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Antimicrobial Agents and Chemotherapy, November 1999, p. 2702-2709, Vol. 43, No. 11
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Competition of Various 
-Lactam Antibiotics for
the Major Penicillin-Binding Proteins of Helicobacter
pylori: Antibacterial Activity and Effects on Bacterial
Morphology
Cindy R.
DeLoney and
Neal L.
Schiller*
Division of Biomedical Sciences, University
of California, Riverside, Riverside, California 92521
Received 15 January 1999/Returned for modification 3 May
1999/Accepted 31 August 1999
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ABSTRACT |
The penicillin-binding proteins (PBPs) of helical (log-phase)
Helicobacter pylori ATCC 43579 were identified by using
biotinylated ampicillin. The major PBPs had apparent molecular masses
of 47, 60, 63, and 66 kDa; an additional minor PBP of 95 to 100 kDa was also detected. The relative affinities of various 
-lactams for these
PBPs were tested by competitive-binding assays. Only PBP63 appeared to
be significantly bound to each of the competing antibiotics, whereas
PBP66 strongly bound mezlocillin, oxacillin, amoxicillin, and
ceftriaxone. Whereas most of the -lactams significantly bound two or
more PBPs, aztreonam specifically targeted PBP63. The influence of
sub-MICs of these -lactams on the morphologies of log-phase H. pylori was observed at both the phase-contrast and transmission electron microscopy levels. Each of the eight -lactams examined induced blebbing and sphere formation, whereas aztreonam was the only
antibiotic studied which induced pronounced filamentation in H. pylori. Finally, studies comparing the PBPs of helical
(log-phase) cultures with those of coccoid (7-, 14-, and 21-day-old)
cultures of H. pylori revealed that the major PBPs at 60 and 63 kDa seen in the helical form were almost undetectable in the
coccoid forms, whereas PBP66 remained the major PBP in the coccoid
forms, although somewhat reduced in level compared to the helical form.
PBP47 was present in both forms at approximately equal concentrations. These studies thus identified the major PBPs in both helical and coccoid forms of H. pylori and compared the relative
affinities of seven different -lactams for the PBPs in the helical
forms and their effects on bacterial morphology.
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INTRODUCTION |
Helicobacter pylori, a
curved, microaerophilic, gram-negative bacterium which colonizes the
mucus layer of the gastric epithelium, is the causative agent of
chronic type B gastritis and has been linked to the development of
peptic ulcers and gastric cancer (see reference 17
for a review). Epidemiologic studies estimate that at least a third of
the world's population is infected with H. pylori,
including about 50% of Americans 60 years old or older, making this
infection one of the most common in the world (11). Although
there are a variety of therapeutic choices for H. pylori infections, most regimens employ various multidrug combinations, including one or more antibiotics and the addition of bismuth, an
H2 receptor antagonist, or a proton pump inhibitor
(17, 22). Therapy often includes the use of one or more
-lactams, including amoxicillin (4, 17, 20, 29). However,
after initial clearance of the infection, there is often a relapse or
recurrence of infection (6, 17). While this recurrence may
be due to the emergence of antibiotic-resistant strains, such as seen
in metronidazole resistance (38), some studies also point to
the importance of coccoid forms of the bacterium (4, 39).
H. pylori has been found to occur in two morphologic forms:
the actively replicating helical or rod form and the round or coccoid
form, which some consider a degenerate form (28) while others consider the coccoid form to be viable but nonculturable (24). While the active helical form is thought to be
responsible for disease production, the nonmotile coccoid form might be
involved in the transmission of H. pylori (33,
39). This coccoid form has been found to be associated with
tissue necrosis (9, 26) and with histopathologic changes in
mouse stomachs (7) and can survive for prolonged periods in
environmental samples such as water (33). The morphologic
conversion from the helical or rod form to the coccoid form has been
examined in various in vitro studies (1, 3, 8, 30, 31, 33,
35), but the pathogenicity of the coccoid form remains
controversial (7, 10, 18, 39, 40).
To identify the antibiotic-binding sites for the various -lactam
antibiotics used in the treatment of H. pylori infection, as
well as to investigate potential factors involved in the dramatic morphological conversion of the helical to the coccoid form, we characterized the penicillin-binding proteins (PBPs) expressed in
helical versus fully coccoid H. pylori. The PBPs are a set of enzymes involved in the synthesis of the peptidoglycan layer of the
bacterial cell wall and include transpeptidases, transglycosylases, endopeptidases, and carboxypeptidases (5, 21). It has been shown that -lactam antibiotics bind covalently to the PBPs, and by
using labeled -lactams, these enzymes can be detected and analyzed
by conventional sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) followed by fluorography (5, 16, 21,
32). Recently, a PBP detection method was developed by Dargis and
Malouin (14), who used biotinylated aminopenicillins in
place of radiolabeled penicillins, providing a method of detection of
these proteins which is both faster and free of hazardous reagents. Using this new approach, we characterized the major PBPs of H. pylori ATCC 43579.
