New chemical tools to probe cell wall biosynthesis in bacteria

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ScienceDirect New chemical tools to probe cell wall biosynthesis in bacteria Robert T Gale and Eric D Brown Some of the most successful drugs in the antibiotic pharmacopeia are those that inhibit bacterial cell wall biosynthesis. However, the worldwide spread of bacterial antibiotic resistance has eroded the clinical efficacy of these drugs and the antibiotic pipeline continues to be lean as drug discovery programs struggle to bring new agents to the clinic. Nevertheless, cell wall biogenesis remains a high interest and celebrated target. Recent advances in the preparation of chemical probes and biosynthetic intermediates provide the tools necessary to better understand cell wall assembly. Likewise, these tools offer new opportunities to identify and evaluate novel biosynthetic inhibitors. This review aims to highlight these advancements and to provide context for their utility as innovative new tools to study cell wall biogenesis and for antibacterial drug discovery. Address Michael G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 Corresponding author: Brown, Eric D ([email protected])

Current Opinion in Microbiology 2015, 27:69–77 This review comes from a themed issue on Antimicrobials Edited by Paul M Dunman and Andrew P Tomaras

http://dx.doi.org/10.1016/j.mib.2015.07.013 1369-5274/# 2015 Elsevier Ltd. All rights reserved.

Introduction Antimicrobial agents that target bacterial cell wall biosynthesis are among the most successful and effective of those in the current antibiotic armamentarium. These agents, notably the b-lactam and glycopeptide classes of antibiotics that disrupt peptidoglycan assembly, have provided the mainstay of treatment regimes for bacterial infections in the clinic. However, rampant antibiotic resistance now threatens the clinical efficacy of these drugs worldwide. Cell wall biosynthesis continues to be an attractive antibacterial target [1–3]; however, modern drug discovery programs have unearthed no new cell-wall active antibacterial drugs. It is clear that the effective utilization of this www.sciencedirect.com

target requires novel tools and a thorough biochemical understanding of biosynthetic events. Recent development of chemical probes and simplified synthetic routes to important biosynthetic intermediates promises to aid these drug discovery efforts. Such resources have enabled both in vitro and in vivo study of cell wall assembly. In addition, they have been used to develop unique highthroughput screens and to evaluate the mode of action for several exciting non-protein targeting antibiotics. Herein we discuss these advancements and highlight their usefulness to modern drug discovery efforts.

Peptidoglycan assembly The biosynthesis of bacterial peptidoglycan is a validated and celebrated target of the clinical antibiotic pharmacopeia. The assembly of this macromolecule — a meshwork consisting of glycan heteropolymers connected via short peptides — begins with the cytoplasmic generation of UDP-N-acetylmuramic acid pentapeptide. This precursor is then coupled to a membrane-embedded undecaprenyl phosphate (C55-P) lipid carrier on the cytoplasmic face of the bacterial membrane and elaborated to Lipid II. Lipid II is flipped across the bacterial membrane and subsequently polymerized and cross-linked through the transglycosylase and transpeptidase activities of penicillin-binding proteins (PBPs). This biosynthetic process is shown in Figure 1 and has recently been reviewed [4]. While many cytoplasmic stages of peptidoglycan assembly are considered viable targets for novel chemotherapeutic agents [1], the majority of successful antibiotics used in the clinic inhibit the later lipid-linked steps of the pathway. The emergence of resistance to these agents has sparked interest to better understand the molecular details of lipid-linked peptidoglycan assembly, and to develop new tools and assays that identify and progress novel biosynthetic inhibitors. Thorough biochemical studies of this nature require access to milligram quantities of peptidoglycan biosynthetic intermediates. Isolation of these compounds, namely Lipid I and Lipid II, from natural bacterial sources is futile due to their low abundance in cells [5]. Thus, research groups have focused efforts on synthetic strategies as a means to obtain these valuable intermediates. Synthesis of lipid-linked peptidoglycan biosynthetic intermediates

Walker and co-workers along with VanNieuwenhze et al. have detailed chemical preparations for Lipid I [6,7]. VanNieuwenhze et al. and Schwartz et al. have outlined Current Opinion in Microbiology 2015, 27:69–77

