Journal of Bacteriology, March 2004, p . 1221-1228, Vol . 186, No . 5

Role of Class A Penicillin-Binding Proteins in PBP5-Mediated ß-Lactam Resistance in Enterococcus faecalis

Ana Arbeloa,¹ Heidi Segal,^1, Jean-Emmanuel Hugonnet,¹ Nathalie Josseaume,¹ Lionnel Dubost,² Jean-Paul Brouard,² Laurent Gutmann,¹ Dominique Mengin-Lecreulx,³ and Michel Arthur^1*

INSERM E0004-LRMA, Université Paris VI, 75270 Paris,¹ Département Régulations, Développement et Diversité Moléculaire, Museum National d'Histoire Naturelle, USM0502-CNRS UMR8041, 75005 Paris,² Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université Paris-Sud, 91405 Orsay, France³

Received 21 July 2003/ Accepted 21 November 2003

 
Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins [PBP] of classes A and B that associatea conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively . In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of ß-lactam antibiotics,such as ceftriaxone . To identify the glycosyltransferase partner[s]of PBP5, combinations of deletions were introduced in all threeclass A PBP genes of E . faecalis JH2-2 [ponA, pbpF, and pbpZ]. Among mutants with single or double deletions, only JH2-2 ponA pbpF was susceptible to ceftriaxone . Ceftriaxone resistancewas restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferaseof Staphylococcus aureus . Thus, PBP5 partners essential forpeptidoglycan polymerization in the presence of ß-lactamsformed a subset of the class A PBPs of E . faecalis, and heterospecificcomplementation was observed with an ortholog from E . faecium.Site-directed mutagenesis of pbpF confirmed that the catalyticserine residue of the transpeptidase module was not requiredfor resistance . None of the three class A PBP genes was essentialfor viability, although deletion of the three genes led to anincrease in the generation time and to a decrease in peptidoglycan cross-linking . As the E . faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase . The latter enzyme was not inhibited by moenomycin, since deletion of thethree class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

 
High-molecular-weight penicillin-binding proteins [PBPs] arethe main determinants of clinically relevant ß-lactamresistance phenotypes in streptococci, staphylococci, and enterococci,although the genetic basis for resistance differs in these bacteria.Resistant isolates of Streptococcus pneumoniae harbor multiplemosaic PBP genes generated by recombination with gene fragmentsacquired from related streptococci by natural transformation[19] . Methicillin-resistant Staphylococcus aureus [MRSA] strains have acquired an additional PBP gene [mecA] encoding PBP2a, which is sufficient for high-level resistance to virtually all ß-lactams in the absence of modification of otherPBPs [34] . Since MRSA strains grow in the presence of ß-lactamantibiotics at concentrations sufficient to rapidly saturateall PBPs except PBP2a, it is assumed that the latter enzymeis sufficient for peptidoglycan cross-linking [26] . Similarly,Enterococcus faecalis and Enterococcus faecium strains producePBP5 that appears sufficient for transpeptidation in the presenceof ß-lactams [7, 29, 30] . Resistance is an intrinsicproperty of these species, as virtually all isolates are resistantto moderate [e.g., ampicillin] or high [e.g., ceftriaxone] levelsof ß-lactams and produce a species-specific PBP5 [24].Acquisition of high-level resistance to ampicillin, mainly inclinical isolates of E . faecium, results from overproductionof PBP5 [12, 13, 29] and from amino acid substitutions thatdecrease the interaction of PBP5 with the drug [27, 35] . Alterationof other as-yet-unknown accessory factors is also involved [18,29].

The high-molecular-weight PBPs fall into two classes based onthe association of the conserved C-terminal transpeptidase modulewith an N-terminal glycosyltransferase module [class A] or amorphogenesis module [class B] devoid of any known catalyticactivity [14] . As the S . aureus PBP2a and the enterococcal PBP5are class B PBPs, peptidoglycan polymerization in the presenceof high concentrations of ß-lactams is thought torequire cooperation between the D,D-transpeptidase module of these PBPs and the glycosyltransferase module of class A PBPs. Evidence for such cooperation has been obtained in MRSA strainsbased on selective inactivation by site-directed mutagenesisof the glycosyltransferase activity of PBP2, which led to aviable mutant susceptible to methicillin [26] . Polymerizationof the glycan chains in the mutant was presumably catalyzedby a monofunctional glycosyltransferase [Mgt], as PBP2 is thesole class A PBP produced by S . aureus [26, 32] . The sets ofpeptidoglycan-polymerizing enzymes of S . aureus and enterococciare significantly different, since the genome of E . faecalisharbors three putative class A PBP genes, no homologue of mgt,and three putative class B PBP genes [Table 1] . To evaluatethe role of the three class A PBP genes of E . faecalis in intrinsicß-lactam resistance, we have developed a method toconstruct multiple deletions in the chromosome of this bacterium.We report the deletion of all combinations of one to three classA PBP genes and their impact on bacterial growth, peptidoglycancross-linking, and susceptibility to cell wall synthesis inhibitors.