We also compared the relative binding affinities of seven different
-lactams in comparison to biotin labelled ampicillin (Bio-Amp) for
this H. pylori strain and determined the concentration of
each antibiotic needed to inhibit the binding of Bio-Amp by 50% or
greater (i.e., the IC50) for each major PBP. MICs were determined for each -lactam on H. pylori, and the
influence of sub-MICs of each antibiotic on bacterial morphology was
observed by both phase-contrast microscopy and transmission electron
microscopy. Finally, the PBP profiles of fully helical (48-h,
log-phase) cultures were compared to those of broth cultures aged for 7 days (mostly coccoid), 14 days (>99% coccoid), and 21 days (100% coccoid).
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MATERIALS AND METHODS |
Bacterial culture conditions.
Stock cultures of H. pylori ATCC 43579 were streaked for isolation on brucella agar
(Becton-Dickinson Microbiology, Cockeysville, Md.) supplemented with
10% defibrinated sheep blood (Colorado Serum Company, Denver) and 1%
IsoVitalex (Becton-Dickinson Microbiology) and cultured at 37°C in a
humidified 12% CO2 incubator. Liquid cultures were
prepared by suspension of H. pylori colonies in brucella
broth (Difco Laboratories, Detroit, Mich.) supplemented with 10% fetal
calf serum (Gibco Bethesda Research Laboratories, Grand Island, N.Y.)
and 1% IsoVitalex and grown at 37°C in a humidified 12%
CO2 incubator. Cultures were routinely passed by dilution into fresh medium at 48-h intervals; however, in some experiments, bacteria were cultured in the same medium for up to 21 days.
MIC and MBC determinations.
The following -lactams were
prepared in accordance with the manufacturer's instructions and filter
sterilized: amoxicillin (Sigma, St. Louis, Mo.), ampicillin
(Fisher-Biotech, Fair Lawn, N.J.), penicillin G potassium (Marsham,
Cherry Hill, N.J.), aztreonam (Azactam; Squibb, Princeton, N.J.),
mezlocillin (Mezlin; Miles, West Haven, Conn.), oxacillin (Squibb),
ceftriaxone (Rocephin; Roche, Nutley, N.J.), and cefuroxime (Zinacef;
Glaxo, Research Triangle Park, N.C.). Twofold serial dilutions of each
antibiotic ranging from 16 to 0.008 µg/ml were prepared in culture
medium (brucella broth with 10% fetal calf serum and 1% IsoVitaleX), inoculated with a 100-fold dilution of a 48-h culture of H. pylori ATCC 43579, and incubated in 96-well Falcon tissue culture
plates (Becton-Dickinson Microbiology) at 37°C in a humidified 12%
CO2 incubator. The plates were examined for the presence of
turbidity at 24 and 48 h, and the antibiotic concentration in the
well containing the lowest concentration of antibiotic which was
nonturbid was determined to be the MIC. Bacterial cultures grown in
sub-MICs were visualized with a Zeiss phase-contrast microscope to
characterize bacterial morphology. Aliquots of each
bacterium-antibiotic mixture were then spread onto brucella-sheep blood
agar plates in order to determine the MBC of each antibiotic. These
plates were monitored for the presence of colonies at 24, 48, and
72 h of incubation, and the lowest antibiotic concentration which
completely prevented bacterial growth was recorded as the MBC.
Bio-Amp labeling of PBPs.
Two liters of a 48-h culture of
H. pylori was centrifuged at 6,000 × g for
10 min, washed, and resuspended in ice-cold 0.01 M phosphate-buffered
saline (PBS), pH 7.2, treated with lysozyme (1 mg/5 ml of bacterial
suspension, 20 min at room temperature), and sonicated until most of
the cells were disrupted as visualized microscopically. Unbroken cells
were removed by centrifugation at 8,000 × g for 20 min. Inner and outer membranes were concentrated by centrifugation at
100,000 × g for 40 min and washed twice in ice-cold
PBS. Membrane fractions from 5-ml aliquots were frozen at 
20°C
until analyzed.
