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Real Time Monitoring of Peptidoglycan Synthesis by Membrane-Reconstituted Penicillin 2 Binding Proteins 3 Víctor M

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Real Time Monitoring of Peptidoglycan Synthesis by Membrane-Reconstituted Penicillin 2 Binding Proteins 3 Víctor M 1 Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin 2 binding proteins 3 Víctor M. Hernández-Rocamora1*, Natalia Baranova2*, Katharina Peters1, Eefjan Breukink3, 4 Martin Loose2#, Waldemar Vollmer1# 5 6 1 Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Richardson 7 Road, Newcastle upon Tyne, NE2 4AX, UK. 8 2Institute for Science and Technology Austria (IST Austria), Klosterneuburg, Austria 9 3Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, 10 University of Utrecht, Padualaan 8, 3584 Utrecht, The Netherlands. 11 12 * Contributed equally. 13 # Correspondence: [email protected]; [email protected]. 14 15 1 16 ABSTRACT 17 Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the 18 cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β- 19 lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding 20 proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan 21 chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in 22 their physiological membrane environment is poorly understood. Here we developed a novel 23 FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in 24 liposomes or supported lipid bilayers and we demonstrate this assay with PBP1B homologues 25 from Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii in the presence 26 or absence of their cognate lipoprotein activator. Our assay allows unravelling the mechanisms 27 of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be 28 used for high throughput screening for new antimicrobials. 2 29 INTRODUCTION 30 Peptidoglycan (PG) is a major cell wall polymer in bacteria. It is composed of glycan strands 31 of alternating N-actetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues 32 interconnected by short peptides. PG forms a continuous, mesh-like layer around the cell 33 membrane to protect the cell from bursting due to the turgor and to maintain cell shape 34 (Vollmer et al., 2008). The essentiality and conservation of PG in bacteria make peptidoglycan 35 metabolism an ideal target of antibiotics. 36 Class A penicillin-binding proteins (PBPs) are bifunctional PG synthases, which use 37 the precursor lipid II to polymerize glycan chains (glycosyltransferase reactions) and crosslink 38 peptides from adjacent chains by DD-transpeptidation (Goffin & Ghuysen, 1998). 39 Moenomycin inhibits the glycosyltransferase and -lactams the transpeptidase function of class 40 A PBPs (Sauvage & Terrak, 2016, Macheboeuf et al., 2006). In E. coli, PBP1A and PBP1B 41 account for a substantial proportion of the total cellular PG synthesis activity (Cho et al., 2016) 42 and they are tightly regulated by interactions with multiple proteins (Egan et al., 2015, Typas 43 et al., 2012, Egan et al., 2020, Egan et al., 2017), including the outer membrane anchored 44 activators LpoA and LpoB (Egan et al., 2018a, Typas et al., 2010, Jean et al., 2014). 45 Historically, in vitro PG synthesis assays have been crucial to decipher the biochemical 46 reactions involved in PG synthesis and determine the mode of action of antibiotics (Izaki et al., 47 1968). However, these studies were limited by the scarcity of lipid II substrate and the inability 48 to purify a sufficient quantity of active enzymes. Lipid II can now be synthesized chemically 49 (VanNieuwenhze et al., 2002, Schwartz et al., 2001, Ye et al., 2001) or semi-enzymatically 50 (Breukink et al., 2003, Egan et al., 2015), or isolated from cells with inactivated MurJ (Qiao et 51 al., 2017). Radioactive or fluorescent versions of lipid II are also available to study PG 52 synthesis in the test tube. However, there are several drawbacks with currently available PG 53 synthesis assays. First, most assays are end-point assays that rely on discrete sampling and 54 therefore do not provide real-time information about the enzymatic reaction. Second, some 55 assays involve measuring the consumption of lipid II or analysing the reaction products by 56 SDS-PAGE (Egan et al., 2015, Barrett et al., 2007, Qiao et al., 2014, Sjodt et al., 2018) or 57 HPLC after digestion with a muramidase (Bertsche et al., 2005, Born et al., 2006). These 58 laborious techniques make assays incompatible with high through-put screening and hinder the 59 determination of kinetic parameters. A simple, real-time assay with dansyl-labelled lipid II 60 substrate overcomes these problems but is limited to assay GTase reactions (Schwartz et al., 61 2001, Offant et al., 2010, Egan et al., 2015). 