 
Growth conditions. Bacterial strains were grown in brain heart infusion [BHI] brothor agar [Becton Dickinson, le Pont de Claix, France] at 37°C.MICs of ampicillin [Bristol-Myers, Paris, France], ceftriaxone[Laboratoires Roche, Neuilly, France], and moenomycin [Hoechst,Mainz, Germany] were determined with 10⁵ CFU per spot on BHIagar after 48 h of incubation.

Replication and transfer properties of suicide vector pHS1. The vector pHS1 was constructed to introduce serial deletionsin the chromosome of E . faecalis JH2-2 [17] by homologous recombination.This vector [Fig . 1A] is composed of [i] the origin of replicationand the repA[Ts] gene encoding the thermosensitive replicationprotein of plasmid pGhost4 [20], [ii] the aph3'-aac6" bifunctionalgene of plasmid pAT392 [2] conferring resistance to all aminoglycosides, including gentamicin, and [iii] the origin of transfer of transposon Tn916 [oriT_Tn916] allowing conjugal transfer between gram-positivebacteria provided that the donor harbors Tn916 [8] . The vectorpHS1 and derivatives were propagated at 37°C in Escherichiacoli EC101 [5] with selection for gentamicin resistance [16 µg/ml] . The plasmids were introduced into E . faecalis JH2Sm::Tn916 [8] by electroporation [10] and maintained in thishost at permissive temperature for replication [28°C] inmedia containing gentamicin [128 µg/ml] . Plasmid pHS1 and derivatives were transferred by mating from JH2Sm::Tn916 [resistant to tetracycline and streptomycin] to JH2-2 [resistant to rifampin and fusidic acid] . Transconjugants were selectedon BHI agar containing rifampin [40 µg/ml], fusidic acid[20 µg/ml], and gentamicin [128 µg/ml] . Typicaltransfer frequencies were in the order of 10^-4 per donor.

 
Recombination events generating chromosomal deletions. DNA fragments [ca . 500 bp] flanking the sequence targeted fordeletion, designated H1 and H2, were independently amplifiedby PCR . The H1 and H2 fragments, separated by an erythromycinresistance cassette [erm], were cloned into pHS1 . The resultingpHS1 derivatives carrying the H1-erm-H2 insertions were introducedinto E . faecalis JH2-2 as described above . Replacement of thesequence targeted for deletion by the erm cassette by a doublecrossover [Fig. 1C] was selected at the nonpermissive temperature for plasmid replication [42°C] on media containing erythromycin[10 µg/ml].

The erm resistance cassette was removed from the chromosomal pbp loci by using derivatives of pHS1 carrying H1 directly fused to H2 [Fig . 1D] . Chromosomal integration of the plasmids bya single crossover was obtained by selecting for gentamicin resistance [128 µg/ml] at nonpermissive temperature forplasmid replication . Plasmid excision was obtained by subculturingclones in the absence of antibiotic at 28°C, as the activityof the Rep[Ts] protein at permissive temperature was reportedto stimulate homologous recombination [5] . The excised plasmids were cured at 42°C, and clones susceptible to gentamicinand erythromycin were screened by replica plating.