Membrane pellets were thawed and resuspended in 500 µl of 0.1 M
phosphate buffer, pH 7.2. Bio-Amp was prepared by the method of Dargis
and Malouin (14). Briefly, solutions of biotinamidocaproic acid 3-sulfo-N-hydroxysuccinimide ester (Sigma) and
ampicillin (Fisher-Biotech) in 0.1 M sodium phosphate buffer, pH 7.2, were incubated at a 5:1 ratio for 30 min at room temperature with
gentle agitation. The reaction was then stopped by addition of a
10-fold molar excess of glycine and incubated for 30 min at room
temperature with gentle agitation. The Bio-Amp was then frozen in
aliquots at 80°C for up to 12 months at a final concentration of
800 µg/ml. This biotinylation procedure did not adversely affect the
activity of ampicillin, since the MIC of Bio-Amp was comparable to that of ampicillin (data not shown).
Approximately 1 mg of total membrane proteins was incubated with
Bio-Amp at a concentration of 40 µg/ml for 30 min at room
temperature. The membranes were then washed in 0.1 M phosphate
buffer,
pH 7.2, and centrifuged at 40,000 ×
g for 40 min. The
pellets were then resuspended in 0.01 M phosphate buffer, pH 7.2,
and
the inner membrane proteins were solubilized in 1%
N-lauroylsarcosine
(Sigma) for 20 min. The insoluble
membranes were then removed
by centrifugation at 40,000 ×
g for 40 min, and the supernatant
was used as described
below. In some experiments, the Bio-Amp
was pretreated for 30 min at
room temperature with increasing
amounts of
-lactamase (0.005, 0.05, 0.5, 5 and 50 U/ml; Sigma)
prior to incubation with
H. pylori membranes. Pretreatment of
Bio-Amp with
-lactamase
cleaves the
-lactam ring of the ampicillin
molecule, thus preventing
ampicillin from binding to PBPs via
the
-lactam ring. Bio-Amp
binding not decreased by pretreatment
with
-lactamase was considered
to be due to non-PBP-specific
binding of Bio-Amp.
The supernatant fractions, containing solubilized, labeled membrane
proteins, were then separated by SDS-10% PAGE using ~100
µg of
protein per well. After electrophoresis, proteins were transferred
to
nitrocellulose by using SDS-PAGE running buffer with 5% methanol
at
400 mA of constant current for 2 h. The nitrocellulose
sheets
were then blocked either for 1 h at room temperature or
overnight
at 4°C in 0.01 M PBS-Tween (0.1% Tween 20) containing 5%
nonfat
dry milk. Membranes were rinsed in PBS-Tween three times and
then
incubated in streptavidin-horseradish peroxidase (Amersham,
Arlington
Heights, Ill.) diluted 1:1,000 in PBS-Tween for 1 h at
room temperature.
The membranes were then rinsed three times in
PBS-Tween and examined
for luminescence by the ECL protocols described
by the manufacturer
(Amersham). Membranes were exposed to ECL Hyperfilm
(Amersham)
for 10 to 60 s until banding patterns appeared.
Molecular weights
were determined by comparison to ECL molecular weight
standards
(Amersham). Reported molecular weights were the averages
determined
from blots from at least three separate experiments. For
comparison
experiments, resulting protein patterns were read for
absorbance
intensities using densitometric tracings with an Ultroscan
XL
laser densitometer (LKB Products, Bromma,
Sweden).
PBP patterns were also compared in aged versus log-phase
H. pylori cultures. Membrane fractions were prepared as described
above from 48-h-old (log-phase) and 7-, 14-, and 21-day-old cultures.
Morphologies of bacteria at all of these time points were observed
by
phase-contrast
microscopy.
Antibiotic competition experiments.
In antibiotic
competition experiments, log-phase H. pylori membrane
fractions were prepared as described above and incubated with each
antibiotic at 1, 10, and 100 times the MIC for 30 min at room
temperature. These samples were then incubated with 40 µg of Bio-Amp
per ml for 30 min at room temperature. Proteins were then prepared as
described above and compared for banding pattern intensity using
densitometric tracings, and the IC50s of each antibiotic
for the major PBPs were approximated by using tracing data.
Transmission electron microscopy.
Cultures were grown in the
presence or absence of antibiotics at sub-MICs for 24 to 48 h.
