Assay for peptidoglycan O-acetyltransferase: A potential new antibacterial target Patrick J. Moynihan, Anthony J. Clarke ⇑ Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 article info Article history: Received 12 February 2013 Accepted 16 April 2013 Available online 7 May 2013 Keywords: Peptidoglycan O-acetylation O-acetyltransferase Transacetylase Muramic acid abstract The O-acetylation of peptidoglycan occurs at the C-6 hydroxyl group of muramoyl residues in many human pathogens, both gram positive and gram negative, such as Staphylococcus aureus and species of Campylobacter, Helicobacter, Neisseria, and Bacillus, including Bacillus anthracis. The process is a matura- tion event being catalyzed either by integral membrane O-acetylpeptidoglycan transferase (Oat) of gram-positive bacteria or by a two-component peptidoglycan O-acetyltransferase system (PatA/PatB) in gram-negative cells. Here, we describe the development of the first in vitro assay for any peptidoglycan O-acetyltransferase using PatB from Neisseria gonorrhoeae as the model enzyme. This assay is based on the use of chromogenic p-nitrophenyl acetate as the donor substrate and chitooligosaccharides as model acceptor substrates in place of peptidoglycan. The identity of the O-acetylated chitooligosaccharides was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rates of transacetylations were determined spectrophotometrically by monitoring p-nitrophenol release after accounting for both spontaneous and enzyme-catalyzed hydrolysis of the acetate donor. Conditions were established for use of the assay in microtiter plate format, and its applicability was demonstrated by determining the first Michaelis–Menten kinetic parameters for PatB. The assay is readily amenable for application in the high-throughput screening for potential inhibitors of peptidoglycan O-acetyltransfer- ases that may prove to be leads for novel classes of antibiotics. Ó 2013 Elsevier Inc. All rights reserved. The peptidoglycan (PG) 1 sacculus completely surrounds the cytoplasmic membrane of nearly all bacterial cells to maintain its integrity (the exceptions are the mycoplasmas). As such, PG repre- sents the major structural element essential to bacteria. PG is a het- eropolymer of alternating N-acetylglucosaminyl (GlcNAc) and N- acetylmuramoyl (MurNAc) residues linked b-(1–4) and short ‘‘stem’’ peptides that are attached to the lactyl moiety of each MurNAc res- idue (Fig. 1). The existence of the peptides permits cross-linking be- tween neighboring glycan strands to provide the continuous three- dimensional PG sacculus. Given its importance to bacterial cell via- bility, PG is the target of the lytic enzymes associated with the innate immune systems of host cells such as lysozymes (muramidases). To protect from this lysis, many bacteria decorate their PG with simple aglycon moieties, particularly acetate (recently reviewed in Refs. [1,2]). The O-acetylation of PG occurs at the C-6 hydroxyl group of muramoyl residues (Fig. 1). This modification is performed by many human pathogens, both gram positive and gram negative, such as Staphylococcus aureus and species of Campylobacter, Helico- bacter, Neisseria, and Bacillus, including Bacillus anthracis [1–3]. The extent of O-acetylation varies with species and strain, as well as culture condition, and ranges between 20% and 70% (relative to MurNAc residues). These levels are significant because the activity of lysozyme is inhibited in a concentration-dependent manner [4– 8] through steric hindrance that precludes productive binding of O- acetylated PG. With B. anthracis, spore PG is heavily O-acetylated [9] in addition to the PG of vegetative cells, as is the PG of VBNC (viable but non-culturable) cells of Enterococcus faecalis [10]. Pre- sumably, this O-acetylation helps to protect these different cell forms from degradation and thereby contributes to the persistence of the potential pathogens. In addition to protecting cells from lysis, O-acetylation leads to persistence of high-molecular-weight fragments of PG circulating within mammalian hosts, thereby causing serious pathobiological effects such as complement activation, pyrogenicity, somnogene- sis, and arthrogenicity (reviewed in Refs. [1,2]). From the physio- 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.04.022 ⇑ Corresponding author. Fax: +1 519 837 1802. E-mail address: a.clarke@exec.uoguelph.ca (A.J. Clarke). 1 Abbreviations used: PG, peptidoglycan; GlcNAc, N-acetyl-D-glucosamine; MurNAc, N-acetyl-D-muramic acid; Oat, O-acetylpeptidoglycan transferase; CoA, coenzyme A; Pat, peptidoglycan O-acetyltransferase; SDS, sodium dodecyl sulfate; IPTG, isopropyl b-D-1-thiogalactopyranoside; SPE, solid phase extraction; NTA, nitrilotriacetic acid; pNP-Ac, p-nitrophenyl acetate; DP, degree of polymerization; DTNB, 5,5 0 -dithiobis-(2- nitrobenzoic acid); HPLC, high-performance liquid chromatography; TFA, trifluoro- acetic acid; MALDI–TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry. Analytical Biochemistry 439 (2013) 73–79 Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio