Penicillin‐Binding Protein Imaging Probes

Ozden Kocaoglu1, Erin E. Carlson1

1 Indiana University, Bloomington, Indiana
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130102
Online Posting Date:  December, 2013
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Abstract

ABSTRACT

Penicillin‐binding proteins (PBPs) are membrane‐associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β‐lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, the apparent functional redundancy of the ∼5 to 15 proteins that most bacteria possess makes determination of their individual roles difficult. Existing techniques to study PBPs are not ideal because they do not directly visualize protein activity and can suffer from artifacts. Therefore, development of new methods for studying the roles of distinct PBPs in cell wall synthesis was compulsory. Due to penicillin's covalent mode of inhibition, fluorophore‐conjugated analogs can be utilized to visualize PBP activity. Herein, we describe a general protocol to label and detect subsets of active PBPs in live, Gram‐positive bacteria using fluorescent β‐lactams. Curr. Protoc. Chem. Biol. 5:239‐250 © 2013 by John Wiley & Sons, Inc.

Keywords: penicillin‐binding proteins; peptidoglycan; fluorescence imaging; activity‐based probes; β‐lactams

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: In‐Gel Detection of in Vivo–Labeled PBPs with β‐Lactam Probes
  • Basic Protocol 2: Fluorescence Imaging of Probe‐Labeled Proteins
  • Support Protocol 1: Dual Labeling with Ceph C‐T and Boc‐FL
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In‐Gel Detection of in Vivo–Labeled PBPs with β‐Lactam Probes

  Materials
  • B. subtilis PY79 cells (gift from Daniel B. Kearns, Department of Biology, Indiana University–Bloomington) grown in Luria‐Bertani (LB) broth at 37°C with shaking at 150 rpm to OD 600 0.4 to 0.5
  • S. pneumoniae IU1945 cells (gift from Malcolm E. Winkler, Department of Biology, Indiana University–Bloomington) grown statically in brain heart infusion broth (BHI; BD Difco) at 37°C in an atmosphere of 5% CO 2 to OD 620 0.15 to 0.20
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.4
  • 2 mg/ml Ceph C‐T probe stock (Kocaoglu et al., ) in DMSO (store at −80°C, protected from light; stable for months)
  • 1 mg/ml Boc‐FL probe stock (Invitrogen, cat. no. B‐13233) in DMSO (store at −80°C, protected from light; stable for months)
  • 10 mg/ml lysozyme stock solution (see recipe)
  • 4× SDS‐PAGE gel loading buffer (see recipe)
  • 10% resolving and 4.5% stacking SDS‐PAGE gel (see recipe)
  • Fluorescent molecular weight standards (see recipe)
  • 10× Tris‐glycine SDS running buffer (Invitrogen, cat. no. LC2675‐5)
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge (e.g., Eppendorf 5424)
  • Branson sonifier 250 with 1/8 in. tapered microtip
  • NanoDrop 1000 spectrophotometer (Thermo Scientific)
  • Analog heat block (VWR)
  • XCell SureLock mini‐cell gel electrophoresis system (Invitrogen, cat. no. EI0001)
  • Typhoon 9210 gel scanner (Amersham Biosciences) with 580‐nm band‐pass filter and 526‐nm short‐pass filter
  • ImageJ software (NIH) for gel data analysis
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, )