After incubation in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH
7.4, for 60 min, the bacteria were washed twice in sterile
double-distilled H2O (ddH2O) with gentle centrifugation and resuspended in a final volume of 50 µl in sterile ddH2O. Copper grids (100-mesh thick bar) were prepared with
a Formvar-coated support film, heavy carbon coated, and then glow discharged treated in an Edwards 306 vacuum evaporator (Edwards High
Vacuum International, Wilmington, Mass.) to make the surfaces of the
grids hydrophilic. After addition of samples to the grids, all of the
liquid was drawn off, the samples were washed three times with sterile
ddH2O, and all of the liquid was again drawn off. For
unidirectional shadowing, samples were prepared by using a tungsten
hairpin filament with 10 mm of 80% platinum-20% palladium metal wire
at a 20°C angle in an Edwards 306 vacuum evaporator. Grids were
observed with a Hitachi H-600 transmission electron microscope (Hitachi
Instruments, Inc., San Jose, Calif.) operating at 75 kV at
magnifications ranging from ×4,000 to ×40,000.
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RESULTS |
Identification of PBPs of H. pylori.
Treatment of
H. pylori membrane fractions with Bio-Amp identified a
number of membrane proteins which were potential PBPs (Fig.
1, lane C). Because of possible
nonspecific binding of Bio-Amp to non-PBP membrane proteins,
additional experiments were conducted with Bio-Amp pretreated
with increasing concentrations of -lactamase. As shown in Fig. 1,
increasing concentrations of -lactamase significantly decreased
Bio-Amp labeling of proteins with molecular masses of 47, 60, 63, and
66 kDa. Although the protein band at 47 kDa did not completely
disappear with -lactamase treatment, the intensity of the band
decreased by greater than 50% in this experiment and almost completely
disappeared in other experiments and in antibiotic competition
experiments (described later). A minor (i.e., less-abundant) PBP which
was also affected by -lactamase treatment was seen at 95 to 100 kDa.
The protein bands seen at 51 and 54 kDa in this gel were not
consistently observed in repeat studies and are unlikely to be major
PBPs. Protein bands which were not significantly reduced in intensity
by the addition of -lactamase were considered to be the result of
nonspecific Bio-Amp binding. Based on this analysis, the major PBPs
identified by labeling with Bio-Amp in helical H. pylori had
apparent molecular sizes of 47, 60, 63, and 66 kDa.
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FIG. 1.
Log-phase H. pylori membrane fractions were
labeled with either Bio-Amp (lane C) or Bio-Amp pretreated with
increasing amounts of -lactamase (lanes 0.05U, 0.5U, 5U, and 50U).
Protein bands which were not decreased in intensity by the use of
Bio-Amp pretreated with increasing -lactamase concentrations were
considered to be proteins nonspecifically bound with Bio-Amp (NSB).
Lane M represents the ECL molecular size markers, while the molecular
sizes on the right reflect estimates for PBPs determined as the
averages from at least three separate blots.
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When decreasing concentrations of
H. pylori membrane
fractions labeled with Bio-Amp were analyzed densitometrically for PBP
band intensities, PBP66 labeling remained the most intense throughout
the dilution series (data not shown). Although less than that
of PBP66,
Bio-Amp labeling of PBP60 and that of PBP63 were roughly
equivalent in
intensity and labeling of PBP47 was clearly less
than that of the other
three PBPs. Therefore, PBP66 appears to
be the most abundant
Bio-Amp-labeled PBP in
H. pylori.
PBP binding study
competition between various -lactams and
Bio-Amp.
The MIC of each antibiotic used was first determined as
described in Materials and Methods, and the results are shown in Table 1. Aztreonam, the only monobactam
examined, had the highest MIC, twice that of oxacillin and more than 10 to 100 times greater than those of the other antibiotics used. As
expected, the MBCs of each of these antibiotics were either the same or
2 to 4 dilutions above the MIC. In the following experiments, each
antibiotic was preincubated with H. pylori membrane
fractions at a concentration equal to 10 or 100 times the MIC for 30 min prior to the addition of Bio-Amp. Reduction in Bio-Amp labeling of
PBPs with increasing concentrations of each -lactam represents
competition due to prebinding of these PBPs by that specific
antibiotic. A representative experiment illustrating the results of
this type of analysis using penicillin G is shown in Fig.
2; in order to demonstrate quantitative differences in PBP binding, laser densitometric tracings were performed
and a summary of these studies is shown in Fig.
3.
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TABLE 1.
Determination of the MIC and MBC of each -lactam
antibiotic for H. pylori ATCC 43579 and the effects of
sub-MICs of these antibiotics on bacterial morphology
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FIG. 2.
