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Antimicrobial Agents and Chemotherapy, June 2000, p. 1745-1748, Vol. 44, No. 6
Infectious Diseases Research, Lilly Research
Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285
Received 19 August 1999/Returned for modification 21 December
1999/Accepted 10 March 2000
To further understand the role of penicillin-binding protein 2a
(PBP 2a) of Streptococcus pneumoniae in penicillin
resistance, we confirmed the identity of the protein as PBP 2a. The PBP
2a protein migrated electrophoretically to a position corresponding to that of PBP 2x, PBP 2a, and PBP 2b of S. pneumoniae and was absent in a pbp2a
insertional mutant of S. pneumoniae. We found that
the affinities of PBP 2a for penicillins were lower than for
cephalosporins and a carbapenem. When compared with other S. pneumoniae PBPs, PBP 2a exhibited lower affinities for
Streptococcus
pneumoniae, one of the major human pathogens of the upper
respiratory tract, has developed resistance to many Results of the recent genetic studies have established that the
pbp2a gene in combination with the pbp1b gene is
essential for viability of S. pneumoniae (15,
22) and that the pbp2a-encoded transglycosylase may
play a major role in peptidoglycan polymerization in the cell
(15). In addition, an active form of PBP 2a lacking its
transmembrane domain has been obtained and shown to contain two
distinct transpeptidase and transglycosylase domains that could be
physically separated (5, 28).
In this study, we report the biochemical identification and
characterization of PBP 2a from S. pneumoniae. The
results of this study suggest that PBP 2a, unlike other PBPs of
S. pneumoniae, has a low affinity for The materials used in this study are as follows: cephalothin,
penicillin G, and cephalexin (Eli Lilly); cefuroxime and
ceftazidime (Glaxo); imipenem (Merck); methicillin,
oxacillin, and cefotaxime (Sigma); piperacillin (American
Cyanamid); BOCILLIN FL (Molecular Probes, Inc., Eugene,
Oreg.); thrombin (Pharmacia LKB Biotechnology, Alameda,
Calif.), and Luria-Bertani (LB) medium (Bio 101, Inc., La Jolla,
Calif.). The remaining materials used were the same as those described
previously (28).
To purify glutathione S-transferase (GST)-PBP 2a* that lacks
its transmembrane domain, Escherichia coli cells harboring
pGEX-2T/pbp2a* (28) were grown, induced,
harvested, and disrupted as described previously (28). The
inclusion bodies of GST-PBP 2a* were then purified, refolded, and
stored at The transpeptidase activity of PBP 2a was assayed by measuring its
ability to hydrolyze S2d, an analog of the bacterial cell wall stem
peptides (1, 16, 17, 30). For 50% inhibitory concentration
(IC50) determinations, reaction mixtures (1 ml each) contained 52 mM potassium phosphate (pH 7), 2.0 mM S2d, 0.5 mM 4,4'-dithiopyridine, 80 µg of GST-PBP 2a*, and various amounts of
antibiotics (30). The increase in
A325 was monitored at 37°C for 2 min with a
double-beam BioSpec-1601 spectrophotometer (Shimadzu Instruments,
Inc.). For Ki determinations, reaction mixtures
(1 ml each) contained 52 mM potassium phosphate (pH 7.0), 0.5 mM 4,4'-dithiodipyridine, various amounts of S2d (0.5 to 8.0 mM), 80 µg
of GST-PBP 2a*, and fixed amounts of each antibiotic.
