Antimicrobial Agents and Chemotherapy, November 1998, p. 2883-2888, Vol. 42, No. 11
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effect of Disruption of a Gene Encoding an
Autolysin of Enterococcus faecalis OG1RF
Xiang
Qin,1,2,3
Kavindra V.
Singh,1,3
Yi
Xu,3,4
George M.
Weinstock,2,3 and
Barbara E.
Murray1,2,3,*
Division of Infectious Diseases, Department
of Medicine,1
Department of Microbiology
and Molecular Genetics,2 and
Department of Biochemistry and Molecular
Biology,4
Center for the Study of
Emerging and Re-emerging Pathogens,3
University of Texas Medical School, Houston, Texas 77030
Received 9 February 1998/Returned for modification 16 June
1998/Accepted 19 August 1998
 |
ABSTRACT |
A mutant (TX5127) of Enterococcus faecalis OG1RF was
generated by disruption mutagenesis of a previously described autolysin gene. TX5127 formed longer chains (2 to 10 cells per chain) than wild-type OG1RF (mainly single cells) during growth in broth even though it had a growth rate similar to that of the parental strain as
measured by turbidity and cell count. Autolysin activity, as defined by
the ability to lyse heat-killed Micrococcus lysodeikticus cells, was absent in TX5127, while this activity was easily detectable in OG1RF. However, disruption of this autolysin gene did not block the
ability of TX5127 to hydrolyze E. faecalis cell walls
compared to that of OG1RF. The autolysis rate of cells of TX5127 in 10 mM sodium phosphate buffer (pH 6.8) was slower than that of wild-type OG1RF. TX5127 also showed a decreased rate of lysis in the presence of
penicillin, as measured by changes in the turbidity of the culture
during 24 h of incubation at 37°C and a slightly decreased effect of penicillin as measured by time-kill curves. The virulence of
TX5127 was similar to that of OG1RF in the mouse peritonitis model,
indicating that the autolysin of E. faecalis is not
important for infection in this model.
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INTRODUCTION |
Enterococci are among the more
common causes of hospital-acquired infections and, among all
enterococcal infections, Enterococcus faecalis is the most
commonly recovered species (22). In recent years, the
treatment of enterococcal infections has become more and more difficult
because of the increasing antibiotic resistance of these organisms. One
of the problems with enterococci is their relative resistance to
penicillin and other 
-lactams. Low-level resistance to -lactams
is intrinsic and appears to be due to the low affinity of enterococcal
penicillin-binding proteins to penicillin (14, 34). In
addition to this resistance, enterococci are often tolerant to
-lactams; that is, -lactams have low bactericidal effects. The
mechanism of tolerance of enterococci to -lactams is still unclear.
However, it has been demonstrated that resistance to penicillin and
tolerance to penicillin are two distinguishable features of E. faecalis, because they could be elicited independently by in vitro
exposure to penicillin (14). The tolerance of E. faecalis to -lactams has been suggested as being associated
with the autolysis system (29). Storch et al.
(29) have shown that an increase in autolytic activity in
clinical isolates was correlated with increases in penicillin-induced
lysis and killing. In addition, Fontana et al. reported that E. faecalis strains which lacked or had diminished autolysin activity
were less susceptible to the bactericidal activity of penicillin
(13).
Autolysins of enterococci have been characterized primarily from
Enterococcus hirae ATCC 9790. Two forms of autolysins have been reported in E. hirae, namely, muramidase-1, defined by
the ability to lyse E. hirae cell walls, and muramidase-2,
defined by the ability to lyse lyophilized Micrococcus
lysodeikticus cells (9, 10, 17, 27). Muramidase-1 is a
-1,4-N-acetylmuramidase with an 87-kDa active form and a
latent form that can be activated by trypsin (10), while
muramidase-2 exists in a 125-kDa active form and a 75-kDa active form
(9). Autolysin activities in E. faecalis have
been reported, including one which could lyse heat-killed M. lysodeikticus cells and another which could lyse heat-killed
E. faecalis cells; the proteins with autolytic activities were shown to have molecular masses and substrate specificities similar
to those of E. hirae (13). An E. faecalis gene encoding an autolysin of unknown specificity has
been cloned and sequenced by Béliveau et al. (1).