H. pylori membrane fractions were
preincubated with penicillin G at 1, 10, or 100 times the MIC prior to
labeling with Bio-Amp, separated by SDS-PAGE, and visualized on Western
blots by chemiluminescence. The control lane (C) represents PBPs
labeled by Bio-Amp in the absence of penicillin G. The locations of
major PBPs at 66, 63, 60, and 47 kDa are shown on the left. The
decreased Bio-Amp labeling intensity of these PBPs in the presence of
increasing concentrations of penicillin G reflects competition between
penicillin G and Bio-Amp for binding sites on these PBPs. Densitometric
tracings of these protein bands are shown in Fig. 3A.
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FIG. 3.
H. pylori membrane fractions were
preincubated with each of the antibiotics shown at 1, 10, or 100 times
the MIC prior to labeling with Bio-Amp, separated by SDS-PAGE, and
visualized on Western blots. These blots were then quantitatively
examined by laser densitometry. Control (Bio-Amp labeling without
preincubation with a competing antibiotic), open bars; MIC, dotted
bars; 10 times the MIC, hatched bars; 100 times the MIC, solid bars.
Abs., absorbance.
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In every experiment, PBP66 was clearly the protein which reacted most
intensely with Bio-Amp whereas the relative concentrations
of the other
three major PBPs varied between membrane preparations.
Of the major
H. pylori PBPs identified, only PBP63 appeared to
be
significantly bound (
75% reduction in Bio-Amp binding) by
each of
the antibiotics examined in this study. Aztreonam, the
only monobactam
used, showed preferential saturation of PBP63
by as little as 10 times
the MIC, with less preference for PBP66
(31% reduction with 100 times
the MIC), PBP60 (47% with 100 times
the MIC), and PBP47 (48% with 100 times the MIC). Of the four
penicillins studied, only mezlocillin
showed almost complete saturation
of each PBP, with PBP63 being 90%
saturated with a concentration
as low as the MIC. Oxacillin showed a
similar pattern, although
with somewhat less affinity for PBP47.
Amoxicillin appeared to
bind preferentially to PBP60, even at the MIC,
and was near saturation
at 100 times the MIC while significantly
binding to PBP63 and
PBP66 at 100 times the MIC. Penicillin G appeared
to bind with
some affinity to all of the PBPs but was less effective at
competing
with Bio-Amp than was oxacillin or mezlocillin and did not
completely
saturate any of the PBPs, even at 100 times the MIC.
Ceftriaxone
appeared to bind to all of the PBPs, with near saturation
of all
of the proteins except PBP47, although Bio-Amp binding to this
protein was reduced by 75% by the MIC. Cefuroxime showed more
specificity for
H. pylori PBPs than did ceftriaxone, with
preferential
binding to PBP63 (saturation using 100 times the MIC) and
PBP66
(53% saturation at 100 times the MIC). Bio-Amp labeling of PBP60
was not significantly affected by cefuroxime, and PBP47 was only
affected (63% reduction) at 100 times the
MIC.
The IC
50s of each antibiotic for the four major PBPs were
estimated by densitometry, and the results are shown in Table
2.
Each value represents the
concentration of
-lactam (at 1, 10,
or 100 times the MIC) required
to inhibit
50% of the binding
of Bio-Amp at 40 µg/ml. Mezlocillin
was most effective at inhibiting
Bio-Amp binding to each major PBP,
with an IC
50 of 0.125 µg/ml
for each PBP, while
ceftriaxone inhibited the binding of Bio-Amp
to PBP63, PBP60, and PBP47
at 0.25 µg/ml. Aztreonam, with the
highest MIC, inhibited the binding
of Bio-Amp to PBP63 at the
MIC (8 µg/ml) but required
800 µg/ml
to inhibit the binding of
Bio-Amp to the other PBPs. Amoxicillin
competed very well with
Bio-Amp for each of the PBPs, with only 0.03 µg/ml required to
inhibit Bio-Amp binding to PBP60 and only 3 µg/ml
required for
PBP66, PBP63, and PBP47. Penicillin G showed a similar
pattern;
however, greater than 3 µg of this antibiotic per ml was
required
to inhibit the binding of Bio-Amp to PBP66. Greater
concentrations
of oxacillin and cefuroxime were required to inhibit
Bio-Amp binding
to the major PBP of
H. pylori, with 20 µg
of oxacillin per ml
and 6 µg of cefuroxime per ml required to inhibit
Bio-Amp binding
to PBP66 and PBP47.