70 Antimicrobials

Figure 1

Transpeptidation D-Ala D-Ala m-DAP D-Glu L-Ala

D-Ala D-Ala m-DAP D-Glu L-Ala

PBPs

L-Ala D-Glu m-DAP

L-Ala D-Glu m-DAP

D-Ala

D-Ala

[D-Ala] D-Ala m-DAP D-Glu L-Ala

L-Ala D-Glu m-DAP D-Ala

D-Ala D-Ala m-DAP D-Glu L-Ala

Cell Wall Transglycosylation

Plasma membrane

Translocation

UDP

(i) MurA, PEP (ii) MurB, NADPH

L-Ala MurG D-Glu UDP-GlcNAc P A m-D D-Ala D-Ala Lipid II

UDP

(i) MurC, L-Ala, ATP (ii) MurD, D-Glu, ATP (iv) MurE, m-DAP, ATP (v) MurF, D-Ala-D-Ala, ATP

Cytoplasm L-Ala D-Glu m-DAP Lipid I D-Ala D-Ala

MraY

Undecaprenyl pyrophosphate

GlcNAc

UDP L-Ala D-Glu m-DAP D-Ala D-Ala

MurNAc

UDP-MurNAc pentapeptide Current Opinion in Microbiology

Gram-negative bacterial peptidoglycan biosynthesis. The soluble cytoplasmic events of peptidoglycan assembly culminate with the preparation of UDP-MurNAc pentapeptide from precursor UDP-GlcNAc. The first step of this pathway involves linkage of an enolpyruvyl residue onto the C(3) hydroxyl moiety of UDP-GlcNAc in a reaction mediated by MurA. The resulting molecule is then reduced via the NADPH-dependent enolpyruvyl reductase MurB to yield UDP-MurNAc. ATP-dependent amino acid ligases (MurC-MurF) subsequently incorporate the stem peptide side chain on the lactate handle of UDP-MurNAc to produce UDP-MurNAc pentapeptide. UDP-MurNAc pentapeptide is anchored to membrane-embedded lipid carrier undecaprenyl phosphate in a pyrophosphate exchange reaction mediated by MraY to yield Lipid I. Lipid I is subsequently transformed to Lipid II via addition of GlcNAc to its C(4) hydroxyl group in a reaction facilitated by MurG. Lipid II is translocated to the extracellular leaflet of the cytoplasmic membrane (in a reaction currently thought to be mediated by either FtsW [23,24] or MurJ [25]), where it is polymerized, cross-linked and processed (shown in brackets) by the transglycosylase, transpeptidase and carboxypeptidase activities of penicillin-binding proteins (PBPs) [4]. The outer membrane is not shown. Abbreviations: GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid.

total syntheses for Lipid II [8,9]. Lipid II can also be prepared chemoenzymatically through MurG-mediated elaboration of Lipid I [6]. While these chemical and chemoenzymatic strategies are dependable and versatile, they involve lengthy chemical transformations that are impractical to conduct in most basic research laboratories. Therefore, a popular route to Lipid I and Lipid II employs a one-pot enzymatic reaction containing MraY and MurG-rich membrane extracts from Micrococcus flavus [10,11]. Braddick et al. used Lipid II prepared in this Current Opinion in Microbiology 2015, 27:69–77

fashion to develop a quantitative assay for Lipid II polymerization by the Staphylococcus aureus transglycosylase MGT [12]. Schneider and co-workers utilized enzymatically prepared Lipid II to recapitulate, in vitro, events of S. aureus pentaglycine interpeptide assembly [11] — a process that maintains methicillin resistance in vivo [13]. Recently, the ligand preferences for the semisynthetic glycopeptide oritavancin (currently approved by the FDA to treat skin infections caused by several pathogenic Gram-positive bacteria) and the novel depsipeptide www.sciencedirect.com

New chemical tools to probe cell wall biosynthesis in bacteria Gale and Brown 71

teixobactin (produced from a previously unculturable Gram-negative bacterium) were elucidated using these biosynthetic intermediates in binding and enzymatic assays [14,15].