These authors reported that two E. coli clones containing
the gene had multiple active forms of autolytic activity which could
lyse lyophilized M. lysodeikticus cells and E. faecalis cell walls that were contained in a renatured sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. This
autolysin has a predicted size of 74 kDa. However, the physiological
functions of the autolysin of E. faecalis are still unknown.
In our initial immunoscreening of a genomic library of E. faecalis OG1RF, a predominant antigen detected by human patient sera and immune rabbit serum (36) was found to be an
autolysin encoded by the gene reported by Béliveau et al.
(1). In this study, we generated an autolysin mutant and
studied both the mutant and parental strains for autolytic activities,
autolysis, penicillin-induced lysis, penicillin resistance, and
virulence to determine whether this dominant antigen is a virulence
factor. To our knowledge, this is the first report of a targeted
disruption of an enterococcal autolysin.
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MATERIALS AND METHODS |
Strains and media.
The E. faecalis strain used in
this study, OG1RF, has been described previously (23).
pBluescript SK(
) was used for routine cloning (Stratagene, La Jolla,
Calif.). Brain heart infusion (BHI) medium (Difco Laboratories,
Detroit, Mich.) was used for growth of E. faecalis unless
otherwise stated. SR medium (8) was used for electroporation
of E. faecalis. The concentrations of antibiotics used for
selection were as follows: for Escherichia coli,
tetracycline, 12.5 µg/ml; kanamycin, 25 µg/ml; for E. faecalis, kanamycin, 2,000 µg/ml; fusidic acid, 25 µg/ml.
Penicillin G was purchased from Sigma Chemical Co. (St. Louis, Mo.).
DNA techniques.
Plasmid DNA from E. coli was
isolated by the alkaline SDS method as previously described
(4). Transformation of E. coli was performed
according to the method described by Calvin and Hanawalt
(5). Transformation of E. faecalis by
electroporation was carried out as described previously
(18). Chromosomal DNA of E. faecalis and Southern
blots were prepared as described previously (26, 35).
Mutagenesis.
In order to generate a targeted autolysin
mutant of E. faecalis OG1RF, an internal fragment (from 564 to 1,617 bp) of the previously described autolysin gene (1)
was released from plasmid YX7 (36) and cloned into plasmid
pTEX4577 (29), which contains a kanamycin resistance gene of
gram-positive origin (12), by using ScaI and
KpnI sites. The resulting construct, pTEX4581, which carries
the internal fragment of the autolysin gene, was electroporated into
E. faecalis OG1RF followed by selection on SR agar plates
with 2,000 µg of kanamycin per ml. Since pBluescript SK() lacks the
origin of replication of a gram-positive plasmid, kanamycin-resistant
colonies should represent mutants which integrated the recombinant
plasmid into the autolysin gene by homologous recombination. The
correct mutation was confirmed by Southern blotting analysis, and the
autolysin mutant was designated TX5127.
Assay for autolysin (muramidase-2-like) activity.
Determination of the ability of E. faecalis OG1RF and TX5127
to produce autolysin, as defined by the ability to lyse heat-killed M. lysodeikticus cells (muramidase-2-like lytic activity)
(1, 16), was carried out as described previously
(13). In brief, 10 µl of the bacteria to be assayed was
spotted onto the surface of Todd-Hewitt (TH) agar containing
heat-killed cells of M. lysodeikticus (Micrococcus
luteus) (Sigma Chemical Co.) adjusted to an optical density at 600 nm (OD600) of 0.5, and the plates were incubated at 37°C
for 48 h. Bacteria that showed a clear lysis zone on heat-killed M. lysodeikticus cell plates were considered autolysin
(muramidase-2-like lytic enzyme) producing (9, 17).
