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Journal of Bacteriology, November 2001, p. 6525-6531, Vol. 183, No. 22
Laboratory of Microbiology, The Rockefeller
University, New York, New York 10021,1 and
Molecular Genetics Unit, Instituto de Tecnología
Química e Biológica, Universidade Nova de Lisboa, Oeiras,
Portugal2
Received 8 May 2001/Accepted 22 August 2001
The essential function of penicillin-binding protein 2 (PBP2) in
methicillin-susceptible Staphylococcus aureus RN4220 was clearly established by placing the pbp2 gene under control
of the inducible Pspac promoter; the resulting bacteria
were unable to grow in the absence of inducer. In contrast, the deficit
in PBP2 caused by inhibition of transcription of the pbp2
gene did not block growth of a methicillin-resistant S. aureus strain expressing the extra penicillin-binding protein
PBP2A, a protein of extraspecies origin that is central to the
mechanism of methicillin resistance. Several lines of evidence indicate
that the essential function of PBP2 that can be compensated for by
PBP2A is the transpeptidase activity. This provides direct genetic
evidence that PBP2A has transpeptidase activity.
The wide-spectrum resistance
of methicillin-resistant Staphylococcus aureus (MRSA) to all
However, some new observations require basic revision of this model.
The structural determinant of staphylococcal PBP2 was shown to be an
auxiliary gene; transposon inactivation of pbp2 resulted in
a massive reduction in the level of resistance in an MRSA strain
despite the fact that the bacteria continued to produce normal amounts
of PBP2A (20). Recent experiments showed that expression
of high-level resistance required the cooperative functioning of PBP2A
and the penicillin-insensitive transglycosylase (TGase) domain of
PBP2 (17).
These observations brought back into focus the possibility that native
staphylococcal PBPs, primarily PBP2, are participants in the mechanism
of Bacterial strains and plasmids.
The bacterial strains and
plasmids used in this study are described in Table
1. S. aureus strains were
grown in tryptic soy broth (TSB) (Difco Laboratories) with aeration at
37°C or on tryptic soy agar (TSA) (Difco Laboratories) plates at
37°C. Escherichia coli strains were grown in Luria-Bertani
broth (Difco Laboratories) with aeration at 37°C.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.22.6525-6531.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Complementation of the Essential Peptidoglycan
Transpeptidase Function of Penicillin-Binding Protein 2 (PBP2) by
the Drug Resistance Protein PBP2A in Staphylococcus
aureus
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactam antibiotics has had a devastating impact on the chemotherapy
of staphylococcal infections ever since the first appearance of MRSA in
clinical specimens in the early 1960s. The key component of this
resistance mechanism is an acquired penicillin-binding protein
(PBP), PBP2A, which has unusually low affinity for all -lactam
antibiotics. The genetic determinant of PBP2A, the mecA
gene, is not native to S. aureus but was imported from an
as-yet-unidentified extraspecies source (2). Until recently, the accepted model of this resistance mechanism implied that
in the presence of -lactam antibiotics the only PBP that remains
functional is the low-affinity PBP2A, since the other four
staphylococcal PBPs had high enough affinities to penicillin type
antibiotics to become rapidly and fully acylated and inactivated even
at low drug concentrations that were several orders of magnitude below
the concentration needed to inhibit the growth of many MRSA strains. In
this model, PBP2A is a surrogate enzyme capable of taking over the
normal functions of staphylococcal PBPs in cell wall biosynthesis.
-lactam resistance. The purpose of the study described in this
paper was to examine in more detail the role(s) PBP2 plays in
staphylococcal wall synthesis in antibiotic-susceptible bacteria as
well as resistant bacteria (i.e., both in the absence and in the
presence of acquired PBP2A). To do this, pbp2 was put under
control of the inducible promoter Pspac, which allowed
testing of the essentiality of this gene in the backgrounds of
methicillin-susceptible and -resistant strains of S. aureus.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and plasmids used in this study
DNA methods. Routine DNA manipulations were performed by using standard methods (1, 21). Restriction enzymes were purchased from New England Biolabs. Plasmid DNA was purified by using Wizard Plus SV Minipreps or Midipreps DNA purification systems (Promega). DNA sequencing was done at The Rockefeller University Protein/DNA Technology Center by the BigDye terminator cycle sequencing method with either a 3700 DNA analyzer for capillary electrophoresis or ABI Prism 377 DNA sequencers for slab gel electrophoresis.
