Alpha-hemolysin promotes internalization of Staphylococcus aureus into human lung epithelial cells via caveolin-1- and cholesterol-rich lipid rafts.

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-β-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5β1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process.