Basic Protocol 2: Fluorescence Imaging of Probe‐Labeled Proteins

  Materials
  • B. subtilis PY79 cells grown in Luria‐Bertani (LB) broth at 37°C with shaking at 150 rpm to OD 600 0.4 to 0.5
  • S. pneumoniae IU1945 cells grown statically in brain heart infusion (BHI) broth (BD Difco) at 37°C in an atmosphere of 5% CO 2 to OD 620 0.15 to 0.20
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.4
  • 2 mg/ml Ceph C‐T probe stock (Kocaoglu et al., ) in DMSO (store at −80°C, protected from light; stable for months)
  • 1 mg/ml Boc‐FL probe stock (Invitrogen, cat. no. B‐13233) in DMSO (store at −80°C, protected from light; stable for months)
  • 100 µg/ml 4′, 6‐diamidino‐2‐phenylindole, dihydrochloride (DAPI; Invitrogen, cat. no. D1306) in Milli‐Q water (store at −80°C, protected from light; stable for months)
  • 0.1% (w/v) poly‐L‐lysine in H 2O (Sigma‐Aldrich, cat. no. P8920)
  • Immersion oil with a 1.512 refractive index
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge (e.g., Eppendorf 5424)
  • Micro cover glasses: 22 × 22 mm, 1.5 thickness (VWR, cat. no. 48366‐227)
  • Glass microscope slides: 25 × 75 mm, 1.0 mm thick (VWR, cat. no. 48300‐025)
  • OMX 3‐D‐structured illumination microscopy (SIM) super‐resolution microscope (Applied Precision)
  • Imaging software: SoftWoRx (Applied Precision) and Imaris 3‐D (Bitplane) for OMX 3‐D‐SIM
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Figures

Videos

Literature Cited

Literature Cited
   Dargis, M. and Malouin, F. 1994. Use of biotinylated beta‐lactams and chemiluminescence for study and purification of penicillin‐binding proteins in bacteria. Antimicrob. Agents Chemother. 38:973‐980.
   Fisher, J.F. , Meroueh, S.O. , and Mobashery, S. 2005. Bacterial resistance to beta‐lactam antibiotics: Compelling opportunism, compelling opportunity. Chem. Rev. 105:395‐424.
   Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 97:10.2A.1‐10.2A.44.
   Galleni, M. , Lakaye, B. , Lepage, S. , Jamin, M. , Thamm, I. , Joris, B. , and Frere, J.M. 1993. A new, highly sensitive method for the detection and quantification of penicillin‐binding proteins. Biochem. J. 291:19‐21.
   Georgopapadakou, N.H. and Liu, F.Y. 1980. Penicillin‐binding proteins in bacteria. Antimicrob. Agents Chemother. 18:148‐157.
   Hao, Z. , Hong, S. , Chen, X. , and Chen, P.R. 2011. Introducing bioorthogonal functionalities into proteins in living cells. Acc. Chem. Res. 44:742‐751.
   Kleppe, G. and Strominger, J.L. 1979. Studies of the high molecular weight penicillin‐binding proteins of Bacillus subtilis . J. Biol. Chem. 254:4856‐4862.
   Kocaoglu, O. , Calvo, R.A. , Sham, L.T. , Cozy, L.M. , Lanning, B.R. , Francis, S. , Winkler, M.E. , Kearns, D.B. , and Carlson, E.E. 2012. Selective penicillin‐binding protein imaging probes reveal substructure in bacterial cell division. ACS Chem. Biol. 7:1746‐1753.
   Lippincott‐Schwartz, J. and Patterson, G.H. 2003. Development and use of fluorescent protein markers in living cells. Science 300:87‐91.
   Macheboeuf, P. , Contreras‐Martel, C. , Job, V. , Dideberg, O. , and Dessen, A. 2006. Penicillin binding proteins: Key players in bacterial cell cycle and drug resistance processes. FEMS Microbiol. Rev. 30:673‐691.
   Sauvage, E. , Kerff, F. , Terrak, M. , Ayala, J.A. , and Charlier, P. 2008. The penicillin‐binding proteins: Structure and role in peptidoglycan biosynthesis. FEMS Microbiol. Rev. 32:234‐258.
   Scheffers, D.J. , Jones, L.J. , and Errington, J. 2004. Several distinct localization patterns for penicillin‐binding proteins in Bacillus subtilis . Mol. Microbiol. 51:749‐764.
   Spratt, B.G. and Pardee, A.B. 1975. Penicillin‐binding proteins and cell shape in E. coli . Nature 254:516‐517.
   Vollmer, W. , Blanot, D. , and de Pedro, M.A. 2008. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32:149‐167.
   Zhao, G. , Meier, T.I. , Kahl, S.D. , Gee, K.R. , and Blaszczak, L.C. 1999. BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin‐binding proteins. Antimicrob. Agents Chemother. 43:1124‐1128.
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