Construction of pPBP2i.
Plasmid pDH88 was digested
with EcoRI and BamHI. The digestion products were
electrophoresed on a 1% low-melting-point agarose gel (Gibco-BRL Life
Technologies), and the 1.6-kb fragment was cut from the gel and
purified by using a QIAquick gel extraction kit. This fragment
contained the IPTG
(isopropyl--D-thiogalactoside)-inducible Pspac promoter followed by a polylinker and by the
lacI gene under control of the constitutive Ppcn
promoter. The 1.6-kb fragment was cloned into pSP64E, resulting in
plasmid pMGPI.
Construction of S. aureus strains with inducible pbp2. Plasmid pPBP2i was introduced into RN4220 by electroporation essentially as previously described (12). The transformation mixture was plated on TSA containing erythromycin (10 µg/ml) and different concentrations of IPTG (0, 0.05, 0.5, and 4 mM).
Plasmid pMGPII was constructed by cloning the BamHI/EcoRI 1.6-kb fragment from pDH88 containing the lacI gene into E. coli-S. aureus shuttle vector pGC2 and was electroporated into RN4220. Two transformants, RNpPBP2iII-1 and RNpPBP2iII-5, were chosen for further study. Plasmids pPBP2i and pMGPII were sequentially transduced to MRSA strain COL by using phage 80
as previously described (16), except that 0.5 mM IPTG was added to the media used for preparation of
the transducing lysate and for transduction. Transductants were
selected with erythromycin (10 µg/ml) for pPBP2i and with chloramphenicol (20 µg/ml) for pMGPII. COLpPBP2iII-1 and
COLpPBP2iII-5 were chosen for further study.
For construction of pPBP2iC, the EcoRI/BamHI
fragment from pPBP2i containing the pbp2 fragment fused with
the Pspac promoter was cloned into pSP64 (Promega). A
1.1-kb PCR fragment encoding the chloramphenicol resistance marker was
amplified from pSPT181c by using primers
5'-CTGTCGACCAGTCATACCAATAAC-3' and
5'-CATGTCGACGCTCAACGTCAATAAAGC-3' and was cloned into the
SalI site of pSP64.
Plasmid pPBP2iC was electroporated into RN4220 in the presence of IPTG
and subsequently transduced to RUSA4 and to COL by using media
supplemented with IPTG. Selection was done with 15 µg of
chloramphenicol per ml.
Isolation of RNA and Northern blot hybridization.
Overnight
cultures of COLpPBP2iII-1 and COLpPBP2iII-5
were grown in TSB supplemented with erythromycin (10 µg/ml) and
chloramphenicol (10 µg/ml). Each strain was diluted 1:40 in two tubes
of TSB supplemented with erythromycin (5 µg /ml) and chloramphenicol
(5 µg /ml), and one of the preparations was also supplemented with
0.5 mM IPTG. After cells were grown to the mid-log phase (optical
density at 620 nm [OD620], ~0.7), they were pelleted
and processed with an RNeasy mini kit (Qiagen) or with a FastRNA Blue
isolation kit (Bio 101 Inc.) in combination with FastPrep FP120
(Bio 101 Savant) used according to the manufacturer's
recommendations. RNA (5 µg) was electrophoresed through a 1.2%
agarose-0.66 M formaldehyde gel in MOPS (morpholine propanesulfonic
acid) running buffer (Sigma). Blotting of RNA onto Hybond N+ membranes
(Amersham) was performed with Turboblotter alkaline transfer systems
(Schleicher & Schuell). For detection of specific transcripts, a
pbp2-specific DNA probe was constructed by PCR amplification
with two primers, 5'-AGCTTGGCAATCAGTTAAGC-3' and
5'-TCCCACCATAAAAGATGAAG-3'. The probe was labeled with a
Ready To Go labeling kit (Pharmacia Biotech) by using
[-32P]dCTP (Amersham Life Sciences) and was hybridized
under high-stringency conditions. The blots were subsequently washed
and autoradiographed.
