Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence

Abstract

This paper describes a highly sensitive new method for the identification of penicillin-binding proteins (PBPs) that is based on the use of an ampicillin-digoxigenin conjugate (DIG-AMP conjugate) which is detected by immunoblotting and chemiluminescence. The sensitivity of chemiluminescence permitted X-ray film exposure times to be decreased to minutes, as opposed to the days or weeks which are requisite when conventionally radiolabeled P-lactams are used. Coupling of ampicillin to digoxigenin yielded a product containing digoxigenin (detected by chemiluminescence) which also was inhibitory for Staphylococcus aureus and Escherichia coli. Unconjugated digoxigenin at concentrations of up to 100 ,ug/ml was not inhibitory for either organism. For S. aureus the MICs of DIG-AMP (0.7 ,ug of conjugated ampicillin per ml) and of free ampicillin (0.5 ,ug/ml) were comparable, indicating that ampicillin retained its bioactivity when coupled to digoxigenin. However, for E. coli the MICs of DIG-AMP (70 ,ug of conjugated ampicillin per ml) and of free ampicillin (8 ,ug/ml) Penicillin-binding proteins (PBPs) are enzymes involved in the terminal biosynthetic reactions of the peptidoglycan layer of the bacterial cell wall. These enzymes, some of which are the lethal targets for penicillins and other P-lactam antimicrobial agents, are involved in various aspects of cell growth, cell division, and the maintenance of cell shape (6,26,27,29).Previous studies to examine the interactions between PBPs and 1-lactam drugs have relied primarily on the use of radiolabeled compounds (10,25,28,29). However, there are major disadvantages associated with the use of radiolabeled 3-lactams for this purpose: (i) they typically are expensive, and some forms are unstable, (ii) they require extended periods for autoradiography or fluorography, and (iii) there are substantial hazards associated with their handling as well as increasing concerns regarding disposal and storage of radioactive waste. To circumvent these problems, we developed a method to conjugate digoxigenin to the free amino group of ampicillin. Following incubation of either cell envelopes or viable bacteria with the conjugate, PBPs were detected by immunoblotting and chemiluminescence. This new procedure should have broad applicability as a rapid and highly sensitive nonradioactive alternative for investigating the interactions of ,-lactams and their targets in bacterial cells. MATERIALS AND METHODSChemical reagents. Benzylpenicillin, sodium ampicillin, Nlauroylsarcosine (Sarkosyl), and dimethylformamide were ob-* Corresponding author. Mailing address:

The Leptospiral Major Outer Membrane Protein LipL32 Is a Lipoprotein Expressed during Mammalian Infection

“…E. coli INV?Ј cells harboring pRZ1132K or pCRII were grown to stationary phase in DYT broth (24). Cells were harvested by centrifugation, and membrane and cytoplasmic fractions were prepared according to the method of Weigel et al (38).…”

Penicillin-Binding Proteins in Leptospira interrogans

“…Pellets of L. interrogans and E. coli harboring plasmids containing the pbpB and ponA genes and their vector controls were suspended in phosphate-buffered saline and sonicated. Aliquots of the sonicated cells (100 g of protein) were incubated at 37°C for 10 min with 2.5 g of AMP per ml conjugated to digoxigenin (DIG) as described by Weigel et al (22). Of each sample, 12.5 g was resolved by SDS-PAGE; PBPs were identified by immunoblotting with an anti-DIG-alkaline phosphatase conjugate (Boehringer Mannheim Corp., Indianapolis, Ind.)…”

Crystal Structure of the 47-kDa Lipoprotein of Treponema pallidum Reveals a Novel Penicillin-binding Protein

“…␤-Lactam Binding to Tp47-Binding of digoxigenin-labeled ampicillin (Dig-Amp) to Tp47 was determined by a chemiluminescent detection method (13,20); the use of Dig-Amp circumvents problems associated with utilizing radiometric methods for assaying ␤-lactam binding (20). ␤-Lactam binding to Tp47 also was examined by ESI-MS; in these experiments, a typical 100-l reaction mixture contained 100 g of protein, 2 mM ZnCl 2 , and 2 mM ␤-lactam (in buffer A) and was incubated at 37°C for various times.…”