Effects of sub-MICs on H. pylori morphology.
Having demonstrated that each of the eight -lactam antibiotics was
active against this H. pylori strain and bound, with
differential preferences, to the major PBPs, we next examined the
effect of each of these antibiotics on bacterial morphology. Table 1
summarizes the results of these experiments, and representative
transmission electron micrographs are shown in Fig.
4. Each of these antibiotics, when used
at one-half to one-fourth of the MIC, induced the formation of
spherical cells (large cells with few cytoplasmic elements) and
membrane blebbing. The addition of aztreonam induced similar morphologies but also led to pronounced filamentation, with many filaments 5 cells or greater in length and some spanning the length of
the focal field (Fig. 4D). Interestingly, none of the penicillins or
cephalosporins used in this study caused significant filamentation (>3
cells in length) at sub-MICs.
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FIG. 4.
Transmission electron micrographs illustrating
representative examples of log-phase H. pylori control
bacteria (A), spherical cell formation typical of that seen after
treatment with various -lactam antibiotics (B), membrane blebbing
(indicated by the arrowhead) seen after treatment with various
-lactam antibiotics (C), and significant filamentation, which was
only observed in cultures exposed to sub-MICs of aztreonam (D). The bar
in each graph represents 1 µm. Note that the spherical imperfections
in the background are artifacts due to the formation of holes in the
Formvar-coated support film.
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Comparison of PBP profiles of helical and coccoid forms of H. pylori.
Continued incubation of H. pylori in liquid
culture led to conversion of the helical form of H. pylori
to the coccoid form within 3 to 5 days of incubation at 37°C, and
after 7 to 14 days, >99% of the bacteria were observed to be coccoid,
whereas by day 21, only coccoid forms were seen. Comparative binding
studies using Bio-Amp and membranes obtained from bacteria grown for 2, 7, 14, and 21 days are shown in Fig. 5.
The intensity of Bio-Amp labeling for both PBP60 and PBP63 was markedly
reduced (by 44% on day 7 and 81% on day 14), and labeling all but
disappeared by day 21 (PBP60 labeling was reduced by 96%, and PBP63
labeling was reduced by 86%). On the other hand, Bio-Amp labeling of
PBP47 did not vary significantly from day 2 to day 21. PBP66, the PBP most abundantly labeled by Bio-Amp in helical H. pylori, was
retained over the course of this 21-day study, with only modest
reductions in intensity on days 7 and 14 and only a 32% decrease by
day 21. Although the results are not shown here, control studies using -lactamase pretreatment of Bio-Amp were performed to confirm that
each of the proteins labeled with Bio-Amp in these aged cultures was a
PBP.
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FIG. 5.
Membrane fractions of H. pylori liquid
cultures harvested after 2, 7, 14, or 21 days of incubation at 37°C
were labeled with Bio-Amp, separated by SDS-PAGE, and visualized on
Western blots. These blots were then quantitatively examined by laser
densitometry, and the results of a representative experiment are shown.
Abs. absorbance.
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DISCUSSION |
Previous studies by Dargis and Malouin (14)
demonstrated that labeling of PBPs with Bio-Amp was comparable to that
in studies using radiolabeled penicillin. Using this approach, we
identified four major PBPs in helical H. pylori ATCC 43579PBP47, PBP60, PBP63, and PBP66. We also noted the presence of an
additional Bio-Amp-labeled protein at a molecular mass of 95 to 100 kDa
which we consider to be a minor PBP. Some additional protein bands
which were occasionally labeled with Bio-Amp proved to be due to
nonspecific binding since these proteins were labeled even after the
Bio-Amp had been treated with -lactamase to destroy the normal
PBP-binding site of ampicillin. Similar nonspecific labeling of
proteins was also noted by Dargis and Malouin (14), who used
several different bacterial species, as well as by Galleni et al.
(19) who used Escherichia coli. To the best of
our knowledge, Ikeda et al. (25) are the only other
investigators who have examined PBPs in H. pylori; they used
14C-labeled penicillin G and identified three PBPs in
H. pylori FP1532, although they did not report the molecular
weights of these proteins.
Examination of the H. pylori 26695 genome sequence reported
by Tomb et al. (37) revealed the presence of three genes
with protein sequence homologies to E. coli PBPs: HP 597, which has 33.7% identity to PBP1A and a molecular mass of 74.23 kDa;
HP 1556, which has 30.6% identity to FtsI (also known as PBP3) and a
molecular mass of 69.07 kDa; and HP 1565, which has 35% identity to
PBP2 and a molecular mass of 67.02 kDa. Based on this information, we
predict that PBP66, PBP63, and PBP60 correspond to these three known
gene sequences whereas the H. pylori gene corresponding to
PBP47 remains to be identified. PBP47 may represent a
"nontraditional" PBP with a sequence which is not closely
homologous to the other three PBPs described above.
