Penicillin-Binding Protein 5 and Expression of Ampicillin Resistance in Enterococcus faecium

Abstract

We report a structural and transcriptional analysis of the pbp5 region of Enterococcus faecium C68. pbp5 exists within a larger operon that includes upstream open reading frames (ORFs) corresponding to previously reported psr (penicillin-binding protein synthesis repressor) and ftsW (whose product is a transmembrane protein that interacts with PBP3 in Escherichia coli septum formation) genes. Hybridization of mRNA from C68, CV133, and four ampicillin-resistant CV133 mutants revealed four distinct transcripts from this region, consisting of (i) E. faecium ftsW (ftsW Efm ) alone; (ii) psr and pbp5; (iii) pbp5 alone; and (iv) ftsW Efm , psr, and pbp5. Quantities of the different transcripts varied between strains and did not always correlate with quantities of PBP5 or levels of ampicillin resistance. Since the psr of C68 is presumably nonfunctional due to an insertion of an extra nucleotide in the codon for the 44th amino acid, the region extending from the ftsW Efm promoter through the pbp5 gene of C68 was cloned in E. coli to facilitate mutagenesis. The psr ORF was regenerated using site-directed mutagenesis and introduced into E. faecium D344-SRF on conjugative shuttle vector pTCV-lac (pCWR558 [psr ORF interrupted]; pCWR583 [psr ORF intact]). Ampicillin MICs for both D344-SRF-(pCWR558) and D344-SRF(pCWR583) were 64 g/ml. Quantities of pbp5 transcript and protein were similar in strains containing either construct regardless of whether they were grown in the presence or absence of ampicillin, arguing against a role for PSR as a repressor of pbp5 transcription. However, quantities of psr transcript were increased in D344-SRF(pCWR583) compared to D344-SRF(pCWR558), especially after growth in ampicillin; suggesting that PSR acts in some manner to activate its own transcription.Penicillin resistance in Enterococcus faecium is associated with production of low-affinity penicillin-binding protein PBP5. The presence of this penicillin-binding protein (PBP) in virtually all clinical E. faecium strains that have been investigated (including those susceptible to clinically achievable levels of penicillin [L. B. Rice, unpublished data]) suggests that it is intrinsic to this species, rather than an acquired gene. Supportive evidence for the role of PBP5 in penicillin resistance is derived from experiments indicating that PBP5-expressing cells replicate when incubated with penicillin at concentrations sufficient to saturate all of the other PBPs, as well as from studies demonstrating that E. faecium strains lacking PBP5 are highly susceptible to penicillin (10-12, 25, 26).Early studies on Enterococcus hirae 9790 (which until 1985 was considered to be a type strain for Enterococus faecalis [8,16]) reported that elevated levels of penicillin resistance (to ca. 64 g/ml) were associated with increased quantities of detectable PBP5. Increased PBP5 production in one resistant mutant (R40) was associated with deletion of the N-terminal portion and some upstream DNA of an open reading frame (ORF) located ca. 1 kb upstream of the pbp5...

Molecular Characterization of Ampicillin-Resistant Enterococcus faecium Isolates from Hospitalized Patients in Norway

“…We also found statistically significant associations between point mutations in ampicillin-resistant and -susceptible isolates in positions that earlier have been related to ampicillin resistance, such as 485M3T, 496N3K, 499A3T, 525E3D, 586V3L, and 629E3V. However, recent studies have indicated that specific point mutations alone do not entirely explain the differences in levels of resistance and that the mechanisms by which ampicillin resistance is expressed are more complex (23,26).…”

“…Ampicillin resistance in E. faecium is associated with the production of low-affinity penicillin-binding protein 5 (PBP5). High-level ampicillin resistance may be due to either increased expression of PBP5 or alterations in the pbp5 gene resulting in lower affinities for ampicillin (23,26).…”

Membrane Topology and DNA-Binding Ability of the Streptococcal CpsA Protein

“…The PSR protein, originally analyzed for Enterococcus hirae, was found to be a repressor for penicillin-binding protein 5 (PBP5) as well as a regulator of other cell surface-related processes (18,28). However, a subsequent report on PSR in Enterococcus faecium demonstrated that neither repressor or activator activities were associated with control of PBP5 but suggested that PSR may activate its own transcription during growth in the presence of ampicillin (40). Expression analysis of the gene encoding MsrR in Staphylococcus aureus revealed transcriptional activation in the presence of multiple cell wall-targeted antimicrobials, including vancomycin and lysostaphin (41).…”

Biochemistry and Comparative Genomics of SxxK Superfamily Acyltransferases Offer a Clue to the Mycobacterial Paradox: Presence of Penicillin-Susceptible Target Proteins versus Lack of Efficiency of Penicillin as Therapeutic Agent

“…The operon also contains a gene that codes for a Psr protein first identified as a possible repressor of transcription. Recent developments argue against the possibility that Psr plays such a role in E. faecium (196) and E. faecalis (58).…”