Amplification of H1 and H2 sequences. The following pairs of oligonucleotides were used to amplifyby PCR the H1 and H2 sequences flanking ponA, pbpF, pbpZ, andpbp5: ponA H1, 5'-TTATCCCAAACGAAGTG-3' and 5'-AGATCTGTGTTGGATGCATGTCT-3'; ponA H2, 5'-AGATCTGCAACCACCTGAAAGTAG-3' and 5'-TTGTGGGCTTAGAAGATG-3';pbpF H1, 5'-TTAAGGTGACACAATCG-3' and 5'-AGATCTTTGTCCATAGTACTCCC-3';pbpF H2, 5'-AGATCTTGGGACAAATTAAAGACG-3' and 5'-TATCACGCACAGGAGTC-3';pbpZ H1, 5'-TGGATCACCAATCATGC-3' and 5'-AGATCTCAAAAGCTTCACCTCA-3';pbpZ H2, 5'-AGATCTATTACGCTTCTTACTGG-3' and 5'-GTTGGTGTGGTATTATC-3'.BglII restriction sites [underlined] were used to ligate theH1, H2, and erm fragments as shown in Fig . 1C and D.

The H1-H2 DNA fragment used for deletion of pbp5 was constructed by two sequential amplifications with partially complementary primers as previous described [1] . In the first step, the H1 and H2 fragments were separately amplified [primers H1F, 5'-AGAATCATTTTTGACTG-3',and H1R, 5'-CAAATGGTTCGCTGGGTTTCAATAATCCCCTAAC-3', for H1; primersH2Fb, 5'-ACCCAGCGAACCATTTGAAAAGAGAAAATGAACG-3', and H2R, 5'-AGGGAAATATGTTGGTC-3',for H2] . Seventeen bases of primers H1R and H2Fb were complementary[underlined] . In the second step, the H1 and H2 fragments weredenatured, annealed, and coamplified with primers H1F and H2R[see above] . The same method was used to obtain the H1-erm-H2fragment with primers H1F, H1R, H2R, and H2F . Primers H1R [seeabove] and H2F [5'-GTAAGTTAAGGGACTGCAAAAGAGAAAATGAACG-3'] containedsequences complementary to the erm resistance cassette [italicized].

Analysis of the structure of the deletions. Genomic DNA was prepared [Wizard genomic DNA purification kit;Promega, Madison, Wis.], separately digested with AccI and XmnI,except for pbp5 [digested with PstI and SspI], and analyzed by Southern blot hybridization . The probes were obtained by labeling DNA of derivatives of plasmid pCRblunt [Invitrogen, Carlsbad, Calif.] harboring the H1 and H2 sequences with [-³²P]dCTP [Megaprime DNA labeling system; Amersham Biosciences, Little Chalfont, England] . For each of the four PBP genes [ponA, pbpF, pbpZ, and pbp5], the hybridization patterns corresponded to the predicted map of the wild-type locus, pbp replaced by erm, and deletion of the gene [data not shown] . In addition, PCRwas performed with oligonucleotides adjacent to the H1 and H2sequences, to confirm the reduction in size of the PCR productsresulting from gene replacement and deletion . Finally, the presenceof the fused H1 and H2 fragments in the chromosome of the mutantswas verified by direct sequencing of the PCR products.

Properties of expression vector pNJ2. Plasmid pNJ2 was constructed to obtain expression of clonedgenes under the control of the heterologous aphA-3p promoter,which is active in E . faecalis [3] . This vector [Fig . 1B] is composed of [i] plasmid pAT28 that replicates both in E . coli [oriR pUC] and gram-positive bacteria [oriR pAMß1], and confers resistance to spectinomycin [31], [ii] the chloramphenicolacetyltransferase gene [cat] and the aphA-3p promoter of plasmidpAT79 [3], and [iii] the origin of transfer of transposon Tn916[oriT_Tn916] [8] . The vector pNJ2 and its derivatives were introduced into E . faecalis JH2Sm::Tn916 by electroporation and transferredby conjugation to derivatives of E . faecalis JH2-2 [frequencyof ca . 10^-4 per donor] . The plasmids were selected with spectinomycin[60 µg/ml] and chloramphenicol [20 µg/ml] . Rifampin[40 µg/ml] and fusidic acid [20 µg/ml] were added to the media for selection of the E . faecalis JH2-2 recipients.