(C35) analogues of authentic Lipid II (Figure 2) to overcome these issues during in vitro study. These compounds have facilitated the in vitro reconstitution of several PBP transglycosylase and transpeptidase reactions. Assays revealing the transpeptidase function for S. aureus PBP4 employed a heptaprenyl-linked Lipid II analogue (Figure 2, Lipid II analogue 1) [16]. Interestingly, this low molecular weight PBP was shown to exchange D-amino acids into nascent cell wall as well

PG biosynthetic intermediates readily aggregate in biochemical systems devoid of detergents and/or lipids due to their long C55 polyisoprenoid groups. The Kahne and Walker labs have prepared soluble heptaprenyl-linked Figure 2

HO R1 O

O

O AcHN O

O

O

O

P

HN

OH

O

O

P

x

R4

OH y

NH

O

H N

HO O

O

O

H N

NH

R2 H N

R3

O OH

O

Compound Name Lipid I (Lys)

R1

R2

R3

H

H

R4

x

y References

H

7

3 [6,7,11,22**]

H

H

7

3 [6,8-11,22**]

H

H

4

2 [16]

H

4

2 [17]

H

4

2 [18]

7

3 [22**]

7

3 [23]

4

1 [26*]

NHAc HO HO

Lipid II (Lys)

O OH NHAc

Lipid II analogue 1 (Lys)

HO HO

O OH NHAc

Lipid II analogue 2 (m-DAP)

HO HO

O

O

OH

O

NH2

OH NHAc HO

Lipid II analogue 3 (m-DAP-NH2) HO

O OH

5 NH

NHAc

Lipid II analogue 4 (Lys-NBD1)

HO HO

O

O

N O

H

N

OH

NO2

NHAc

Lipid II analogue 5

(Lys-NBD2)

HO HO

O

N O

H

N

OH

NO2

Lipid II analogue 6 (Lys)

O

H O N S O

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NHAc HO HO

H

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O O

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OH

Current Opinion in Microbiology

Lipid-linked peptidoglycan biosynthetic intermediates and analogues. www.sciencedirect.com

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as intermediate building blocks Lipid I and Lipid II. Studies involving the heptaprenyl analogue of canonical Gram-negative Lipid II (Figure 2, Lipid II analogue 2) could capture both transglycosylase and transpeptidase activities of Escherichia coli PBP1A and PBP1B in vitro [17]. Following a similar synthetic route to the Gramnegative Lipid II analogue, Lebar et al. prepared a Bacillus subtilis Lipid II analogue containing an amidated m-DAP residue in the pentapeptide chain (Figure 2, Lipid II analogue 3) [18]. Procurement of this compound enabled studies aimed at understanding how stem peptide variations affect PBP-mediated peptidoglycan crosslinking [18].

Fluorescent substrates

Lipid-linked intermediates of peptidoglycan biosynthesis contain no natural chromophore or unique absorption spectrum in the UV–Vis region. Thus, these molecules are routinely evaluated from biochemical assays using lengthy analytical techniques such as: thin-layer chromatography [10,15,19], LC/MS [10,16,17,18] and NMR spectroscopy [20,21]. Huang et al. recently described a flexible membrane-free enzymatic preparation of Lipid I, Lipid II and analogues; including fluorescent Lipid II analogues bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) moieties (Figure 2, Lipid II analogue 4) [22]. These tools were amended to a fluorescence-based HPLC assay to assess substrate specificities for the transglycosylase reactions of E. coli PBP1b and Clostridium difficile PBP. Mohammadi et al. used a similar NBDcontaining Lipid II molecule (Figure 2, Lipid II analogue 5) to demonstrate flippase activity — the translocation of lipid-linked peptidoglycan biosynthetic intermediates across a membrane bilayer — in proteoliposomes enriched with recombinant FtsW proteins [23,24]. Although these studies provide direct biochemical evidence supporting FtsW for this function, in vivo data support MurJ [25] in E. coli, and thus, the debate over the identity of the Lipid II flippase continues at this time. Other fluorescent Lipid II analogues have been prepared; Huang et al. chemically synthesized truncated Lipid II analogues containing a pentapeptide-linked coumarin fluorophore and an isoprenoid-linked dabsyl quencher (example compound: Figure 2, Lipid II analogue 6) [26]. The presence of a Fo¨rster Resonance Energy Transfer (FRET) donor/acceptor pair on these Lipid II analogues facilitated the construction of a continuous FRET-based HPLC in vitro assay capable of monitoring transglycosylase reactions. Kinetic parameters using these substrates were collected for transglycosylases from several clinically relevant pathogens including: Acinetobacter baumannii PBP1b, Klebsiella pneumoniae PBP1b and C. difficile PBP. The authors highlighted the utility of this quantitative assay to drug discovery programs by conducting a high-throughput screen (1536-well format, 120 Current Opinion in Microbiology 2015, 27:69–77