Depletion of PBP2. Strains with pbp2 under control of IPTG-inducible promoter Pspac were tested on solid medium by using TSA plates containing the appropriate antibiotic(s) (10 µg of erythromycin per ml and 10 µg of chloramphenicol per ml for strains with pPBP2i and pMGPII or 10 µg of chloramphenicol per ml for strains with pPBP2iC) and supplemented or not supplemented with 0.5 mM IPTG.
Experiments in liquid culture were performed by using the following protocol. Strains were grown overnight at 37°C in TSB supplemented with 0.5 mM IPTG and the appropriate antibiotic(s) (see above). These starting cultures were then each diluted 1:100 in 50 ml of TSB containing 5 µg of erythromycin per ml and 5 µg of chloramphenicol per ml and supplemented with 0.5 mM IPTG, and they were allowed to grow until the OD620 was approximately 0.2. Each culture was centrifuged at room temperature, washed with TSB, centrifuged, resuspended in 50 ml of medium lacking IPTG, and divided into two portions, one of which was supplemented with 0.5 mM IPTG. The cultures were again incubated with agitation at 37°C, and OD620 was monitored. To measure the growth rate of COLpPBP2iII-1 or COLpPBP2iII-5, an overnight culture was grown in TSB containing 10 µg of erythromycin per ml and 10 µg of chloramphenicol per ml and supplemented with 0.5 mM IPTG. This starting culture was washed and resuspended in TSB without IPTG to remove traces of the inducer before the culture was diluted 1:200 in TSB containing 5 µg of erythromycin per ml and 5 µg of chloramphenicol per ml and supplemented or not supplemented with 0.5 mM IPTG. Each culture was incubated with agitation at 37°C, and the OD620 was monitored.Cell wall analysis. COL, COLpPBP2iII-1, and COLpPBP2iII-5 were grown overnight at 37°C in TSB supplemented with 10 µg of erythromycin per ml, 10 µg of chloramphenicol per ml, and 0.5 mM IPTG. Each starting culture was washed to remove traces of the inducer before it was diluted 1:5,000 in 500 ml of TSB containing 5 µg of erythromycin per ml and 5 µg of chloramphenicol per ml and supplemented or not supplemented with 0.5 mM IPTG. Cells were grown until the OD620 was approximately 0.3. Also included was a culture of strain COL grown in the presence of 1 µg of oxacillin per ml. Isolation of cell wall peptidoglycan and analysis of the family of enzymatically released muropeptides by reversed-phase high-pressure liquid chromatography (HPLC) were carried out essentially as previously described (3).
Electron microscopy. COL was grown in TSB. COLpPBP2iII-5 was grown in TSB containing 5 µg of erythromycin per ml and 5 µg of chloramphenicol per ml and supplemented or not supplemented with 0.5 mM IPTG from a preinoculum that had been grown in the presence of the same antibiotics at concentrations of 10 µg/ml in medium that was supplemented or not supplemented with the inducer. When the OD620 reached 0.6, 5 ml of the culture was harvested by low-speed centrifugation and fixed with 1 ml of 2.5% glutaraldehyde. Electron microscopy was done at the Electron Microscopy Service of The Rockefeller University.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PBP2 is essential for growth of an MSSA strain but not for growth
of an MRSA strain.
To determine if PBP2 is essential for survival
of methicillin-susceptible S. aureus (MSSA) strain RN4220,
we constructed a pbp2 conditional mutant in which
pbp2 is regulated by the IPTG-inducible, LacI-repressible
Pspac promoter (26). A fragment containing the
ribosome-binding site and the first 171 codons of pbp2 was fused to the inducible promoter Pspac, creating plasmid
pPBP2i, which was electroporated into RN4220. As a result of the
integration of pPBP2i into the chromosome by a Campbell type of
mechanism, a full-length copy of pbp2 was placed under
control of Pspac, which is repressed by the product of the
lacI gene that is also present in pPBP2i. Transformants were
picked, and after correct insertion of the plasmid was confirmed by
PCR, they were plated on selective plates in the presence or absence of
IPTG. Supplementing the plates with 0.5 mM IPTG resulted in normal
growth of the transformants, so this concentration was routinely used
to propagate the mutants. In the absence of IPTG there was some growth
in the area where there was heavy inoculum, where some colonies seemed
to grow over a lawn of lysed cells. It has been found previously that
in S. aureus tight regulation of genes under the
control of Pspac can be obtained if lacI is
present in a multiple-copy plasmid while a single copy of
Pspac is present in the chromosome (11).