Shuttle plasmids for pbp and mgt expression in E . faecalis. The pbp5 open reading frame and ribosome binding site of E.faecalis JH2-2 [pbp5_fs] was amplified with primers n-PB1 [5'-ATAGGTGAAACACAAGC-3']and PB2 [5'-ACAGAAACCTGTTTCG-3'] and cloned under the controlof the aphA-3p promoter to generate pNJ2pbp5_fs . The same approachwas used to express the pbpF genes from E . faecalis JH2-2 [pbpF_fs]and E . faecium D344S [22] [pbpF_fm] and the mgt gene of S . aureusNCTC 8325 [mgt_Sa] . The following primers containing SacI andXbaI sites [underlined] were used for amplification: pbpF_fs, primers pbpF1S [5'-GGTGGTGAGCTCTAGACTTAGCCAAGAAACG-3'] and PBPF4S [5'-GGTGGTCTGCAGTCTAGACAACTAATTTCCTAATAAG-3']; pbpF_fm, primersD344-F-1 [5'-TTGAGCTCACTACAACTTAAGCAGGA-3'] and D344-F-2 [5'-TTTCTAGAGTAGTTACTCTCTATTGT-3'];mgt_Sa,primers MGT1 [5'-TTGAGCTCAAGGTATATACTAAGTGAG-3'] andMGT2 [5'-TTTCTAGAGCAAGTATTTAACGATTTAA-3'] . DNA sequencing was performed for all recombinant plasmids used in this study to confirm the accuracy of the PCR.

Site-directed mutagenesis of E . faecalis pbpF. The codon specifying the catalytic serine residue [TCG, Ser₄₀₂] was replaced by a GGA glycine codon . The 5' portion of pbpF_fs was amplified with pbpF1S [see above] and PBPF2S [5'-GGTGGTGGATCCGCCTGGTGAACGTTTTGTT-3'] to introduce the GGA codon [bold] and a BamHI site [underlined]. The 3' portion of pbpF_fs was amplified with PBPF4S [see above]and PBPF3S [5'-GGTGGTGGATCCTTAAAACCAATTTCTG-3'] . The PCR fragmentswere digested with BamHI, ligated, and introduced into pNJ2to generate plasmid pNJ2pbpF_fs[S₄₀₂-G].

Peptidoglycan structure analysis. Bacteria were grown at 37°C to an optical density of 0.8in BHI broth . Peptidoglycan was extracted with 4% sodium dodecylsulfate at 100°C, treated with pronase [200 µg/ml]and trypsin [200 µg/ml], and digested with lysozyme [200µg/ml] and mutanolysin [200 µg/ml] . Muropeptides were reduced with sodium borohydride and separated by reverse-phase high-performance liquid chromatography on a C₁₈ column [3 µm, 4.6 by 250 mm; Interchrom, Montluçon, France] at a flowrate of 0.5 ml/min with a 0 to 20% gradient applied between10 and 90 min [buffer A, 0.05% [vol/vol] trifluoroacetic acidin water; buffer B, 0.035% [vol/vol] trifluoroacetic acid inacetonitrile] . The relative abundance of muropeptides was estimatedby the percentage of the integrated area of peaks detected bythe absorbance at 210 nm . Mass spectral data were collectedwith an electrospray time-of-flight mass spectrometer operatingin the positive mode [Qstar Pulsar I; Applied Biosystems, Courtaboeuf,France] directly connected to the C₁₈ column [flow rate, 0.5ml/min] . The data were acquired with a capillary voltage of5,200 V and a declustering potential of 20 V . The mass scanrange was from m/z 400 to 2,500, and the scan cycle was 1 s.Structure assignment of muropeptides based on mass determinationwas performed as previously described [6].

Analysis of PBPs. The technique used for the analysis of PBPs of the differentstrains was as previously described [33], except that labelingwas performed with 40 µg of benzyl[¹⁴C]penicillin potassium[2.11 GBq/mmol; Amersham Pharmacia Biotech]/ml.

 
Deletion of the pbp5 gene from the chromosome of E . faecalis JH2-2. The vector pHS1 was constructed to delete chromosomal PBP genes by homologous recombination as outlined in Fig . 1 . E . faecalisJH2-2 was highly resistant to the expanded-spectrum cephalosporinceftriaxone, and deletion of pbp5 led to a 4,000-fold reductionin the MIC of this drug [Table 2] . The strain was less resistantto ampicillin, and deletion of pbp5 produced only a fourfoldreduction in the MIC of this antibiotic . The pbp5 gene of E.faecalis JH2-2 cloned under the control of the aphA-3p promoterof the shuttle vector pNJ2 [Fig. 1B] restored wild-type ß-lactamresistance in JH2-2 pbp5.

