. aureus persistent infections in the future.application/pdfenMicrobiologyThesis or Dissertation2026-07-070000-0003-4744-7478