000 compounds) looking for transglycosylase inhibitors of C. difficile PBP [26]. Probes to study peptidoglycan biosynthesis in vivo

Study of peptidoglycan assembly has largely been limited to in vitro biochemical assays. Therefore, the dynamics of peptidoglycan biosynthesis in vivo is not well understood. Recently, fluorescent D-amino acids have been prepared that can probe real-time cell wall biosynthesis in a number of diverse bacterial species [27]. These probes efficiently label sites of active peptidoglycan biosynthesis when added to growing bacterial cultures and are readily visualized using fluorescence microscopy. Thus, fluorescent D-amino acids have enormous potential to address outstanding questions pertaining to bacterial growth and division. Kuru et al. have provided a facile procedure for the synthesis of four fluorescent D-amino acids of various colour (Figure 3; HADA, FDL, NADA and TDL) from commercially available precursors [28]. By pulselabeling growing Streptomyces venezuelae with multiple fluorescent D-amino acids, Kuru and co-workers were remarkably able to visualize the polar growth dynamics of the bacterium [27]. Walker and Kahne have also prepared a fluorescent D-amino acid: an amidated derivative of FDL (Figure 3, FDL-NH2) that enables improved labeling of B. subtilis in vivo [18]. An approach that utilizes bioorthogonal chemical reporters has also been developed to label peptidoglycan in vivo. This application involves the use of small, nonfluorescent azide or alkyne-functionalized D-amino acids for metabolic incorporation. These functionalized D-amino acids can be captured through click chemistry with complementary fluorescent probes after cells have been fixed and permeabilized. Several groups have used this approach with D-alanine analogues (Figure 3; EDA and ADA) to label peptidoglycan in a broad range of bacterial species [27,29], including the intracellular pathogen Listeria monocytogenes during macrophage infection [29]. Additionally, Liechti et al. has shown that D-alanyl-D-alanine analogues (Figure 3; EDA-DA) are also effective in this application by metabolically labeling the peptidoglycan of Chlamydia trachomatis [30]. Together, fluorescent D-amino acids and small D-amino acid analogues bearing bioorthogonal handles provide a means to understand spatial and temporal dynamics of peptidoglycan assembly and turnover in vivo. These tools could be used to assess changes in cell wall biosynthesis upon introduction with chemotherapeutic agents, and thus, could be of great utility to modern drug discovery and development programs.

Wall teichoic acid biosynthesis The peptidoglycan meshwork in most Gram-positive bacteria is heavily modified with long anionic glycopolymers called wall teichoic acids (WTAs). WTAs are anchored to www.sciencedirect.com

New chemical tools to probe cell wall biosynthesis in bacteria Gale and Brown 73

Figure 3

Compound name/structure

References

OH

+ N

HO

O

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NO2 O

CO2H

N CO2H

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O

Kuru et al., 2012 Kuru et al., 2014*

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Kuru et al., 2012 Seigrist et al., 2013

