Bacterial vesicle production scales with outer membrane to cell wall crosslink density

Extracellular vesicles are produced by a wide variety bacteria. These small spheres of membrane are likely produced via multiple pathways. One pathway is blebbing of the membrane, which requires separation of the membrane from the cell wall. Gram negative bacteria contain inner and outer membranes tethered to an internal cell wall by crosslinker proteins. Past experiments have demonstrated that removal of these crosslinker proteins leads to an increase in vesicle production. In this work we examine how vesicle production scales with the number of crosslinkers tethering the outer membrane to the cell wall. Building on prior theoretical work concerning the ability of a membrane to bleb through holes a cell wall, we model how the size of outer membrane regions untethered from the cell wall influence vesicle production. We consider several possible distributions of crosslinkers in the outer membrane and find, surprisingly, that whether crosslinkers are distributed randomly, uniformly, or in clusters, the qualitative predictions of our model are unchanged. We also allow for the possibility that membrane mechanical properties depend on crosslinker concentration, thus changing the relationship between the size of untethered membrane regions and vesicle formation. We test our model against experimental measurements of vesicle production with varied crosslinker protein expression. Our results suggest that bacterial vesicle production is regulated by proteins that tether the outer membrane to the cell wall.