The ability to produce autolysin was further studied by SDS-PAGE
(1). Briefly, 10 ml of overnight cultures of E. faecalis OG1RF and TX5127 was harvested by centrifugation and
resuspended in 1 ml of denaturing buffer (2% dithiothreitol, 15%
sucrose, 3.8% SDS). The culture supernatants were mixed with an equal
volume of 2× denaturing buffer. Samples were then placed in a boiling water bath for 3 min. Twenty-five-microliter aliquots from cell or
supernatant preparations were applied to an SDS-PAGE (10%
polyacrylamide) gel containing 0.2% lyophilized M. lysodeikticus cells. After electrophoresis, the gel was renatured
by incubation for 48 h in 25 mM Tris (pH 8) buffer containing 1%
Triton at room temperature. Lytic activity could be visualized as clear
bands on the opaque SDS-PAGE gel.
Assay for muramidase-1-like lytic activity.
In order to
determine whether the disruption of the gene coding for the cloned
autolysin would alter the expression of other lytic enzymes, we also
measured the ability of the wild type and the mutant strain to lyse
E. faecalis cell walls (analogous to muramidase-1 activity
of E. hirae, which was defined by the ability to lyse
E. hirae cell walls) (10, 13). The preparation of enzyme and E. faecalis OG1RF cell walls and the assay of
enzymatic activity were conducted as previously described
(16).
Autolysis assay.
Cell autolysis was determined by a
modification of the method of Massidda et al. (20).
Six-milliliter cultures of OG1RF or TX5127 grown in THGB (TH broth
[Difco Laboratories] supplemented with 2% glucose) were removed at
different growth phases (exponential phase, late exponential phase, and
stationary phase), chilled on ice, and filtered (0.45-µm pore size;
Millipore Corp., Bedford, Mass.), washed three times with distilled
water at 4°C, and resuspended in 6 ml of 10 mM sodium phosphate
buffer (pH 6.8) with or without trypsin (0.5 µg/ml). Trypsin was used
because it has been reported that in E. hirae, muramidase-1
is present in two forms, the active form and the latent form, which
could be activated by trypsin (25, 27). The suspension was
then incubated at 37°C, and the OD675 was measured at
15-min intervals for up to 6 h.
Penicillin effects.
Penicillin-induced lysis was measured by
the methods previously described (13). In brief, bacterial
cells from a late-exponential-phase culture were diluted 1:20 in fresh
BHI medium to an OD660 of 0.08 to 0.10. Penicillin (stock
solution, 1,024 µg/ml) was added to obtain the desired
concentrations. Cultures were incubated at 37°C, and 1-ml aliquots
were removed at different time points to measure the OD660.
MICs were determined by agar dilution as described previously
(24) with increments of 0.25 µg/ml, and E. faecalis ATCC 29212 was employed as a control. Counts for
time-kill assays (n = 6) were performed according to
the method previously described (21) to compare the
bactericidal activities of penicillin against E. faecalis
OG1RF and TX5127. Statistical analysis was performed with Student's
two-tailed t test.
Morphological examination and growth rate.
To determine
whether mutation of the gene encoding autolysin caused any change in
E. faecalis OG1RF morphologically, we examined the cells of
OG1RF and TX5127 by light microscopy. Cells were stained with Gram
stain (Difco Laboratories) according to the protocol supplied and were
examined with an American Optical microscope.
To determine the growth rate, overnight cultures of E. faecalis OG1RF and TX5127 were diluted 1:20 in BHI and grown at
37°C with shaking. The turbidity was measured at different time
points with a Manostat turbidometer (Manostat, New York, N.Y.). CFU
were determined by serial dilution of the cultures in saline and
plating them onto BHI agar plates in duplicate.
Mouse peritonitis model.