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ADA O

Liechti et al., 2013

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AcHN

EDA-DA

O

HO HO

Müller et al., 2012 Gale et al., 2014*

–

O

O

O P

O

OH

O P

O 7

OH

Lipid α

3 Current Opinion in Microbiology

Compounds used to probe in vivo peptidoglycan biosynthesis and in vitro WTA assembly. Fluorescent molecules are displayed against the color of their fluorophore (red, green and blue). Abbreviations: HADA, HCC-amino-D-alanine; FDL, fluorescein-D-lysine; NADA, NBD-amino-D-alanine; TDL, TAMRA-D-lysine; EDA, ethynyl-D-alanine; ADA, azido-D-alanine; DA, D-alanine.

the C(6) hydroxyl group of peptidoglycan N-acetylmuramic acid and generally consist of repeating polyol phosphate residues tailored with D-alanyl and glycosyl groups. While the precise biological function of WTAs remains unclear, these polymers are known to participate in cell-shape determination [31], the modulation of sensor www.sciencedirect.com

kinase activity [32] and coordination of peptidoglycan biosynthesis [33]. Exciting work in pathogenic S. aureus and Streptococcus pneumoniae reveals a critical role for WTAs in mechanisms of host infection [34–36] and blactam resistance [37,38]; thus, WTAs are projected as promising antibacterial [39,40] and/or pathogenesis targets Current Opinion in Microbiology 2015, 27:69–77

74 Antimicrobials

[1]. Indeed, both the pharmaceutical sector [38] and academic institutions [37,41] have conducted screening campaigns to identify WTA biosynthetic inhibitors. Like peptidoglycan biosynthesis, WTAs are assembled on membrane-embedded undecaprenyl-linked precursors (Figure 4 highlights the biosynthetic pathway). Our lab and the Walker lab have extensively studied the WTA assembly process in vitro, in systems employing soluble analogues of biosynthetic intermediates. These analogues contain truncated polyisoprenoid tails [42] or short aliphatic chains [43,44] in place of the C55 moiety found in authentic WTA biosynthetic substrates. Engineering these modifications into intermediate analogues simplified their preparation and prevented aggregation in biochemical assays. Access to WTA substrate analogues has

facilitated the characterization of several WTA biosynthetic enzymes from B. subtilis and S. aureus, including glycosyltransferases involved in: linkage unit formation [42,43], priming [43,45], polymerization [44,46] and modification [47,48]. However, many of these glycosyltransferases are membrane-associated and catalytically operate on undecaprenyl-linked substrates at the lipid–water interface in vivo. Thus, the membrane-free soluble systems used to study these enzymes in vitro are unable to capture true physiological events of WTA assembly. Understanding the influence of the membrane interface on aspects of catalysis for WTA biosynthetic enzymes may provide key insights for their effective utilization as novel antibacterial/ pathogenesis targets. Biochemical studies of this nature require access to authentic WTA substrates that can be reconstituted into interfacial systems.

Figure 4

Peptidoglycan

Plasma membrane

TagO

Lipid α

TagA Lipid β

TagB TagF Lipid φ.1

TagGH

Undecaprenyl-1-P

TagE GlcNAc-1-P ManNAc

Lipid φ.n

Glycerol-3-P Glu

Lipid γ Current Opinion in Microbiology

Wall teichoic acid biosynthesis in B. subtilis 168. WTA assembly occurs at the inner leaflet of the cytoplasmic membrane on an undecaprenyl phosphate lipid carrier. The first step of the pathway involves the transfer of GlcNAc-1-P to this lipid carrier, in a reaction catalyzed by transmembrane protein TagO, to produce intermediate Lipid a. TagA subsequently catalyzes the addition of ManNAc to the C(4) hydroxyl group of Lipid a to yield Lipid b. TagB catalyzes the incorporation of a single sn-glycerol-3-phosphate residue to the lipid-linked disaccharide to form Lipid f.1. TagF polymerizes roughly 25–35 glycerol phosphate monomers directly on Lipid f.1 to generate poly(glycerol phosphate) WTA polymers. These polymers are modified with a-linked glucose residues via TagE (to generate Lipid g) and are subsequently transported to the extracellular leaflet of the plasma membrane by the ABC transporter TagGH. Lipid-linked polymers are then thought to be modified with D-alanyl substituents before being transferred to the C(6) hydroxyl group of peptidoglycan N-acetylmuramic acid (in reactions likely mediated by LCP enzymes) [39,51,52]. Abbreviations: GlcNAc, N-acetylglucosamine; ManNAc, N-acetylmannosamine; Glu, glucose. Current Opinion in Microbiology 2015, 27:69–77