 
Identification of class A PBP genes essential for PBP5-mediated ß-lactam resistance. Single deletion of any of the three class A PBP genes of E.faecalis [ponA, pbpF, or pbpZ] had no impact on the MICs ofß-lactam antibiotics [Table 2] . Among the three combinationsof double deletions, only the deletion of ponA and pbpF resultedin a large decrease [1,000-fold] in ceftriaxone resistance.Thus, either ponA or pbpF was required for intrinsic ceftriaxoneresistance mediated by PBP5 . Overexpression of the pbp5 geneunder the control of the aphA-3p promoter of pNJ2 did not restore ceftriaxone resistance in JH2-2 ponA pbpF [Table 2].

Deletion of the three class A PBP genes. Previous analyses showed that the self-transferable plasmidpIP964[hly] can mobilize chromosomal genes between E . faecalisstrains by conjugation [4] . To determine whether this system could be used to generate combinations of chromosomal deletions, E . faecalis JH2-2 pbpZ erm/pIP964[hly], obtained by replacingpbpZ with erm, was mated with JH2Sm . Transfer of the hemolysinmarker [hly] of pIP964 occurred at a frequency of ca . 10^-1,as determined on blood agar indicator plates . The chromosomal erythromycin resistance cassette located at the pbpZ locus was also transferable but at a lower frequency [ca . 10^-8] . An E. faecalis JH2Sm transconjugant harboring the pbpZ erm alleleand pIP964[hly] was in turn used as a donor in mating experiments,with E . faecalis JH2-2 ponA pbpF as a recipient . The ponA, pbpF,and pbpZ loci of five transconjugants obtained on selectivemedia containing erythromycin, rifampin, and fusidic acid wereanalyzed by PCR . As expected, all of them had received the pbpZ erm allele . Cotransfers were observed in two transconjugants that received pbpF alone or ponA and pbpF in addition to pbpZ erm . The remaining three transconjugants acquired the pbpZ ermallele and retained the ponA and pbpF alleles of the recipient.The erm cassette was removed from the chromosome of one of thesetransconjugants to generate JH2-2 ponA pbpF pbpZ. Deletion ofthe three PBP genes was confirmed by Southern blot and PCR analyses[data not shown] . This result indicates that none of the threeclass A PBPs is essential for viability.

Growth rate. Deletion of the three class A PBP genes led to an increase ofthe generation time [70.0 ± 4.6 min for JH2-2 ponA pbpF pbpZ versus 37.7 ± 1.0 min for JH2-2] . The increase inthe generation time was marginal [<12%] for the other mutantslacking one or two class A PBP genes.

Susceptibility to moenomycin. The impact of the deletion of class A PBP genes on the activityof the glycosyltransferase inhibitor moenomycin was studiedby the agar dilution method [Table 2] . Deletion of ponA andpbpF or of all three class A PBP genes resulted in high-levelresistance to moenomycin . This surprising observation indicatesthat the antibacterial activity of moenomycin requires ponA,pbpF, or both genes.

Analysis of PBP patterns labeled with benzyl[¹⁴C]penicillin. The chromosome of E . faecalis harbors six genes encoding putative multimodular PBPs belonging to class A [ponA, pbpF, and pbpZ]and class B [pbp5, pbpA, and pbpB] [Table 1] . Sodium dodecylsulfate-polyacrylamide gel electrophoresis resolved five high-molecularpenicillin-labeled protein bands in membrane extracts from E.faecalis JH2-2 [Fig . 2, lane 1] in addition to the low-molecular weight D,D-carboxypeptidase DacA [data not shown] . Based onthe analysis of mutants constructed in the present study, threeof the five penicillin-labeled protein bands could be assignedto class A PBPs . The band with the lower electrophoretic mobilityshould correspond to the ponA gene product, since it was absentfrom JH2-2 ponA [lane 2], JH2-2 ponA pbpF [lane 6], JH2-2 ponA pbpZ [lane 7], and JH2-2 ponA pbpF pbpZ [lane 8] . The PBP encodedby ponA had a much lower electrophoretic mobility than expectedfrom its calculated molecular mass [Table 1], as previouslyshown for putative orthologs from other gram-positive bacteria[25] . The second protein band by order of electrophoretic mobilityshould be the pbpZ gene product, as it was absent from JH2-2 pbpZ [lane 4], JH2-2 pbpF pbpZ [lane 5], JH2-2 ponA pbpZ [lane7], and JH2-2 ponA pbpF pbpZ [lane 8] . The third protein banddisappeared totally in the triple mutant JH2-2 ponA pbpF pbpZ [lane 8] and may therefore contain the pbpF gene product . However, this band cannot solely correspond to the PBP encoded by pbpF, since the band was present in JH2-2 pbpF [lane 3], JH2-2 pbpF pbpZ [lane 5], JH2-2 ponA pbpF [lane 6], and JH2-2 ponA pbpZ [lane 7] . These observations may imply that the third band contained truncated forms of PBPs encoded by ponA and pbpZ, in additionto the PBP encoded by pbpF . Deletion of pbp5 was associatedwith loss of the fifth PBP band [lane 9] . This band was moreintense in a JH2-2 derivative containing a copy of pbp5 clonedinto pNJ2 [Fig . 2, lane 11], confirming that it correspondedto PBP5 . The remaining PBP band [fourth band] may contain theputative class B PBPs encoded by pbpA and pbpB, since it wasunaffected by the ponA, pbpF, pbpZ, and pbp5 deletions.