To express the full-length pbp2a gene of S. pneumoniae (hex) R6 in E. coli for
Western blotting analysis, the gene was cloned and sequenced as
previously described (15) (accession no. AF101780). PCR
amplification of the pbp2a gene was carried out as described below. The 5' PCR primer
(5'-TGATGAAG ATGATAAGGATCCCATATGAAATTAGATAAATTA-3') was
designed to begin at the ATG start codon of pbp2a based upon our known sequence (15) (accession no. AF101780) and
contains added BamHI and NdeI sites for cloning
purposes. The 3' PCR primer (5'-GCTTTGACAAGCTTCTTAGCGAAAT-3')
was designed to end at the stop codon of pbp2a and
contains an added HindIII site after the stop codon for
cloning into the vector. The PCR mixtures contained ~100 ng of
S. pneumoniae (hex) R6 genomic DNA
(15), 10 µl of 10× ThermoPol buffer containing 20 mM
Mg2SO4 (New England Biolabs, Beverly, Mass.),
10 µl of 1-mg/ml bovine serum albumin, 8 µl of deoxynucleoside
triphosphates (2.5 mM), 1 µl of each primer (40 pmol), 3 U of Vent
DNA polymerase (New England Biolabs), and 66.5 µl of water. The
following PCR amplification conditions were used: 94°C for 30 s,
annealing at 50°C for 30 s, and polymerization at 72°C for 3 min for a total of 30 cycles. Resulting PCR products were purified
using a QIAquick spin column (Qiagen, Valencia, Calif.), and 1 µg of
product was subjected to DNA sequence analysis (15).
Three identical PCR products were generated using the conditions
described above and then digested with BamHI. This fragment was cloned into an expression vector, pET-11b (Novagen, Madison, Wis.).
To verify that no errors were introduced during the PCR process, the
DNA sequence of the entire pbp2a gene was determined following its cloning into pET-11b.
To analyze PBP 2a in the cell, Western blotting analysis was performed
by using polyclonal antibodies against sodium dodecyl sulfate
(SDS)-denatured PBP 2a* which were prepared by Harlan Bioproducts for
Science, Inc. (Indianapolis, Ind.). A purified and refolded GST-PBP 2a*
protein preparation was digested with thrombin according to the
manufacturer's instructions (Pharmacia), SDS denatured, and subjected
to SDS-polyacrylamide gel electrophoresis (PAGE) (19).
Protein bands containing PBP 2a* were visualized by incubating
the gel in 0.5 M KCl and 50 mM potassium phosphate, pH 7.2, and excised
(30). Each protein band containing approximately 100 µg of
PBP 2a* was injected into New Zealand White rabbits.
To prepare membrane fractions for Western blotting analysis, the
following insertional mutants of S. pneumoniae
(hex) R6 were constructed as described previously
(15): nanA::Eryr,
pbp1b::Eryr,
pbp2a::Eryr, and
pbp1a::Eryr and grown in brain heart
infusion medium (Difco) without shaking at 37°C. The cultures were
collected at an optical density at 660 nm of 0.4 by centrifugation at
8,000 × g for 10 min and washed with 20 mM potassium
phosphate, pH 7.0.
E. coli BL21(DE3) [F The collected cells of E. coli and S. pneumoniae were resuspended in 20 mM potassium phosphate
(pH 7.5)-140 mM NaCl and disrupted by passing twice through a French
pressure cell (Aminco Laboratories, Inc., Rochester, N.Y.). The
resulting suspensions were centrifuged. Supernatants were collected and
centrifuged at 100,000 × g for 45 min. Pellets
(membrane fractions) were collected, washed with, and resuspended in
the same phosphate buffer (1 ml each). The membrane fractions were
denatured by SDS and subjected to Western blotting analysis as
described previously (30). The polyvinylidene difluoride
membranes were blocked in 1× phosphate-buffered saline (Gibco BRL)
containing 5% dry milk at 4°C overnight, incubated with primary
antibodies (1:500 dilutions) and secondary antibodies (1:2,000) for 2 to 3 h at room temperature.
To detect PBPs, we performed penicillin-binding assays (29)
by using BOCILLIN FL, a fluorescent penicillin, as a labeling reagent
(Molecular Probes). S. pneumoniae nanA
(hex) R6 and pbp1a mutants, and E. coli BL21(DE3) strains carrying pET-11b and
pET11b-pbp2a plasmids, were grown, collected, disrupted, and
centrifuged as described above. For detection of PBPs, 75 µl of each
membrane fraction was mixed with 25 µl of 100 µM BOCILLIN FL
(final concentration, 25 µM), incubated at 35°C for 20 min, and
denatured with 100 µl of SDS denaturing solution (19). The
denatured reaction mixtures (5 to 10 µl each) were loaded onto a
polyacrylamide gel (10%; Bio-Rad). The presence of PBPs was detected
using a FluorImager 575 (excitation at 488 nm and emission at 530 nm) (Molecular Dynamics, Inc., Sunnyvale, Calif.) (29).