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New chemical tools to probe cell wall biosynthesis in bacteria Gale and Brown 75

Preparation of authentic WTA substrates

Tanja Schneider’s group first reported on an enzymatic route to authentic WTA intermediate substrates. Remarkably, her team solubilized S. aureus TagO — a protein with 13 transmembrane spans belonging to the Nacetylhexosamine-1-phosphate transferase family that includes MraY of the peptidoglycan pathway — and further reconstituted its activity in N-lauroylsarcosine mixed-micelles to provide the WTA precursor Lipid a (Figure 3) [49]. Lipid a could be elaborated to Lipid b through the addition of UDP-GlcNAc and recombinant proteins TagA and MnaA to the system. Access to these WTA precursors has facilitated studies to elucidate the mechanism of action for several cell-wall active antibiotics. For example, Mu¨ller et al. demonstrated that the type A lantibiotic nisin, previously thought to bind primarily Lipid I and Lipid II, could also interact with these WTA precursors in vitro [49]. Similarly, Munch et al. found that the lantibiotic NAI-107 (a natural product effective against numerous drug resistant Gram-positive pathogens) has the ability to inhibit TagO and TagA-mediated WTA biosynthetic reactions [21]. In addition, the newly discovered natural product teixobactin was shown to bind Lipid a in vitro [15]. These compounds target multiple undecaprenyl-containing glycolipids used to assemble peptidoglycan and WTA — such a mechanism may curb resistance development [50]. Our lab has recently published a chemoenzymatic route to authentic WTA intermediates. We devised a semisynthetic method to obtain Lipid a that utilizes Laurus nobilis leaves as a source of undecaprenol precursor [51]. Undecaprenol can be transformed to the universal lipid carrier undecaprenyl phosphate in a single chemical phosphorylation step. Thus, our approach is unique in that it does not rely on the commercial availability of undecaprenyl phosphate (which is expensive for purchase in useful quantity). Undecaprenyl phosphate can then be transformed to Lipid a through a two-step chemical synthesis that involves coupling to a chemically prepared a-phosphate derivative of N-acetylglucosamine. With access to Lipid a, we reconstituted the intracellular steps of B. subtilis poly(glycerol phosphate) biosynthesis using a mixed-micelle in vitro system. Consequently, we obtained access to authentic substrates for all WTA enzymes in the biosynthetic pathway (Figure 4, Lipid a–Lipid g) [51]. This work marked the first attempt to study WTA assembly using authentic substrates in a defined system that provides an interface for catalysis. Such substrates will facilitate experiments aimed at a thorough understanding of the molecular details of interfacial WTA biosynthesis. Indeed, it is plausible that the membrane influences enzyme catalytic efficiency, processivity and mechanism.

Conclusion Significant progress has been made to supply substrates and novel biochemical tools for detailed study of cell wall www.sciencedirect.com

assembly. These tools have enabled several fundamental steps of peptidoglycan and WTA biosynthesis to be reconstituted in vitro. In addition, they have been used to: track active peptidoglycan biosynthesis in a variety of bacteria; elucidate the mode of action for several promising antibiotics; develop novel HTS strategies; and characterize proteins of unknown function. While the procurement of these tools is a significant advancement to modern drug discovery and development programs, a paucity of resources still exists for the effective utilization of this old target. It is imperative that we continue to move forward and bolster the repertoire of tools that promise to aid our efforts against antibiotic resistance.

Acknowledgements RTG acknowledges support from a Canadian Institute of Health Research graduate scholarship. EDB acknowledges salary support from Canada Research Chairs program and operating funds from the Canadian Institute of Health Research (MOP-15496).

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2.

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7.

