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Journal of Clinical Microbiology, March 2001, p. 995-1001, Vol. 39, No. 3
Department of Oral Microbiology, GKT Dental
Institute, King's College London,1
Department of Oral Microbiology,
SBRLHT,2 and Department of Medical
Microbiology, King's College Hospital,3 London,
United Kingdom
Received 21 August 2000/Accepted 28 December 2000
The production of mannosidase activity by all currently recognized
species of human viridans group streptococci was determined using an
assay in which bacterial growth was dependent on the degradation of the
high-mannose-type glycans of RNase B and subsequent utilization of
released mannose. RNase B is an excellent substrate for the
demonstration of mannosidase activity since it is a glycoprotein with a
single glycosylation site which is occupied by high-mannose-type glycoforms containing five to nine mannose residues. Mannosidase activity was produced only by some members of the mitis group (Streptococcus mitis, Streptococcus oralis, Streptococcus
gordonii, Streptococcus cristatus, Streptococcus infantis,
Streptococcus parasanguinis, and Streptococcus
pneumoniae) and Streptococcus intermedius of the
anginosus group. None of the other species within the salivarius and
mutans groups or Streptococcus peroris and
Streptococcus sanguinis produced mannosidase activity.
Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, it was demonstrated that the Man5 glycan
alone was degraded while Man6 to Man9, which
contain terminal The viridans group streptococci,
which comprise a significant proportion of the normal flora of the
oropharyngeal tract (8), form a highly heterogenous group
of organisms (13, 37). Over the last decade,
identification of these organisms at the species level has been
facilitated by the development of a range of rapid phenotypic tests,
some of which rely, at least in part, on the detection of preformed
glycosidase activities, enzymes with the potential to cleave sugar
residues from a range of glycoconjugates, including glycoproteins. A
number of schemes for the identification of individual species of the
viridans group streptococci have included phenotyping for the
production of a range of glycosidases using synthetic substrates,
glycosides with a fluorogenic (4-methylumbelliferone [4MU]) or
chromogenic (p-nitrophenol [pNP]) leaving group, some of
these being supplied as part of commercially available kits, such as
the API-20STREP system (2, 5, 9, 18, 19, 20, 36, 39).
These schemes for the classification of the viridans group streptococci
have enabled associations between the isolation of a particular species
and specific extra-oral diseases to be established (15, 16,
38), which is of major importance due to the emergence of these
bacteria as significant pathogens of immunocompromised patients and
neonates (1, 3, 29, 35). Little is known regarding the
mechanisms by which these streptococci grow at sites removed from the
oral cavity, but it has been suggested that glycosidase production may
play a nutritional role by releasing carbohydrates which are essential for proliferation from host glycoproteins (1).
In a recent investigation it was demonstrated that the acute-phase
human serum glycoprotein,
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.3.995-1001.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mannosidase Production by Viridans Group
Streptococci
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(1
2) mannose residues in addition to the
(13), (16), and 
(14) residues present in
Man5, remained intact. Investigations on mannosidase production using synthetic (4-methylumbelliferone- or
p-nitrophenol-linked) - or -mannosides as substrates
indicated that there was no correlation between degradation of these
substrates and degradation of the Man5 glycan of RNase B. No species degraded these -linked mannosides, while degradation of
the -linked synthetic substrates was restricted to strains within
the Streptococcus anginosus, S. gordonii, and S. intermedius species. The data generated using a native
glycoprotein as the substrate demonstrate that mannosidase production
within the viridans group streptococci is more widely distributed than had previously been considered.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1-acid glycoprotein, supported in vitro growth of Streptococcus oralis (4), an
important pathogen within the viridans group streptococci which is the
cause of a large number of cases of infective endocarditis and
septicemia in neutropenic patients (1, 6, 7).
1-acid glycoprotein, which comprises five fully
sialylated complex-type oligosaccharide chains, was degraded to release
all nonterminal sugar residues, including those mannose residues which,
in addition to two N-acetylglucosamine residues, form part
of the conserved pentasaccharide core
(GlcNAc2Man5) of N-linked glycans. Thus, novel
mannosidase activities were demonstrated in S. oralis and
these enzymes were not detected using synthetic substrates. These
observations were supported by demonstration of in vitro growth of
S. oralis strain AR3 on RNase B (30). This
glycoprotein contains a single glycosylation site, and
posttranslational modification results in the addition of
high-mannose-type glycans containing five to nine mannose residues
attached to the chitobiose core (Man5 to Man9)
(10, 24). Using a combination of high-pH anion-exchange
chromatography with pulsed amperometric detection and matrix-assisted
laser desorption ionization-time of flight mass spectrometry
(MALDI-TOF MS), it was demonstrated that S. oralis produced
-(13), -(16), and -(14) mannosidase activities which
resulted in the degradation of the Man5 glycoform only
(Fig. 1), leaving the remaining
glycoforms (Man6 to Man9), which contain additional -(12)-linked mannose residues, intact
(30).
