Septins are eukaryotic cytoskeletal proteins involved in numerous cellular processes including host-pathogen interactions. During infection by the Gram-negative pathogen Shigella flexneri, bacteria are entrapped in septin cages for host defense. Previous work suggested that septins associate with the Gram-positive pathogen Staphylococcus aureus, but determinants of these interactions remained unknown. To address this, we investigated septin-S. aureus interactions by using infection of human epithelial HeLa cells and our cell-free in vitro reconstitution platform based on purified septin complexes. S. aureus invasion is a crucial step to successfully establish infection. In Chapter 3 we discovered that septins are recruited with actin to S. aureus engaging the receptor integrin α5β1 during host cell invasion. We also showed that septin depletion affected bacterial adhesion and invasion, as well as total protein levels of integrin α5β1 and focal adhesion kinase. Following invasion, intracellular S. aureus must evade host defense mechanisms. In Chapter 4 we used HeLa cells expressing a cytosolic S. aureus escape marker to investigate septin recruitment to cytosolic or vacuolar bacteria, and tested a wide range of experimental parameters on septin-S. aureus interactions. In Chapter 5, our cell-free in vitro system revealed that septins can directly bind the S. aureus surface. We also showed that wall teichoic acids (WTA), the major autolysin Atl and Sortase A restricted septin binding in vitro and during HeLa cell infection. WTAs and Atl are known to restrict peptidoglycan recognition by host immune sensors. We performed peptidoglycan pulldown assays and showed that purified septins bind to purified S. aureus peptidoglycan, highlighting a novel role for septins as a sensor of peptidoglycan. 3Overall, our findings identified new ways to block S. aureus invasion that may also apply to other pathogens engaging integrin α5β1 for cellular invasion, and further characterisation of peptidoglycan sensing by septins may inspire new host-directed antibacterial strategies.