In order to further characterize the PBPs in H. pylori, we
examined the relative binding affinities of each of the -lactam antibiotics for the four major H. pylori PBPs and the
subsequent effects of these antibiotics at sub-MICs on bacterial
morphology. This approach mimics that of other investigators who
characterized the various PBPs in E. coli and identified
their roles in peptidoglycan formation. These investigators examined
the varied effects of -lactam antibiotics on cellular division,
elongation, and shape in E. coli by comparing the binding
affinities of different -lactams for the known E. coli
PBPs. These studies revealed that PBP1 of E. coli is
involved in bacterial elongation, PBP2 is responsible for the rod
shape, and PBP3 is involved in septum formation (12, 13,
36). These proteins have been called the "essential" PBPs of
E. coli, while those with lower molecular weights appear to be dispensable (12, 13, 21, 36). The inhibition of these PBPs results in the various morphologies seen at the MICs of the respective -lactamse.g., inhibition of PBP2 results in round E. coli cells and inhibition of PBP3 results in
filamentation (13, 36).
Kamimura et al. (27) noted that low concentrations of
cefixime caused filamentation in E. coli by binding to PBP3
and bacteriolysis at high concentrations by binding to PBP1A and PBP1B.
In contrast, Ikeda et al. (25) found that the same treatment
of H. pylori caused rounded cells at low concentrations,
with cefixime bound primarily to the protein they refer to as PBP B
(molecular weight unreported). Armstrong et al. (2) treated
Campylobacter pyloridis (since renamed H. pylori)
with amoxicillin, benzylpenicillin, and cephalexin at concentrations
above the determined MICs and found the normal bacilliform morphology
to be replaced by bulging and dumbell-like profiles with cell wall
blebbing and vesiculation and eventually by swollen forms with
incomplete cell walls undergoing lysis. Berry et al. (4)
noted that amoxicillin at 10 times the MIC was bactericidal for
H. pylori but also induced the formation of coccoid forms.
In the present study, we compared relative binding intensities of
various -lactam antibiotics for the four major H. pylori PBPs with their effects on bacterial morphology. Each of the
antibiotics was found to be bacteriocidal for H. pylori,
although the concentrations required to achieve this effect differed
significantlythese differences may be accounted for in part by the
relative abilities of the antibiotics to diffuse across the outer
membrane of H. pylori. This would explain the major
difference in the MICs and MBCs of oxacillin and mezlocillin, while
their relative abilities to bind each of the major PBPs were almost
identical (note that the labeling studies were done with membrane
fractions and not intact bacteria).
From these studies, we conclude that PBP66, PBP63, and PBP60 are the
major PBPs bound by the -lactams used in this study and that the
interaction of these PBPs with sub-MICs of these antibiotics was
responsible for producing the observed morphological changes in
H. pylori. PBP63 was the only PBP to be significantly bound
by each of the -lactams studied (75% binding) and therefore may
be an essential PBP for helical H. pylori. It is of note
that aztreonam was the only -lactam to preferentially bind to a
single PBP, PBP63, and this antibiotic was the only one in the study which induced pronounced filamentation in H. pylori at
sub-MICs. Satta et al. (32) found aztreonam to also saturate
a single PBP, PBP3 (60 kDa) in E. coli, which has been
determined to be involved in septum formation (12, 13, 36).
Therefore, by analogy, it is possible that PBP63's main function in
H. pylori is in septum formation.
The four penicillins examined here showed rather similar binding
patterns. The results obtained with mezlocillin are particularly impressive, with an IC50 of only 0.125 µg/ml for each
PBP. The IC50s of amoxicillin and penicillin G were
similar, except that amoxicillin bound more competitively to PBP66 than
did penicillin G. With the exception of amoxicillin, the
IC50s of all of the -lactams were the lowest for PBP63,
indicating that it is the preferred target of these antibiotics. As
expected, unlabeled ampicillin competed very effectively with Bio-Amp
for binding to each of the four major PBPs (data not shown).