E. faecalis OG1RF and TX5127
were grown overnight in BHI broth. The cells were harvested by
centrifugation, washed once with 0.9% saline, and then resuspended in
saline. Serial dilutions were made in saline and were mixed (1:10) with
50% sterile rat fecal extract (SRFE). Groups of six outbred (ICR)
female mice 4 to 6 weeks old (weighing 22 to 25 g) were challenged
intraperitoneally with different inocula (15). A control
group of mice was injected with 50% SRFE only. Survival was monitored
every 12 h. Determination of Kaplan-Meier survival curves and log
rank analysis were performed as described previously (28).
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RESULTS AND DISCUSSION |
Mutagenesis.
After transformation of 100 µl of E. faecalis cells with 5 µl of pTEX4581, the construct carrying the
internal fragment of the autolysin gene, 28 Kanr colonies
were recovered on SR medium-kanamycin (2,000 µg/ml) plates.
Hybridization of Southern blots of the chromosomal DNA from one of
these colonies by using the internal fragment of the autolysin gene as
a probe showed the correct chromosomal insertion in the autolysin gene
(data not shown); this insertion mutant derivative was designated TX5127.
Assay for autolysin (muramidase-2-like) activity.
On plates
containing heat-killed M. lysodeikticus cells, OG1RF showed
a clear zone of lysis around the colonies, while the autolysin mutant
failed to show clearing (data not shown), indicating that the gene
product had been inactivated or was not readily secreted. Similarly,
with renatured SDS-PAGE gels containing lyophilized M. lysodeikticus cells, no band of clearing was shown for whole cells
of TX5127, while OG1RF showed clear bands similar to those previously
reported (Fig. 1) (1). Similar
results were shown for supernatants of TX5127 and OG1RF, respectively
(data not shown). The fact that TX5127 lost the ability to hydrolyze
the lyophilized cells of M. lysodeikticus would classify
this gene product as a muramidase-2-like enzyme based on the
definitions used for E. hirae (17).
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FIG. 1.
Cell wall lytic activity of OG1RF and TX5127 against
M. lysodeikticus cells in renatured SDS-PAGE. Lane 1, whole
cells of TX5127; lane 2, whole cells of OG1RF.
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Assay for muramidase-1-like lytic activity.
To determine
whether the inactivation of this autolysin would alter the effect of
other autolytic enzymes, we measured muramidase-1-like activity in
OG1RF and TX5127. Hydrolysis of E. faecalis cell walls by
TX5127 from different growth phases was similar to that of wild-type
OG1RF (data not shown).
Autolysis assay.
In order to detect whether inactivation of
this autolysin gene affected the autolysis of E. faecalis
cells, cultures of OG1RF and TX5127 from different growth phases (early
exponential phase, late exponential phase, and stationary phase) were
used in an autolysis assay. As shown in Fig.
2, there is a partial inhibitory effect
of the mutation on exponential-phase cells' autolysis but a much more
dramatic inhibitory effect on late-log or stationary-phase cells'
autolysis. One interpretation is that other enzymes are involved in the
early exponential phase but that the muramidase-2-like enzyme mainly
works in the late log or stationary phase. Perhaps the expression of
lytic enzymes is regulated in different growth phases. Another possible
explanation is that the substrates (cell wall) of this autolysin and
other lytic enzymes are different in different growth phases. The
effect of trypsin on autolysis was also investigated. TX5127 showed a
slower autolysis rate than OG1RF in the presence or absence of trypsin,
although in the presence of trypsin, there was more rapid autolysis of
both OG1RF and TX5127 (Fig. 2), similar to that shown by Cornett et al.
with both a wild-type strain and a muramidase-2 mutant strain of
E. hirae (7). This suggests that this autolysin
and other lytic enzymes are involved in autolysis and that other lytic
enzymes may be activated by trypsin.
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FIG. 2.
Autolysis of OG1RF (circles) and TX5127 (squares). (A)
Cells from early exponential phase. (B) Cells from late exponential
phase. (C) Cells from early stationary phase. Cells were collected on
filters and resuspended in 10 mM sodium phosphate (pH 6.8). The changes
in turbidity were measured by a spectrophotometer at 675 nm. Open
symbols, without trypsin; solid symbols, with trypsin. Standard errors
were in a range of between 0 and 6% at OD675.