VanNieuwenhze MS, Mauldin SC, Zia-Ebrahimi M, Aikins JA, Blaszczak LC: The total synthesis of Lipid I. J Am Chem Soc 2001, 123:6983-6988.

8.

VanNieuwenhze MS, Mauldin SC, Zia-Ebrahimi M, Winger BE, Hornback WJ, Saha SL, Aikins JA, Blaszczak LC: The first total synthesis of Lipid II: the final monomeric intermediate in bacterial cell wall biosynthesis. J Am Chem Soc 2002, 124:3656-3660.

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10. Breukink E, van Heusden HE, Vollmerhaus PJ, Swiezewska E, Brunner L, Walker S, Heck AJR, de Kruijff B: Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes. J Biol Chem 2003, 278:19898-19903. 11. Schneider T, Senn MM, Berger-Ba¨chi B, Tossi A, Sahl H-G, Wiedemann I: In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus. Mol Microbiol 2004, 53:675-685. Current Opinion in Microbiology 2015, 27:69–77

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This paper describes the synthesis of several truncated Lipid II analogues containing FRET donor/acceptor pairs that can be used to study transglycosylase activity in vitro and conduct high-throughput screens. 27. Kuru E, Hughes HV, Brown PJ, Hall E, Tekkam S, Cava F, de Pedro MA, Brun YV, VanNieuwenhze MS: In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent Damino acids. Angew Chem Int Ed Engl 2012, 51:12519-12523. 28. Kuru E, Tekkam S, Hall E, Brun YV, Van Nieuwenhze MS:  Synthesis of fluorescent D-amino acids and their use for probing peptidoglycan synthesis and bacterial growth in situ. Nat Protoc 2014, 10:33-52. This publication outlines the facile synthesis of four fluorescent D-amino acids capable of probing real-time peptidoglycan biosynthesis in a broad range of bacteria. 29. Siegrist MS, Whiteside S, Jewett JC, Aditham A, Cava F, Bertozzi CR: D-Amino acid chemical reporters reveal peptidoglycan dynamics of an intracellular pathogen. ACS Chem Biol 2013, 8:500-505. 30. Liechti GW, Kuru E, Hall E, Kalinda A, Brun YV, VanNieuwenhze M, Maurelli AT: A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis. Nature 2013, 506:507-510. 31. D’Elia MA, Millar KE, Beveridge TJ, Brown ED: Wall teichoic acid polymers are dispensable for cell viability in Bacillus subtilis. J Bacteriol 2006, 188:8313-8316. 32. Botella E, Devine SK, Hubner S, Salzberg LI, Gale RT, Brown E.D, Link H, Sauer U, Code´e JD, Noone D et al.: PhoR autokinase activity is controlled by an intermediate in wall teichoic acid metabolism that is sensed by the intracellular PAS domain during the PhoPR-mediated phosphate limitation response of Bacillus subtilis. Mol Microbiol 2014, 94:1242-1259. 33. Atilano ML, Pereira PM, Yates J, Reed P, Veiga H, Pinho MG, Filipe SR: Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus. Proc Natl Acad Sci U S A 2010, 107:18991-18996. 34. Xu H, Wang L, Huang J, Zhang Y, Ma F, Wang J, Xu W, Zhang X, Yin Y, Wu K: Pneumococcal wall teichoic acid is required for the pathogenesis of Streptococcus pneumoniae in murine models. J Microbiol 2015, 53:147-154. 35. Weidenmaier C, Peschel A, Xiong Y-Q, Kristian SA, Dietz K, Yeaman MR, Bayer AS: Lack of wall teichoic acids in Staphylococcus aureus leads to reduced interactions with endothelial cells and to attenuated virulence in a rabbit model of endocarditis. J Infect Dis 2005, 191:1771-1777. 36. Weidenmaier C, Kokai-Kun JF, Kristian SA, Chanturiya T, Kalbacher H, Gross M, Nicholson G, Neumeister B, Mond JJ, Peschel A: Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections. Nat Med 2004, 10:243-245. 37. Farha MA, Leung A, Sewell EW, D’Elia MA, Allison SE, Ejim L, Pereira PM, Pinho MG, Wright GD, Brown ED: Inhibition of WTA synthesis blocks the cooperative action of PBPs and sensitizes MRSA to b-lactams. ACS Chem Biol 2013, 8:226-233. 38. Wang H, Gill CJ, Lee SH, Mann P, Zuck P, Meredith TC, Murgolo N, She X, Kales S, Liang L et al.: Discovery of wall teichoic acid inhibitors as potential anti-MRSA b-lactam combination agents. Chem Biol 2013, 20:272-284. 39. Sewell EW, Brown ED: Taking aim at wall teichoic acid synthesis: new biology and new leads for antibiotics. J Antibiot 2014, 67:43-51. 40. Pasquina LW, Santa Maria JP, Walker S: Teichoic acid biosynthesis as an antibiotic target. Curr Opin Microbiol 2013, 16:531-537. 41. Czarny TL, Perri AL, French S, Brown ED: Discovery of novel cell wall-active compounds using PywaC, a sensitive reporter of cell wall stress, in the model Gram-positive bacterium Bacillus subtilis. Antimicrob Agents Chemother 2014, 58:3261-3269. 42. Zhang Y-H, Ginsberg C, Yuan Y, Walker S: Acceptor substrate selectivity and kinetic mechanism of Bacillus subtilis TagA. Biochemistry 2006, 45:10895-10904. www.sciencedirect.com