 
Complementation of class A PBP gene deletions. The pbpF gene of E . faecalis JH2-2 [pbpF_fs] was cloned under the control of the aphA-3p promoter of the shuttle plasmid pNJ2 and introduced into JH2-2 ponA pbpF and JH2-2 ponA pbpF pbpZ strains . As expected, the resulting plasmid restored ceftriaxone resistance in these mutants [MIC of 1,000 µg/ml for both hosts] . The active-site serine of the transpeptidase moduleencoded by pbpF_fs was replaced by a glycine by site-directed mutagenesis . Expression of the resulting gene [pbpF_fsS₄₀₂-G] cloned into pNJ2 also restored a wild-type level of ceftriaxone resistance in the same hosts [MIC of 1,000 µg/ml] . Thus,the glycosyltransferase module of the PBP encoded by pbpF_fs was sufficient for ceftriaxone resistance in the absence ofa functional C-terminal D,D-transpeptidase module.

Heterologous expression of the pbpF ortholog of E . faecium [pbpF_fm] in JH2-2 ponA pbpF and JH2-2 ponA pbpF pbpZ led to high-levelresistance to ceftriaxone [MIC of 1,000 µg/ml for bothhosts], indicating that the glycosyltransferase module of the E . faecium PBP is functional when expressed in E . faecalis. In contrast, expression of mgt_Sa encoding the monofunctionalglycosyltransferase of S . aureus had no effect on the MIC ofthe antibiotic.

Peptidoglycan structure. In E . faecalis, the peptidoglycan is polymerized from a subunitconsisting of two sugars [GlcNAc and MurNAc], a linear pentapeptidestem [L-Ala₁-D-isoglutamine [iGln]₂-L-Lys₃-D-Ala₄-D-Ala₅] linkedto MurNAc, and a side chain [L-Ala-L-Ala] branched to the -amino group of L-Lys [Fig . 3] [6, 28] . The D,D-transpeptidase activityof PBPs catalyzes formation of Lys₃-[L-Ala-L-Ala]-D-Ala₄ crossbridges by cleavage of the D-Ala₄-D-Ala₅ bond of a donor stempeptide and linkage of D-Ala₄ to the extremity of the L-Ala-L-Ala side chain of an acceptor stem peptide.

 
The diversity of peptidoglycan fragments [muropeptides] obtainedby digestion of the peptidoglycan of E . faecalis JH2-2 by muramidases had three main origins [Table 3], as previously described [6].First, the muropeptides differed by the number of disaccharidepeptide subunits linked together by the transpeptidases [from1 to 4 for the monomers and tetramers, respectively] . Second,the most abundant muropeptides contained a pentapeptide stem[L-Ala-D-iGln-L-Lys-D-Ala-D-Ala], whereas less-abundant formscontained tripeptide stems [L-Ala-D-iGln-L-Lys] lacking theD-Ala residues at the free C-terminal end . The latter muropeptidesmay be generated by hydrolysis of the L-Lys₃-D-Ala₄ peptidebond by L,D-carboxypeptidases [6] . Third, a fraction of themuropeptides contained O-acetylated sugars.

