Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes

Abstract

36Infections with Gram-negative bacteria form an increasing risk for human health, which is 37 mainly due to the increase in antibiotic resistance. The cell envelope of Gram-negative 38 bacteria consists of an inner membrane, a peptidoglycan layer and an outer membrane, which 39 forms an impermeable barrier to many antibiotics and antimicrobial proteins. The 40 complement system is an important factor of the human immune system that can efficiently 41 kill Gram-negative bacteria via the formation of large pores in the bacterial outer membrane, 42called Membrane Attack Complexes (MACs). To better understand how these MAC pores 43 damage the complex cell envelope of Gram-negative bacteria, we recently developed a 44 fluorescent reporter system to study membrane damage in E. coli. Here, we used a similar 45 experimental setup in combination with flow cytometry, confocal microscopy and 46 conventional plating assays to elucidate how different components of the immune system act 47 synergistically to effectively clear invading bacteria. We demonstrate how MAC-dependent 48 outer membrane damage enhances the susceptibility of E. coli to further degradation of the 49 cell envelope by lysozyme, leading to drastic changes in the morphology of these bacteria. 50Furthermore, we elucidate that the MAC enhances the susceptibility of E. coli to 51 phospholipases, and to degradation and killing inside human neutrophils. Altogether, this 52 study provides a detailed overview on how different players of the human immune system 53 work closely together to degrade the complex cell envelope of Gram-negative bacteria. This 54 knowledge may facilitate the development of new antimicrobials that could stimulate, or work 55 synergistically with the immune system. 56 57 58 59 60 61 62 63 64 65 66 67 68Infections with Gram-negative bacteria form a major problem for human health, which is 71 mainly due to the increase in antibiotic resistance. According to the World Health 72Organization there is an urgent need for alternative strategies to treat infections with Gram-73 negative bacteria such as Acinetobacter baumanni, Pseudomonas aeruginosa and Escherichia 74 coli (E.coli), which are at the top of the priority list of antibiotic resistant bacterial species 1 . 75The cell envelope of Gram-negative bacteria consists of an inner membrane, a peptidoglycan 76 layer and an additional outer membrane 2 . The peptidoglycan layer of bacteria is important for 77 the maintenance of osmotic balance and to maintain the bacterial cell shape, for example rod-78shaped for E. coli 3 . The outer membrane forms a physical barrier to a large number of 79 antimicrobial compounds 4 , which makes it challenging to develop new antibiotics against 80 these bacteria. Combination therapy of antibiotics and outer membrane permeabilizing agents 81 have become more attractive over the last decades 5-9 . Furthermore, there is increased 82 awareness that antibiotics may be more effective in the presence of the human immune 83 system 10 , which has developed strategies to d...

Real-Time Flow Cytometry to assess qualitative and quantitative responses of oral pathobionts during exposure to antiseptics

“…Both fluorescence intensity and scatter played a role in the separation of the phenotypes. This is in accordance with previous studies that have shown that stress, which results in cell wall damage, can alter the morphology of a bacterial cell 21,25 .…”

“…In addition, the subpopulations of intact and damaged cells were based on the 2-dimensional space of green vs red fluorescence intensity and were designed manually. Nonetheless, manual gating is subjected to bias 20 and antimicrobial stress can affect not only cell permeability but also cell morphology 21 . Moreover, by splitting the cells into two subpopulations, intact or damaged, we enforced a discretization of the data, which in reality represented a continuous spectrum of phenotypes with intermediate levels of cell permeability.…”

Lysins breaking down the walls of Gram-negative bacteria, no longer a no-go

“…Also the OM composition may be substantially different in vivo due to differential gene expression [21]. In addition, the Membrane Attack Complex (MAC) composed of complement components of the innate immunity forms OM pores large enough for passage of human lysozyme [49] and may thus also potentiate lysins in vivo. Finally, lysins should not remain limited to human clinical applications, but have the potential to be widely applied wherever Gram-negative bacteria cause a problem.…”

Mammals' humoral immune proteins and peptides targeting the bacterial envelope: from natural protection to therapeutic applications against multidrug‐resistant Gram‐negatives

“…This remains controversial however, since some studies suggest that this self‐translocation allows lysozyme to reach the Gram‐negative peptidoglycan, enabling its enzymatic breakdown. This activity would obviously be enhanced in the presence of immune elements that act as outer membrane permeabilizers, as happens in vivo (Ellison & Giehl, 1991 ; Callewaert & Michiels, 2010 ; Heesterbeek et al ., 2021 ).…”