Western blotting and penicillin-binding assay analyses of PBP 2a in
the cell.
As the pbp2a gene of S. pneumoniae was recently cloned and sequenced (13,
15), we wanted to confirm the identity of its gene product.
Membrane preparations of S. pneumoniae
(hex) R6, pbp1a and pbp2a mutants, and
a pbp2a overexpresser strain of E. coli were
subjected to Western blotting analysis (Fig.
1). A protein band with a molecular mass
of approximately 82 kDa was detected in the membrane fractions of the
nanA::Eryr and
pbp1a::Eryr mutants of S. pneumoniae (lanes 2 and 4), but this protein band was
absent in the membrane fraction of the
pbp2a::Eryr mutant (lane 3). A protein
with an identical molecular mass was also detected in the membrane
fraction of E. coli BL21 (DE3) that contained multiple
copies of the S. pneumoniae pbp2a expression plasmid
(lane 5), but it was absent in the membrane fraction of E. coli control cells containing the vector only (lane 6). The results of our Western blotting analysis showed that the
pbp2a gene was expressed in S. pneumoniae, that PBP 2a is a membrane protein consistent
with the presence of a transmembrane domain at the N terminus of the
protein (15), and that the pbp2a mutant is indeed
missing PBP 2a.
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification and Characterization of the
Penicillin-Binding Protein 2a of Streptococcus
pneumoniae and Its Possible Role in Resistance to
-Lactam Antibiotics
ABSTRACT
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Abstract
Text
References
-lactam antibiotics, especially penicillins. Therefore, PBP 2a is a
low-affinity PBP for -lactam antibiotics in S. pneumoniae.
TEXT
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Abstract
Text
References
-lactam
antibiotics (2, 3, 6-9, 12-14) due to the formation of
high-molecular-mass penicillin-binding proteins (PBPs) with reduced
affinity for these antibiotics (2, 3, 6-9, 12-14, 21, 25,
26). S. pneumoniae contains five detectable
high-molecular-mass PBPs: PBP 1a, PBP 1b, PBP 2a, PBP 2x, and PBP 2b
(7-9, 13-15, 21). The role of PBP 1a, PBP 2b, and PBP 2x
in their resistance to -lactam antibiotics is well understood
(2, 3, 6-9, 12-14, 21, 25, 26). In contrast, the
involvement of PBP 2a and in particular PBP 1b in penicillin resistance
is not well characterized (13, 24-26). The genes encoding
PBP 1b and PBP 2a have recently been sequenced (13, 15).
Results of genetic and molecular analyses suggested that PBP 2a
contributed to cefotaxime resistance in some clinical isolates and
laboratory strains of S. pneumoniae (13,
24).
-lactam antibiotics.
70°C as described previously (28). Protein
concentrations were determined, also as described (28).
dcm ompT hsdS
(rB mB)
gal 
(DE3) (Camr)] (Stratagene, La Jolla,
Calif.) containing pET-11b-pbp2a (a pbp2a expression plasmid) and pET-11 was grown overnight at 32°C with vigorous shaking in LB medium (Bio 101) supplemented with 100 µg of
ampicillin/ml. The overnight cultures were inoculated into LB
medium (1:30) containing ampicillin and induced with 1 mM IPTG (isopropyl--D-thiogalactopyranoside) at an optical
density at 600 nm of 0.5 to 0.6 for 2 h. Cells were collected by
centrifugation and washed with 20 mM potassium phosphate, pH 7.2.
View larger version (70K):
[in a new window]
FIG. 1.
Western blotting analysis of PBP 2a from S. pneumoniae (hex) R6. Lane 1, prestained
molecular mass markers (from top to bottom: myosin, 250 kDa; BSA, 98 kDa; glutamic dehydrogenase, 64 kDa; alcohol dehydrogenase, 50 kDa;
carbonic anhydrase, 36 kDa; and myoglobin, 30 kDa); lanes 2 through 4, membrane fractions of S. pneumoniae
(nanA) (parent strain) and pbp2a and
pbp1a insertional mutants, respectively; lanes 5 and 6, membrane fractions of E. coli BL21
(DE3)/pET-11b-pbp2a and E. coli BL21
(DE3)/pET-11b, respectively.
