Role of penicillin-binding protein 2 (PBP2) in the antibiotic susceptibility and cell wall cross-linking of Staphylococcus aureus: evidence for the cooperative functioning of PBP2, PBP4, and PBP2A.
Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus, was used to select a spontaneous resistant mutant of S. aureus strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The pbpB gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of pbpD, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of pbpD also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne mecA gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native S. aureus transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.
Łeski
Tomasz A
TA
The Rockefeller University, 1230 York Ave., New York, NY 10021, USA.
Tomasz
Alexander
A
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015296:Ceftizoxime / Q000494:pharmacology; D002473:Cell Wall / Q000502:physiology*; D024881:Drug Resistance, Bacterial / Q000502:physiology; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D046915:Penicillin-Binding Proteins / Q000502:physiology*; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000502:physiology*
D000900:Anti-Bacterial Agents; D046915:Penicillin-Binding Proteins; D015296:Ceftizoxime
J Bacteriol. 1999 Dec;181(24):7566-70
10601215
J Biol Chem. 1999 Jul 2;274(27):18942-6
10383392
J Bacteriol. 2001 Apr;183(8):2417-24
11274099
Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10886-91
11517340
J Bacteriol. 2001 Nov;183(22):6525-31
11673420
J Biol Chem. 2004 Jan 30;279(5):3398-407
14613936
Microb Drug Resist. 2003 Winter;9(4):329-36
15000739
Nature. 1970 Aug 15;227(5259):680-5
5432063
Eur J Biochem. 1981 Oct;119(2):389-93
7308191
Antimicrob Agents Chemother. 1987 Sep;31(9):1423-5
3499862
Antimicrob Agents Chemother. 1991 Jan;35(1):124-9
2014967
J Biol Chem. 1992 Jun 5;267(16):11248-54
1597460
J Bacteriol. 1992 Aug;174(15):4952-9
1321119
FEMS Microbiol Lett. 1994 Apr 1;117(2):131-6
8181715
Microbiology. 1996 Aug;142 ( Pt 8):1911-8
8760905
J Biol Chem. 1997 Nov 14;272(46):29053-9
9360979
J Bacteriol. 1998 May;180(10):2759-65
9573165
J Bacteriol. 1998 Dec;180(23):6077-81
9829914
Microbiol Mol Biol Rev. 1998 Dec;62(4):1079-93
9841666
J Bacteriol. 2000 Feb;182(4):1074-9
10648534