Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A.

B.L De Jonge,Y.S Chang,D Gage,A Tomasz

doi:10.1016/s0021-9258(19)49903-1

Abstract

All clinical isolates of methicillin-resistant Staphylococcus aureus contain an extra penicillin binding protein (PBP) 2A in addition to four PBPs present in all staphylococcal strains. This extra PBP is thought to be a transpeptidase essential for the continued cell wall synthesis and growth in the presence of beta-lactam antibiotics. As an approach of testing this hypothesis we compared the muropeptide composition of cell walls of a highly methicillin-resistant S. aureus strain containing PBP2A and its isogenic Tn551 derivative with reduced methicillin resistance, which contained no PBP2A because of the insertional inactivation of the PBP2A gene. Purified cell walls were hydrolyzed into muropeptides which were subsequently resolved by reversed-phase high-performance liquid chromatography and identified by chemical and mass spectrometric analysis. The peptidoglycan composition of the two strains were identical. Both peptidoglycans were highly cross-linked mainly through pentaglycine cross-bridges, although other, chemically distinct peptide cross-bridges were also present including mono-, tri-, and tetraglycine; alanine; and alanyl-tetraglycine. Our experiments provided no experimental data for a unique transpeptidase activity associated with PBP2A.

Highlights

From the $Laboratoryof Microbiology, Rockefeller University, NewYork, New York 10021 and $Department of Biochemistry, Michigan State University-National Institutes of Health Mass Spectrometry Facility, Michigan State University, East Lansing, Michigan 48824
All clinical isolates of methicillin-resistant Staphy- mec gene indicates the presence of structural motifs characlococcus aureus containan extra penicillinbinding teristic of peptidoglycan transpeptidases [8].it is protein (PBP) 2A in addition to four PBPs present in generallyassumed, there niso experimental evidence all staphylococcal strains
Enzymatic hydrolysates of peptidoglycan were analyzed by Methicillin-resistant strains of Staphylococcus aureus a combination of reversed-phase high-performance liquid (MRSA)' have becomemost important nosocomial pathogens chromatography (HPLC) and fast atom bombardment mass worldwide [1].While the mechanism of resistance of these spectrometry, enabling us to assign chemical structures to 16 strains is not fully understood, there is general agreement muropeptide peaks

Summary

EXPERIMENTAL PROCEDURES

Tibiotics with low affinity [5,6,7]. analysis of the cloned Strains andGrowth Conditions-All strains listed in Table I were grown in tryptic soy broth (Difco,Detroit, MI) at 37 "Cwith aeration. The cells were harvested by centrifugation for 10min at 16,000 X g (4 "C) and subsequently transferred into 4% (final concentration) boiling sodium dodecyl sulfate (SDS).The cells were boiled for 0.5 h and the cell walls were concentrated by centrifugation for 10 min at 30,000g. The walls were washed with water until no more '10021.Tel.: 212-570-8277F;ax: 212-570-8688. The abbreviations used are: MRSA, methicillin-resistant S. au- broken with glass beads (0.2mm) on a Vortex a t maximal speed at reus; PBP, penicillin binding protein; HPLC, high-performance liquid 4 "C for 15 min. The suspension was centrifuged at 2,000X g, and chromatography; SDS, sodium dodecyl sulfate; FAB, fast atom bom- after removal of the supernatant, the pellet was treated with glass bardment; MS, mass spectrometry; MIC, minimum inhibitory com- beads as described above. At 40,000X g (15min), and thepellet was treated a t 37 "C in 100 mM stance Origin

Yes No

MuropeptideComposition of theHighlyResistant Strain

Muropeptide compositionb

In n