Therefore, RN4220 with integrated pPBP2i was transformed with pMGPII, a
plasmid encoding LacI, and two transformants, RNpPBP2iII-1
and RNpPBP2iII-5, were selected for further study. These
transformants did not grow when they were plated on solid medium in the
absence of IPTG, although the plates still had some colonies in
the zone where there was heavy inoculum (Fig.
1a), indicating that PBP2 was essential for growth of RN4220.
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and 
, COLpPBP2iII-5
grown with and without IPTG, respectively; 
and 
,
RNpPBP2iII-5 grown with and without IPTG, respectively.
Cultures were grown until the OD620 was approximately 0.2. At that point (indicated by grey and black arrows for
RNpPBP2iII-5 and COLpPBP2iII-5, respectively),
the IPTG was removed by washing, the cells were resuspended in fresh
medium without inducer, and each culture was divided into two portions,
only one of which was supplemented with 0.5 mM IPTG. We continued to
monitor the OD620 after this.
1; COLpPBP2iII-1 without IPTG, 0.60 ± 0.06 h1; COLpPBP2iII-5 with IPTG, 0.74 ± 0.04 h1; and COLpPBP2iII-5 without IPTG,
0.60 ± 0.06 h1. These data showed that the deficit
of PBP2, although not lethal, had a cost for the cells, as indicated by
the reduced growth rates of the bacteria.
Replacement of the essential function of PBP2 by PBP2A.
RN4220
and COL have unrelated genetic backgrounds. We assumed that the reason
why PBP2 was essential in antibiotic-susceptible strain RN4220 but not
in drug-resistant strain COL was the presence of PBP2A in the latter.
In order to verify this, the experiments described above were
repeated with strain RUSA4, an isogenic derivative of COL in which the
mecA gene was inactivated by Tn551 insertion, which prevented expression of PBP2A (15). We constructed a
RUSA4 strain with the pbp2 gene under control of the
Pspac promoter by inserting plasmid pPBP2iC into its
chromosome (plasmid pPBP2i could not be transduced to RUSA4
because its resistance marker, erm, is the same as the
resistance marker present in Tn551). Transformants RUSA4pPBP2iC-2 and RUSA4pPBP2iC-3 were chosen
for further study. Plasmid pPBP2iC was also inserted into the COL
chromosome, and COLpPBP2iC-2 and COLpPBP2iC-3
were used for experiments. When these strains were plated on solid
medium, RUSA4pPBP2iC was dependent on IPTG for growth,
while COLpPBP2iC grew both in the presence and in the
absence of IPTG (Fig. 3).
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Cell wall composition and morphology of S. aureus
constructs grown with and without PBP2.
Cell walls of
COLpPBP2iII-1 and COLpPBP2iII-5 were
purified from cultures grown in the presence and in the absence of
IPTG. The cell walls of parental strain COL grown with and without IPTG were also analyzed to control for a possible effect of IPTG on cell
wall composition; no evidence of such an effect was detected (data not
shown). A comparison of the muropeptide compositions of strains with
inducible pbp2 showed that the absence of PBP2 had only a
minor, if any, effect on the muropeptide profile of the cell wall
peptidoglycan (Fig. 5). Similar results
were obtained for COLpPBP2iII-5 (data not shown).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The results described in this paper clarify several aspects of the function of staphylococcal PBP2, in both antibiotic-susceptible and antibiotic-resistant cells.
Contradictory reports have been published previously about the essentiality of PBP2 based on correlations between antibiotic affinities for different PBPs and antibacterial activities (5, 6, 8). It has also been suggested that PBP2 is not essential in MRSA strains (14) based on data obtained with a spontaneous mutant that was not genetically characterized.
Our observations clearly demonstrate that PBP2 is essential for growth of drug-susceptible strains of S. aureus; in a construct in which PBP2 of MSSA strain RN4220 was placed under control of IPTG-inducible promoter Pspac (an approach used previously to show the essentiality of murE in this organism [11]) bacterial growth depended on the presence of the IPTG inducer in both solid and liquid growth media.