 
To analyze the impact of the deletion of class A PBP genes oncell wall cross-linking, the muropeptides of strains JH2-2,JH2-2 ponA pbpF, and JH2-2 ponA pbpF pbpZ were compared [Table3] . The double and triple deletions were associated with a decreasein the relative abundance of the trimers and tetramers . Theseresults indicate that class A PBPs contribute to peptidoglycancross-linking in JH2-2 . The relative abundance of muropeptideswith O-acetylated sugars and incomplete tripeptide stems wasnot altered.

 
The role of PBP5 in intrinsic ß-lactam resistanceof enterococci has been well established by the analysis ofvarious spontaneous mutants of E . faecium obtained in the laboratoryor isolated from patients, as susceptibility testing provideda powerful screen for monitoring alterations of pbp5 expression[7, 18, 29, 30] . In contrast, the role of other PBPs remaineduncharacterized due to insufficiently developed genetic tools.In particular, electroporation is inefficient and certain naturalisolates and mutants of E . faecium and E . faecalis are refractoryto transformation by this method . For this reason, we have constructed the expression vector pNJ2 and the suicide plasmid pHS1 [Fig. 1], which were mobilizable between enterococcal strains by Tn916 at a frequency of ca . 10^-4 per donor . Derivatives of the thermosensitivereplicon pHS1 were designed to serially introduce deletionsinto the chromosome of E . faecalis by homologous recombination[Fig . 1C and D] . The deletions obtained by this approach areprecise, as they are delineated by the oligonucleotides usedto amplify the H1 and H2 sequences flanking the chromosomalregion targeted for deletion . In this study, the mutants containeddeletions removing at least 95% of the open reading frames, and two of them were in-frame deletions to minimize the riskof polar effects on expression of downstream genes after deletionof the erm cassette . The transfer properties of pHS1 combinedto direct mobilization of chromosomal markers by plasmid pIP964are powerful tools to generate combinations of chromosomal deletionsin E . faecalis.

Deletion of pbp5 led to a 4,000-fold reduction in the MIC of ceftriaxone for E . faecalis JH2-2, whereas the MIC of ampicillin was only reduced 4-fold [Table 2] . A spontaneous deletion ofpbp5 in E . faecium led to a larger decrease in the MIC of ampicillin[800-fold] in addition to the loss of resistance to cephalosporins[29] . Therefore, the contribution of PBP5 to intrinsic ampicillinresistance appears smaller in E . faecalis than in E . faecium.This difference is worth noting, since the emergence of high-level ampicillin resistance by modification of PBP5 has been mostly,if not exclusively, reported for clinical isolates of E . faecium[24].

The bacterial cell wall is polymerized by large complexes that include glycosyltransferases and transpeptidases for insertionof new material in the murein layer [15] . In the presence of high concentrations of ceftriaxone, the transpeptidase module of all PBPs, except that of PBP5, is thought to be inactivatedby the antibiotic [7, 29, 30] . Resistance to ceftriaxone wasused as a screen to identify the glycosyltransferases that cooperatewith the transpeptidase module of PBP5 for peptidoglycan polymerizationin the presence of the drug [Table 2] . The screen identifiedthe class A PBPs encoded by ponA and pbpF as essential partnersof PBP5 . Site-directed mutagenesis of pbpF confirmed that thecatalytic activity of the transpeptidase module of the PBP didnot play a role in resistance . S . aureus produces a single classA PBP [PBP2] that is similarly essential for ß-lactamresistance mediated in this organism by PBP2a [26] . The ponAgene of E . faecalis and the gene encoding PBP2 in S . aureusare putative orthologs, as are the genes encoding low-affinityclass B PBP5 and PBP2a [Table 1] [14] . The same functional interactionsmight therefore occur between subclasses of A- and B-type PBPsin different bacteria . In agreement, expression of the putativepbpF ortholog from E . faecium restored ceftriaxone resistancein the ponA pbpF mutant, whereas pbpZ of E . faecalis and mgtof S . aureus had no effect . The peptidoglycan precursors ofE . faecalis and E . faecium contain side chains consisting of the sequence L-alanyl-L-alanine and a single -D-asparaginyl or -D-aspartyl residue, respectively [28] . In spite of this difference, the glycosyltransferase module of the E . faecium PBP was functional in the heterologous host, indicating forthe first time that heterospecific complementation can be usedto get insight into the function of class A PBPs.

