Antibiotics with pleiotropic activities: Daptomycin and amphiphilic glycopeptides
Final Report Abstract
Daptomycin, the first approved member of the lipopeptide antibiotic class is considered a reserve antibiotic in the treatment of systemic and life-threatening infections caused by Gram-positive organisms. Despite the clinical importance, the MoA of DAP has been controversially discussed for decades and remains elusive. Importantly, since the lipopeptide strongly interacts with the cytoplasmic bilayer of the cell, our studies and the studies of others showed that DAP induces a multiplicity of highly interlinked and partially overlapping cellular events, such as inhibition of cell wall biosynthesis and aberrant cell septation, which hampered the identification of a specific molecular target for DAP so far. Corroborating the observed pleiotropic cellular effects induced by DAP, resistance towards DAP can be multifactorial and can largely differ depending on the strain background. As revealed by the comprehensive comparative analysis of two DAPR S. aureus strain pairs, mechanisms of resistance (MoR) resemble those underlying the VISA phenotype, including thickening of the cell wall and membrane lipid composition, stabilized by diverse background mutations. Although, the MoR did not provide hints for a molecular target, the study highlighted the enormous flexibility of bacterial cells to develop DAP resistance by entirely different routes. In attempt to dissect the observed pleiotropic effects on membrane and membrane-associated processes, we initiated two comprehensive MoA studies focusing on the model organisms B. subtilis and S. aureus, using a diverse set of in vivo and in vitro approaches. Using a fluorescence microscopy driven approach in collaboration with L. Hamoen, we could show that DAP targets regions of increased fluidity (RIFs), thereby massively affecting the biophysical properties of the cell membrane and ultimately membrane-bound processes occurring at these regions. Moreover, we found a detachment of the membrane-associated lipid II synthase MurG, that preferably localizes to RIFs, explaining the observed overriding effects on cell wall biosynthesis. Notably, lipid II likely accumulates at regions of increased fluidity (RIFs), since the long bactoprenyl moiety prefers a fluid lipid environment. Furthermore, molecular dynamics simulations of Chugunov et al. suggest that lipid is localized in “atoll”-like regions on the membrane surface in the presence of anionic PG, supporting the notion that these regions are closely associated with cell wall biosynthesis forming distinct “biosynthetic islands”. In line, DAP specifically localizes to the septum region of S. aureus. Within the first funding period, we did not observe interference with lipid II biosynthesis in vitro. However, these detergent-based soluble assay systems lacked a membrane, which may be crucial for oligomerization as well as for the interaction with a target. Therefore, we reconstituted membranebound peptidoglycan reactions into phospholipid micelles to mimic a more natural membrane environment. In this system, DAP interfered with the MraY catalyzed synthesis of lipid I in vitro only when the integral membrane protein MraY, together with its substrate C55-P, was reconstituted in the presence of PG and Ca2+, indicating a specific interaction of DAP with bactoprenyl-coupled precursors. Substantiated by binding studies to supported membrane bilayers loaded with several bactoprenyl-coupled lipid intermediates and by in vivo binding studies, we provide first evidence that DAP forms a specific tripartite complex with PGN precursors and PG in the presence of Ca2+. Our findings result in a DAP mode of action model, which explains its predominant effects on cell wall biosynthesis and integrates the characteristic pleiotropic membranerelated activities. The intensive search for a DAP target, throughout both funding periods produced numerous assays for testing the impact of antibiotics on individual reactions of cell wall polymer biosynthesis and enabled us to unravel the mode of action of several novel antibiotic compounds.
Publications
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(2012). Identification and in vitro analysis of the GatD/MurT enzyme-complex catalyzing lipid II amidation in Staphylococcus aureus. PLoS Pathog. 8(1):e1002509
Münch D, Roemer T, Lee SH, Engeser M, Sahl HG, Schneider T
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(2012). The lipodepsipeptide empedopeptin inhibits cell wall biosynthesis through Ca2+-dependent complex formation with peptidoglycan precursors. J. Biol. Chem. 287(24):20270-80
Müller A, Münch D, Schmidt Y, Reder-Christ K, Schiffer G, Bendas G, Gross H, Sahl HG, Schneider T, Brötz-Oesterhelt H
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(2013). Cyclic Lipopeptides as Antibacterial Agents – potent antibiotic activity mediated by intriguing mode of actions. Int. J. Med. Microbiol. 287(24):20270-80
Schneider T, Müller A, Gross H
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(2013). Mechanisms of Daptomycin Resistance in Staphylococcus aureus: Role of the Cell Membrane and Cell Wall. Review. Ann. N.Y. Acad. Sci. 1277:139-58
Bayer AS, Schneider T, Sahl HG
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(2015). Killing of pathogens by teixobactin without associated resistance. Nature. 517(7535):455-9
Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Hughes DE, Epstein S, Jones M, Poullenec K, Steadman V, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K
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(2015). Structural variations of the cell wall precursor lipid II - Influence on binding and activity of the lipoglycopeptide antibiotic oritavancin. Antimicrob Agent Chemother. 59(2):772-81
Münch D, Engels I, Müller A, Reder-Christ K, Falkenstein-Pau H, Bierbaum G, Bendas G, Grein F, Sahl HG,, Schneider T
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(2016) Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains. Proc. Natl. Acad. Sci. USA. pii: 201611173
Müller A, Wenzel M, Strahl H, Grein F, Saaki TN, Kohl B, Siersma T, Bandow JE, Sahl HG, Schneider T, Hamoen LW
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(2017). Differential daptomycin resistance development in Staphylococcus aureus strains with active and mutated gra regulatory systems. Int. J. Med. Microbiol.
Müller A, Grein F, Otto A, Gries K, Orlov D, Zarubaev V, Girard M, Sher X, Shamova O, Roemer T, François P, Becher D, Schneider T, Sahl, HG
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(2017). Targeting a cell wall biosynthesis hot spot. Nat. Prod. Rep. 34(7): 909-932
Müller A, Klöckner A, Schneider T
