Outer Membrane Permeabilization By The Membrane Attack Complex Sensitizes Gram-Negative Bacteria To Antimicrobial Proteins In Serum And Phagocytes

Author summaryIn this paper we identified how different players of the human immune system cooperate to degrade the complex cell envelope of Gram-negative bacteria. The outer membrane of Gram-negative bacteria forms an impermeable barrier for various antimicrobial proteins of the immune system. Here we show that complement-dependent Membrane Attack Complex (MAC) formation permeabilizes this barrier, allowing otherwise impermeable antimicrobial proteins to reach their targets underneath the outer membrane. Specifically, we show that outer membrane damage by the MAC allows lysozyme to degrade the peptidoglycan layer, and secreted phospholipase A(2)-IIA to hydrolyze the bacterial inner membrane. MAC formation also sensitizes Gram-negative bacteria to more efficient degradation and killing inside human neutrophils. Altogether, this knowledge may guide the development of new antimicrobial strategies to treat infections caused by Gram-negative bacteria.Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.