Deletion of ponA, pbpF, and pbpZ led to a viable mutant, indicatingthat the class A PBPs are unessential . The chromosome of E.faecalis does not encode a monofunctional glycosyltransferase[MGT] or any additional protein displaying similarity to theglycosyltransferase module of class A PBPs . In the triple mutant,transglycosylation is therefore performed by a distinct classof proteins that do not display similarity with known glycosyltransferases.A similar observation was recently reported for a mutant ofBacillus subtilis lacking all four genes encoding class A PBPsin this organism [23] . In contrast, at least one class A PBPis required for viability in E . coli [PBP1a or PBP1b] [11] andS . pneumoniae [PBP1a or PBP2a] [16, 25] . Although unessential,the class A PBPs contribute to peptidoglycan cross-linking inE . faecalis, since deletion of the three class A PBP genes ledto an increase in the proportion of monomers to the detrimentof trimers and tetramers [Table 3] . Except for this difference,the mode of cross-linking and structure of the muropeptideswere essentially unaltered in comparison to the parental strainJH2-2 . These observations indicate that the transpeptidationreaction catalyzed by the entire set of PBPs, or solely by theclass B PBPs, may involve the same precursors with respect tothe presence of tripeptide or pentapeptide at the free C terminusof the acceptor stems.

Moenomycin was recently shown to inhibit the glycosyltransferase activity of purified PBP1b of E . coli in vitro, although the drug was not competitive with respect to the lipid II substrate[9] . The mutants lacking ponA and pbpF or the three class A PBP genes were resistant to moenomycin, whereas the parentalstrain and all other single and double deletion mutants weresusceptible to this antibiotic [Table 2] . Thus, susceptibilityto moenomycin in E . faecalis depends upon production of at least one of the class A PBPs encoded by ponA and pbpF . This observation implies that binding of moenomycin to its targets has a toxic effect despite the fact that the PBPs encoded by ponA and pbpF are not essential for viability . The antibacterial activityof moenomycin appears, therefore, to result from poisoning ofthe polymerization complexes containing class A PBPs ratherthan simply inhibiting their glycosyltransferase active site.This complex mode of action has important implications for thediscovery of new drugs targeting the transglycosylation reactionand the improvement of existing molecules, such as biphenylderivatives of vancomycin and moenomycin [9] . In particular,in vitro inhibition of the transglycosylase activity of purifiedclass A PBPs is not expected to strictly correlate with antibacterialactivity, since poisoning of the peptidoglycan polymerizationcomplexes and inhibition of enzyme activity may occur independently.Moreover, class A PBPs and the related monofunctional glycosyltransferasesmay no longer be considered essential targets in human gram-positive pathogens, since the E . faecalis JH2-2 ponA pbpF pbpZ null mutantwas viable.

In conclusion, neither the D,D-transpeptidase nor the glycosyltransferaseactivity of E . faecalis class A PBPs is essential for peptidoglycansynthesis . Complete bypass of the D,D-transpeptidase activity of the PBPs by an L,D-transpeptidase insensitive to ß-lactaminhibition has been recently reported for E . faecium [21, 22].The L,D transpeptidase is responsible for the synthesis of newpeptidoglycan cross bridges [L-Lys₃D-Asx-L-Lys₃] that replacethe cross bridges formed by the D,D-transpeptidases [D-Ala₄D-Asx-L-Lys₃]. These complementary observations indicate that peptidoglycan polymerization in the total absence of multimodular PBPs is theoretically possible in enterococci.

 
This work was supported by Wyeth Research, by the Programmede Recherche Fondamentale en Microbiologie et Maladies Infectieuseset Parasitaires [MENRT], and by the Fondation pour la Recherche Médicale . A.A . was the recipient of a fellowship fromthe Gobierno Vasco.

E . faecalis genome sequence data were kindly provided by The Institute for Genomic Research, as publicly released at http://www.tigr.org.

 
* Corresponding author . Mailing address: LRMA, Université Paris VI, 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France . Phone: 33 [0]1 43 25 00 33 . Fax: 33 [0]1 43 25 68 12 . E-mail: michel.arthur@bhdc.jussieu.fr.

Present address: Department of Medical Microbiology, MedicalSchool, University of Cape Town, 7925 Cape Town, South Africa.

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