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Penicillin effects.
It has been suggested that autolysins of
E. faecalis are associated with the bactericidal activity of
penicillin (13)
that is, that suppression of the activity
of a cell's autolytic activity could protect bacteria from the
bacteriolytic antibiotics like penicillin. To test this possibility,
penicillin at concentrations of 4, 16, and 64 µg/ml was added to
E. faecalis OG1RF and TX5127 cultures, and the turbidities
of the culture were monitored. With penicillin at 4 and 16 µg/ml,
TX5127 showed less decrease in OD660 than OG1RF after
24 h of incubation (Fig. 3). When a
higher concentration of penicillin was used, the difference in
OD660 of these two strains was less pronounced, as was the
decrease in OD660, suggestive of an Eagle effect
(11). Overall, a concentration of 4 µg/ml generated the
greatest decrease in OD660 for both strains. These results
suggest that inactivation of this autolysin may partially suppress
penicillin-induced lysis. However, mutation of this autolysin gene did
not change the MIC for E. faecalis. The MICs of penicillin for both OG1RF and TX5127 are the same (3.5 µg/ml with 0.25-µg/ml increments), indicating that disruption of the autolysin gene does not
increase penicillin resistance.
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FIG. 3.
Penicillin-induced lysis of OG1RF (open symbols) and
TX5127 (solid symbols). Penicillin stock solution was added at time
zero to obtain final concentrations of 4 (circles), 16 (triangles), and
64 (squares) µg/ml. Turbidity was monitored at OD660.
Insert, OG1RF (open diamonds) and TX5127 (solid diamonds) without
penicillin. Standard errors are indicated by error bars.
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Time-kill curves were used to test whether inactivation of this
autolysin would protect bacteria from the killing effect of penicillinthat is, whether inactivation would increase the tolerance of TX5127 to penicillin compared to that of OG1RF. OG1RF was killed moderately well by 5 µg of penicillin per ml with a decrease of 2.8±0.2 log10 CFU/ml at 24 h. Both OG1RF and TX5127
could still grow slowly in 2 µg of penicillin per ml, but OG1RF
showed about 0.6 log less growth than TX5127 at 24 h (P < 0.001) (Table 1). In 5 and 10 µg of penicillin per ml, TX5127 showed about 0.6 less log reduction
than OG1RF (P = 0.03 and 0.04, respectively) (Table 1),
a small but statistically significant difference. These data indicate
that inactivation of this autolysin may partially protect the bacterial
cells from the lytic effect of penicillin, but does not convert the
mutant into a strain as tolerant as those in vitro-selected tolerant
strains reported by Hodges et al., which did not show a decrease in CFU
per milliliter after 24 h of exposure to 10× the MICs of
penicillin (14).
Morphological examination and growth rate.
Examination of
OG1RF and TX5127 by light microscopy showed that TX5127 formed chains
compared with parental strain OG1RF. Figure
4 shows the distribution of the number of
cells in the chains of OG1RF and TX5127. The majority of the chains of
TX5127 had 2 to 10 cells per chain, while OG1RF mainly existed as
single cells. Morphological change in E. hirae has been
previously reported by Lleò et al. for thermosensitive mutants,
which showed great reduction in the production of muramidase-1 and
formed elongated cells at nonpermissive temperature (19).
Llèo et al. postulated that muramidase-1 might be associated with
the formation of septa, while muramidase-2 might be involved in
separation of daughter cells. Our results in E. faecalis are
consistent with their hypothesis for E. hirae. The formation
of chains of TX5127 indicates that this autolysin (muramidase-2-like
enzyme) is required for appropriate cell separation in E. faecalis.
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FIG. 4.
Distribution of the number of cells in the chains of
OG1RF and TX5127. The chains counted were randomly chosen. Standard
errors are shown by error bars.