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43. Ginsberg C, Zhang Y-H, Yuan Y, Walker S: In vitro reconstitution of two essential steps in wall teichoic acid biosynthesis. ACS Chem Biol 2006, 1:25-28. 44. Pereira MP, Schertzer JW, D’Elia MA, Koteva KP, Hughes DW, Wright GD, Brown ED: The wall teichoic acid polymerase TagF efficiently synthesizes poly(glycerol phosphate) on the TagB product Lipid III. ChemBioChem 2008, 9:1385-1390. 45. Brown S, Zhang Y-H, Walker S: A revised pathway proposed for Staphylococcus aureus wall teichoic acid biosynthesis based on in vitro reconstitution of the intracellular steps. Chem Biol 2008, 15:12-21. 46. Sewell EWC, Pereira MP, Brown ED: The wall teichoic acid polymerase TagF is non-processive in vitro and amenable to study using steady state kinetic analysis. J Biol Chem 2009, 284:21132-21138. 47. Allison SE, D’Elia MA, Arar S, Monteiro MA, Brown ED: Studies of the genetics, function, and kinetic mechanism of TagE, the wall teichoic acid glycosyltransferase in Bacillus subtilis 168. J Biol Chem 2011, 286:23708-23716. 48. Brown S, Xia G, Luhachack LG, Campbell J, Meredith TC, Chen C, Winstel V, Gekeler C, Irazoqui JE, Peschel A et al.: Methicillin

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resistance in Staphylococcus aureus requires glycosylated wall teichoic acids. Proc Natl Acad Sci U S A 2012, 109:18909-18914. 49. Mu¨ller A, Ulm H, Reder-Christ K, Sahl H-G, Schneider T: Interaction of type A lantibiotics with undecaprenol-bound cell envelope precursors. Microb Drug Resist 2012, 18:261-270. 50. Wright G: Antibiotics: an irresistible newcomer. Nature 2015, 517:442-444. 51. Gale RT, Sewell EW, Garrett TA, Brown ED: Reconstituting  poly(glycerol phosphate) wall teichoic acid biosynthesis in vitro using authentic substrates. Chem Sci 2014, 5:3823-3830. Here, the authors outline the semisynthesis of undecaprenyl phosphate and the chemoenzymatic synthesis of several authentic WTA substrates that can be used for detailed studies of WTA biosynthesis at the lipidwater interface. 52. Kawai Y, Marles-Wright J, Cleverley RM, Emmins R, Ishikawa S, Kuwano M, Heinz N, Bui NK, Hoyland CN, Ogasawara N et al.: A widespread family of bacterial cell wall assembly proteins. EMBO J 2011, 30:4931-4941.

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