When similar experiments were performed with drug-resistant strain COL and its isogenic transposon mutant RUSA4, in which the mecA gene was inactivated, strikingly different results were obtained. When pbp2 was made inducible in the background of strain COL expressing the mecA gene product PBP2A, growth of the bacteria did not depend on the presence of IPTG in the medium. When pbp2 was made inducible in the background of strain RUSA4, in which production of PBP2A is inhibited, bacterial growth occurred only in the presence of IPTG. These experiments clearly show that acquired drug resistance protein PBP2A can replace an essential function of native PBP2 in the absence of antibiotics in the surrounding medium.
PBP2 is a bifunctional protein that has both a transpeptidase (TPase) domain and a TGase domain (7, 13). The essential nature of this protein demonstrated in the experiments described above does not reveal which of these two activities is critical for survival and growth of antibiotic-susceptible S. aureus. A combination of the results of several experiments suggests that the critical function of PBP2 is the TPase function. This conclusion is based on the following observations. (i) In a recent study we showed that mutants with mutations in the TPase domain of PBP2 could not be obtained for MSSA while mutants with mutations in the TGase domain could be isolated, indicating that the TGase activity of PBP2 is not essential in the background of an MSSA strain (17). (ii) The experiments described in this paper demonstrate that suppression of production (transcription) of PBP2 is lethal in MSSA strains and also in an MRSA strain with inactivated PBP2A (strain RUSA4) but not in an MRSA strain with active PBP2A (strain COL). It follows that the essential activity of PBP2 replaced by PBP2A must be the TPase activity.
Our data also provide direct genetic evidence that the enzymatic activity of PBP2A is a TPase activity. Until now, the TPase activity for PBP2A has been assumed based on homology with other PBPs and current models of methicillin resistance in S. aureus (22). The exquisite dependence of antibiotic resistance on an intact TGase activity of PBP2 (17) also implies that PBP2A lacks such catalytic activity, which is in accordance with the current classification of this protein as a class B PBP with a TPase domain and a penicillin-insensitive second domain whose function is not known (7).
The muropeptide profiles of cell walls isolated from strain
COLpPBP2iII grown in the presence and in the absence of
IPTG (i.e., with and without PBP2) were very similar, if not identical,
supporting the genetic evidence that the TPase function of PBP2 can be
replaced by PBP2A. Under both conditions, HPLC analysis of enzymatic
peptidoglycan hydrolysates revealed the multicomponent muropeptide
profile with a high proportion of extensively cross-linked components
typical of S. aureus cell walls. Such cell walls differ
profoundly from the cell walls consisting of poorly
cross-linked, monomer- and dimer-rich peptidoglycan produced by
resistant strains of S. aureus grown in the presence of
-lactam antibiotics (Fig. 5), in which the TPase activity of PBP2A
must play a major role (4). What factors determine the
highly specific muropeptide composition of the staphylococcal
peptidoglycan are not clear at present. Previously published data
(25) clearly indicate that there is at least one
additional PBP, namely, PBP4, that plays a major role in production of
highly cross-linked staphylococcal peptidoglycan. It is likely that
PBPs act in concert, i.e., in a manner possibly similar to that of the
hypothetical multienzyme complex involved in biosynthesis of the cell
wall of E. coli (10). If such complex exists in
S. aureus, our results indicate that one of its
characteristics is enough plasticity to enable integration of new
components, such as PBP2A. However, the modest but frequent
morphological abnormalities and slower growth rates detected for the
staphylococci in which the deficit of PBP2 was compensated for by PBP2A
suggest that integration of the foreign PBP2A into the cell wall
synthetic apparatus may not be perfect.
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ACKNOWLEDGMENTS |
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Mariana Pinho was supported by grant PRAXIS XXI/BD/9079/96. Sérgio Filipe was supported by grant PRAXIS XXI/BD/9071/96.
We thank Adriano Henriques and Shangwei Wu, who kindly provided plasmids pDH88 and pSPT181c, respectively.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Microbiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021. Phone: (212) 327-8278. Fax: (212) 327-8688. E-mail: tomasz{at}mail.rockefeller.edu.
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Abstract
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Materials and Methods
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Discussion
References
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Journal of Bacteriology, November 2001, p. 6525-6531, Vol. 183, No. 22
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.22.6525-6531.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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[Abstract] [Full Text] -
Lemaire, S., Fuda, C., Van Bambeke, F., Tulkens, P. M., Mobashery, S.
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