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Since it appeared that this autolysin affected cell separation, we also
examined the growth rate of the TX5127 in comparison with that of
OG1RF. The growth rate of TX5127 in BHI broth was similar to that of
OG1RF measured in Klett units (data not shown). The number of CFU of
TX5127 was somewhat lower than that of OG1RF (data not shown), probably
due to the formation of chains. However, this autolysin of E. faecalis clearly was not essential for cell growth, and there were
still some single cells in the culture of TX5127, suggesting that a
muramidase-1-like autolysin of E. faecalis or other lytic
enzymes may substitute for the function of this autolysin. Double
mutation of the muramidase-1-like autolysin and this autolysin may be
able to further address this question.
Mouse peritonitis model.
Berry et al. have reported that
following the interruption of the major autolysin gene,
Streptococcus pneumoniae type 2 and type 3 strains were less
virulent (2, 3). However, Tomasz et al. reported that
interruption of the autolysin gene in type 3 pneumococci had no effect
on virulence (31). This led to the hypothesis that the role
of autolysin in pneumococcal infections might vary from serotype to
serotype (3). It has been suggested that the possible role
of autolysin in infections by pneumococci may be to facilitate the
release of toxins and/or inflammatory cell wall breakdown products
(6, 32). Using a mouse peritonitis model, we studied the
virulence of TX5127 in comparison to that of OG1RF. The 50% lethal
dose (LD50) of TX5127 was 3.0 × 108 CFU,
similar to the LD50 of OG1RF (3.2 × 108
CFU). The time course of death for TX5127 was also similar to that of
OG1RF (e.g., P = 0.2735 by log rank test for survival after inoculation of 3.2 × 108 CFU of OG1RF versus
3.0 × 108 CFU of TX5127 [data not shown]),
suggesting that this enterococcal autolysin does not play an important
role in infection in the model that we used or that inactivation of
autolysin could be compensated for by other factors.
Conclusions.
It has been proposed that -lactam-induced
lysis of bacteria is the result of inhibition of biosynthesis of cell
walls and the hydrolysis of cell walls by cellular autolytic enzymes
(30, 33). Tomasz et al. showed that suppression of or a
defect in autolytic enzyme(s) in pneumococci was associated with a
simultaneous increase in viability of pneumococci cells in the presence
of penicillin (30). Storch et al. have shown that increases
in penicillin-induced lysis in clinical isolates of E. faecalis were correlated with an increase in autolytic activity
(29). It was also shown by Fontana et al. that in E. faecalis, reduction of the ability to lyse E. faecalis
cell walls was associated with decreased bactericidal activity of
penicillin against E. faecalis clinical isolates
(13). In our study, even though we found that interruption
of the muramidase-2-like hydrolytic activity gene decreased the rates
of autolysis and penicillin-induced lysis, we did not detect an
increase in penicillin resistance (by MIC), and interruption produced,
at most, only a small increase in tolerance by time-kill assay. These
data suggest that alterations in the autolysin gene previously
characterized by Béliveau et al. (1) or its expression
are not responsible for the tolerance of some clinical or in
vitro-derived isolates of E. faecalis to -lactam killing
effects. We have also shown here, by using intraperitoneal challenge of
mice, that this autolysin gene had no significant effects on the
virulence of E. faecalis. Further study would be needed to
determine whether this autolysin is involved in other enterococcal
infections or contributes to the virulence of other strains of E. faecalis.
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ACKNOWLEDGMENT |
This work was supported in part by NIH grant AI33516 from the
National Institute of Allergy and Infectious Diseases to B.E.M.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for the
Study of Emerging and Re-emerging Pathogens, Division of Infectious Diseases, Department of Medicine, University of Texas Medical School,
6431 Fannin St., Houston, TX 77030. Phone: (713) 500-6767. Fax: (713)
500-5495. E-mail:
iminfdis{at}heart.med.uth.tmc.edu.
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Antimicrobial Agents and Chemotherapy, November 1998, p. 2883-2888, Vol. 42, No. 11
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