3 62 Recently two types of real-time TPase assays have been described. The first uses non- 63 natural mimics of TPase substrates such as the rotor-fluorogenic 470 D-lysine probe Rf470DL, 64 which increases its fluorescence emission upon incorporation into PG (Hsu et al., 2019). The 65 second assay monitors the release of D-Ala during transpeptidation in coupled enzymatic 66 reactions with D-amino acid oxidase, peroxidases and chromogenic or fluorogenic compounds 67 (Frere et al., 1976, Gutheil et al., 2000, Catherwood et al., 2020b). Coupled assays are often 68 limited in the choice of the reaction conditions, which in this case must be compatible with D- 69 amino acid oxidase activity. Hence, each of the current assays has its limitations and most 70 assays exclusively report on either the GTase or TPase activity, but not both activities at the 71 same time. 72 Another major drawback of many of the current assays is that they include detergents 73 and/or high concentration (up to 30%) of the organic solvent dimethyl sulfoxide (DMSO) to 74 maintain the PG synthases in solution (Offant et al., 2010, Biboy et al., 2013, Huang et al., 75 2013, Lebar et al., 2013, Qiao et al., 2014, Egan et al., 2015, Catherwood et al., 2020b). 76 However, both detergents and DMSO have been shown to affect the activity and interactions 77 of E. coli PBP1B (Egan & Vollmer, 2016). Importantly, a freely diffusing, detergent- 78 solubilised membrane enzyme has a very different environment compared to the situation in 79 the cell membrane where it contacts phospholipids and is confined in two dimensions (Gavutis 80 et al., 2006, Zhdanov & Höök, 2015). Here we sought to overcome the main limitations of 81 current PG synthesis assays. We established the sensitive Förster Resonance Energy Transfer 82 (FRET) detection technique for simultaneous monitoring of GTase and TPase reactions. The 83 real-time assay reports on PG synthesis in phospholipid vesicles or planar lipid bilayers. We 84 successfully applied this assay to several class A PBPs from pathogenic Gram-negative 85 bacteria, demonstrating its robustness and potential use in screening assays to identify PBP 86 inhibitors. 87 88 RESULTS 89 Real time assay for detergent-solubilised E. coli PBP1B 90 To develop a FRET-based real time assay for PG synthesis using fluorescently labelled lipid 91 II, we prepared lysine-type lipid II versions with high quantum yield probes, Atto550 (as FRET 92 donor) and Atto647n (as FRET acceptor), linked to position 3 (Figure 1 – figure supplement 93 1A-B) (Mohammadi et al., 2014, Egan et al., 2015). For assay development we used E. coli 94 PBP1B (PBP1BEc) (Egan et al., 2015, Bertsche et al., 2005, Biboy et al., 2013) solubilized 95 with Triton X-100 and a lipid-free version of its cognate outer membrane-anchored lipoprotein 4 96 activator LpoB (Typas et al., 2010, Egan et al., 2014, Egan et al., 2018a, Lupoli et al., 2014a, 97 Catherwood et al., 2020b). 98 PBP1BEc can utilize fluorescently labelled lipid II to polymerize long glycan chains 99 only when unlabelled lipid II is also present in the reaction (van't Veer, 2016). We therefore 100 included unlabelled meso-diaminopimelic acid (mDAP)-type lipid II into reactions of PBP1BEc 101 with lipid II-Atto550 and lipid II-Atto647n (Figure 1A). Both probes were incorporated into 102 the produced PG or glycan chains as indicated by SDS-PAGE analysis (Figure 1B, i). After the 103 reaction, fluorescence spectra taken at the excitation wavelength of the donor fluorophore 104 (Atto550, λabs=552 nm) showed a reduced donor emission intensity (λfl=580 nm) and an 105 increased emission of the acceptor fluorophore (Atto647n, λfl=665 nm) (Figure 1C, i) indicative 106 of FRET between the two fluorophores. Analysis of the fluorescence spectra allowed to 107 calculate FRET efficiencies which we found to be 29 ± 6 % (Figure 1D, Figure 1 – figure 108 supplement 2). Ampicillin, which inhibits the TPase, blocked the formation of cross-linked PG 109 (Figure 1B, ii) and reduced the FRET efficiency by a third (Figure 1C, ii, 1D). Moenomycin, 110 which blocks the GTase and, indirectly, TPase activities completely abolished the 111 incorporation of fluorescent lipid II and the associated signal (Figure 1B, iii, 1D) (Bertsche et 112 al., 2005). These results demonstrate that incorporation of the labelled probes into PG by 113 PBP1BEc, produces a signal that depends on the GTase and TPase activity, with the latter being 114 the major contributor. 115 Next, we monitored reactions in real time by measuring fluorescence emission of the 116 donor and acceptor fluorophores (FIdonor and FIacceptor, respectively) after excitation of the donor 117 (540 nm) in a microplate reader for 60 min (Figure 1E, Figure 1 – figure supplement 3A). The 118 ratio between both signals (FIacceptor/FIdonor) was used as indicative of FRET. Without LpoB, 119 FRET appeared after ~5 min and slowly increased until it plateaued after 50-60 min (Figure 120 1E, left panel). By contrast, reactions with LpoB(sol) showed an immediate and rapid increase 121 in FRET which reached the plateau after 10-20 minutes, consistent with faster PG synthesis 122 (Figure 1E, left panel).
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