Affinities of GST-PBP 2a* for different -lactam
antibiotics.
To examine the affinities of GST-PBP 2a* for
different -lactam antibiotics, first we analyzed its kinetic
parameters using standard steady-state methods with S2d as a substrate.
The apparent Km and Vmax
(kcat) values obtained for the GST-PBP 2a*
enzyme were 3 mM and 210 nmol/min/mg (0.35 s1),
respectively. Similar results were obtained for PBP 2a* (data not
shown). Thus, the kcat/Km
value for GST-PBP 2a* was determined to be 120 M1
s1, which is in agreement with the value (220 M1 s1) obtained for PBP 2a* that was
purified and refolded by using different methods (5). In
light of these results, our kinetic analyses were performed with
GST-PBP 2a* rather than with PBP 2a*, because both proteins appeared to
exhibit similar kinetic properties, optimal activities at pH 6.0 and
50°C, and similar levels of binding activities to BOCILLIN FL, a
derivative of penicillin V (28, 29).
-lactam antibiotics are in good
agreement with each other (Table 1).
Using the IC50s obtained and the
k2/K value (acylation efficiency) of the enzyme
for cefotaxime (5), we derived the
k2/K values of the enzyme for other antibiotics
tested (10, 11) (Table 1). When compared with other PBPs in
S. pneumoniae (4, 16, 17, 29), clearly,
the k2/K values obtained indicate that GST-PBP 2a* had lower affinities for all -lactam antibiotics tested, in
particular for penicillins (Table 1). In addition, the affinities of
GST-PBP 2a* for penicillins were, in general, lower than for cephalosporins (except ceftazidime) and imipenem (Table 1).
|
1
s1 as determined by a fluorescence-quenching method
(5). Using this fluorescence-quenching method, Di Guilmi et
al. (5) failed to obtain a k2/K value
of PBP 2a* for penicillin G due to its poor affinity for the enzyme.
This observation is consistent with our finding that the affinity of
this enzyme for penicillin G is significantly (approximately 12-fold)
lower than for cefotaxime (Table 1). Furthermore, the
k2/K values of the GST-PBP 2a* protein for
penicillins (Table 1) were significantly lower than the values (58,000, 53,000, 21,000, and 4,900 M1 s1,
respectively) obtained for the penicillin-sensitive PBP 2x of S. pneumoniae (17, 29), which is known as a
primary penicillin-resistance determinant in the cell (12-18, 20,
23, 25, 26). The k2/K values of GST-PBP
2a* for penicillin G and cefotaxime were also significantly lower than
the values (34,200 to 70,100 and 11,900 to 25,800 M1
s1, respectively) obtained for PBP 1a (4).
Finally, the k2/K values of GST-PBP 2a* for
imipenem, a carbapenem, and cefotaxime, a cephalosporin (Table 1), were
also lower than the values (107,000 and 162,000 M1
s1) obtained for the penicillin-sensitive PBP 2x
(17, 29). The k2/K value of PBP 2a
for cephalexin was similar to that (1,600 M1
s1) of PBP 2x (17). Together, the results of
this study suggest that PBP 2a is a low-affinity PBP compared with
other PBPs in S. pneumoniae. These results also
suggest that PBP 2a is a naturally resistant form of PBP and therefore
probably is not a principal target of -lactam antibiotics. It is
also possible that PBP 2a might be able to take over the activity of
other PBPs in the presence of clinically relevant concentrations of
-lactam antibiotics, which may therefore have accounted for its role
in resistance to -lactam antibiotics.
| |
ACKNOWLEDGMENTS |
|---|
We thank P. Treadway for providing S. pneumoniae cells used in this study.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Infectious Diseases Research, Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Drop Code 0438, Indianapolis, IN 46285-0438. Phone: (317) 276-2040. Fax: (317) 276-1743. E-mail: Zhao_Genshi{at}Lilly.Com.
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Antimicrobial Agents and Chemotherapy, June 2000, p. 1745-1748, Vol. 44, No. 6
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46: 1273-1280
[Abstract] [Full Text]
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