Alteration of cholesterol content and oxygen level in intestinal organoids after infection with Staphylococcus aureus

The pathogenicity elicited by Staphylococcus (S.) aureus, one of the best-studied bacteria, in the intestine is not well understood. Recently, we demonstrated that S. aureus Infection induces alterations in membrane composition that are associated with concomitant impairment of intestinal function. Here, we used two Organoid models, induced pluripotent stem cell (iPSC)-derived intestinal organoids and colonic intestinal stem cell-derived intestinal organoids (colonoids), to examine how sterol metabolism and oxygen levels change in response to S. aureus Infection. HPLC quantification showed differences in lipid homeostasis between infected and uninfected cells, characterized by a remarkable decrease in total cellular Cholesterol. As the altered sterol metabolism is often due to oxidative stress response, we next examined intracellular and extracellular oxygen levels. Three different approaches to oxygen measurement were applied: (1) cell-penetrating nanoparticles to quantify intracellular oxygen content, (2) sensor plates to quantify extracellular oxygen content in the medium, and (3) a sensor foil system for oxygen distribution in Organoid cultures. The data revealed significant intracellular and extracellular oxygen drop after Infection in both intestinal Organoid models as well as in Caco-2 cells, which even 48 h after elimination of extracellular bacteria, did not return to preinfection oxygen levels. In summary, we show alterations in sterol metabolism and intra- and extracellular hypoxia as a result of S. aureus Infection. These results will help understand the cellular stress responses during sustained Bacterial infections in the intestinal epithelium.

HPLC; S. aureus Newman; intestinal organoids; oxygen measurement; oxysterols; stem cells.