With the exception of aztreonam, the other seven -lactams bound
significantly to more than one PBP and induced sphere formation and
blebbing without significant filamentation at sub-MICs. While aztreonam
also induced some sphere formation and blebbing at sub-MICs, filamentation was the dominant altered morphology observed. Thus, with
most of the -lactams, binding to one or more of the PBPs leads to
inhibition of one or more of the enzymes involved in peptidoglycan
biosynthesis, resulting in destabilization of cell structure and
membrane disruption, as seen with membrane blebbing and sphere
formation. In the case of aztreonam, the dominant enzymatic activity
being inhibited is most likely that of PBP63, resulting in
filamentation, while the other peptidoglycan-building enzymes are only
somewhat inhibited, which resulted in some blebbing and sphere
formation. PBP47 may not be as essential to the growth of H. pylori as PBP60, PBP63, and PBP66, since this protein was significantly bound by only two of the antibiotics studied, while the
higher-molecular-weight PBPs were significantly bound by at least four
of the -lactam antibiotics. We also found an additional minor PBP of
95 to 100 kDa, which was somewhat affected by competitive binding with
one or more of the -lactam antibiotics (data not shown). The
significance of this putative minor PBP for the growth and morphology
of helical H. pylori is not known.
Aging of liquid cultures of H. pylori resulted in conversion
of the helical form to the coccoid form. Examination of the Bio-Amp labeling of PBPs in these aged cultures demonstrated major reductions in PBP60 and PBP63, whereas PBP47 was only slightly affected. PBP66
seemed to be the only major PBP to be significantly retained, with only
a 32% reduction in Bio-Amp staining intensity by day 21. A prominent
band appeared in older cultures with a molecular mass of 28 kDa (data
not shown); however, this band was not reduced in intensity with
-lactamase treatment of Bio-Amp and was therefore determined to be
either the result of nonspecific binding or possibly a degradative
product of one of the PBPs. We cannot distinguish between the
possibilities that the decrease in the PBPs of H. pylori
that accompanies aging is due to a decrease in the expression of these
proteins or to degradation of the proteins. If the coccoid form is
simply a degenerative form of the bacterium, then protein degradation
would certainly be expected; however, the retention of PBP47 and PBP66
argues against this being the only explanation for these findings.
Alternatively, since the coccoid form appears to be dormant, enzymes
needed for septum formation and elongation are no longer needed.
Conserving energy by no longer synthesizing unneeded enzymes would be a
prudent strategy.
-Lactam antibiotics, particularly amoxicillin, play a major role in
the treatment of H. pylori infections. However, several investigators (23, 34) have found that continued exposure to
amoxicillin can increase the MIC of amoxicillin, and Dore et al.
recently (15) reported that one reason for the failure of amoxicillin-omeprazole treatment of H. pylori infection is
the presence of amoxicillin-resistant strains. These studies point to
the importance of continued surveillance for the presence and emergence
of amoxicillin-resistant H. pylori strains. In addition, it
is possible that relapses or recurrences of H. pylori
infection arise because of the presence of dormant forms of the
bacterium (coccoids) which are unlikely to be sensitive to -lactam
antibiotics because these coccoids are not actively dividing and have
different PBP profiles than the helical, dividing forms. Additional
studies focusing on the interaction of -lactam antibiotics with the
PBPs of H. pylori (both helical and coccoid forms) will
provide more information important for guiding therapeutic
interventions to prevent recurrent H. pylori infections.
| |
ACKNOWLEDGMENTS |
We thank Sacred Heart Medical Center of Spokane, Wash., for
supplying the antibiotics at cost and F. Malouin of Microcide Pharmaceuticals for help with the biotinylation studies. We also greatly appreciate the assistance of J. Kitasako and P. Desjardins of
the Plant Pathology Department at the University of California, Riverside, in preparing the transmission electron micrographs and the
photographic assistance of R. Hatch.
| |
ADDENDUM IN PROOF |
After the manuscript was submitted for publication, Krishnamurthy
et al. (J. Bacteriol. 181:5107-5110, 1999) reported the
presence of four PBPs in H. pylori, including a novel PBP with significantly increased expression during mid- to late-log-phase growth, and Dore et al. (Helicobacter 4:154-161, 1999) reported the presence of four PBPs in amoxicillin-sensitive
H. pylori, but only three of these PBPs were found in
amoxicillin-resistant strains, suggesting a role for the small PBP in
the amoxicillin-resistant phenotype of H. pylori.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Biomedical Sciences, University of California, Riverside, Riverside, CA 92521. Phone: (909) 787-4569. Fax: (909) 787-5504. E-mail:
neal.schiller{at}ucr.edu.
| |
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Top
Abstract
Introduction
