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.

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...

In the study of glycosidases, a class of activity-based probes (ABPs), that are carbocyclic mimics of natural carbohydrates and can covalently bind the enzyme, have proven to be useful tools. This type of ABP has however not yet been reported for sialidases, glycosidases involved in various important biological processes in both health and disease, which hydrolyse terminal sialic acids. Here we present our study towards the synthesis of a carbocyclic sialic acid suitable for conversion into ABPs. We developed a route starting from a chiral furanone that includes a key early stage nitrone [3 + 2] cycloaddition to install most of the chiral centres present in N-acetylneuraminic acid. The final stereocentre is installed via a Barbier alkylation, after which a ring closing metathesis forms the pivotal carbocyclic intermediate. Due to challenges in the final stretch, we were not able to convert this intermediate into an N-acetylneuraminic acid ABP. However, the work presented here still represents a versatile route to potential future carbocyclic sialic acid derivatives.