View larger version (12K):
[in a new window]
FIG. 1.
The oligosaccharide structure of the Man5
glycoform of RNase B. Asn represents the asparagine of the polypeptide
backbone. N-Acetylglucosamine and mannose are represented by
GlcNAc and Man, respectively, and numbers indicate the positions of the
glycosidic linkages for mannose residues. The Man6 to
Man9 glycoforms of RNase B are formed by the addition of
further mannose units [all in the -(12) configuration] to the
outer three residues occurring in the Man5 glycoform.
In the present study, we have extended our studies on mannosidase production by S. oralis strain AR3 to all currently recognized human species of viridans group streptococci. We have used assays in which bacterial growth on the model glycoprotein RNase B is dependent on the production of mannosidase activity and utilization of liberated mannose and have monitored changes in RNase B glycoforms by MALDI-TOF MS.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains and culture.
Representative strains,
including type strains, of all currently recognized species of human
viridans group streptococci were used (Table
1). Isolates were stored in vials
containing cryopreservative (TSC Ltd., Heywood, Lancashire, United
Kingdom) at 
70°C and were routinely maintained by subculture onto
Fastidious Anaerobe Agar (LabM, Bury, Lancashire, United Kingdom)
supplemented with 5% (vol/vol) defibrinated horse blood (FAA).
Cultures were incubated in an anaerobic cabinet (80% N2,
10% H2, 10% CO2; Don Whitley, Shipley, West
Yorkshire, United Kingdom) for 24 h prior to use in growth
studies.
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Growth of bacteria in minimal media. Bacterial colonies were removed from FAA plates into prereduced, filter-sterilized (0.2-µm-pore-size filters; Pall Gelman Sciences, Northampton, United Kingdom) 50 mM sodium phosphate buffer (pH 7.5) to yield a uniform suspension with an A620 of approximately 0.5. These suspensions were used as inocula for minimal media.
Minimal medium was prepared based on a modification of previously described methods (21, 32), with all sources of fermentable carbohydrates omitted. Preliminary investigations had demonstrated that growth of viridans group streptococci in this sugar-depleted medium was low unless supplemented with a source of fermentable carbohydrate. RNase B (from bovine pancreas; Sigma Chemical Co., Poole, Dorset, United Kingdom) was reconstituted and dialyzed against two changes of distilled water at 4°C. The glycoprotein was lyophilized and reconstituted to yield a concentration of 10 mg/ml. Basal medium was mixed with an equal volume of either 10 mg of RNase B/ml, 20 mM mannose, 20 mM glucose, 20 mM N-acetylglucosamine, or water and was filter sterilized (0.2-µm-pore-size membrane filtration unit). Media were dispensed into a sterile 96-well flat-bottom microtiter tray (Sterilin) in 200-µl aliquots, and the tray was prereduced for ca. 1 h in an anaerobic cabinet. An aliquot of bacterial suspension (10 µl) was added to each well, and 10 µl of sterile sodium phosphate buffer (pH 7.5) was added to control wells. Trays were sealed and incubated for up to 40 h at 37°C in a shaking-plate-reading spectrophotometer (iEMS; Labsystems, Life Sciences International, Hampshire, United Kingdom) with automated data acquisition. A620 was recorded every 30 min with a 10-s period of shaking (300 rpm) prior to each absorbance reading. For strains of viridans group streptococci which would not grow under these conditions, which were all members of the anginosus group (Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius), alternative growth conditions were employed. Supplemented basal media were dispensed into sterile bijou bottles (Sterilin) in 1-ml aliquots, prereduced under anaerobic conditions and inoculated with 50 µl of bacterial suspension prepared in sterile 50 mM sodium phosphate buffer. An aliquot of culture was removed for the determination of A620, and the remaining culture was incubated anaerobically. At the end of the growth period, the absorbance of the each culture was determined as a measure of growth. At the end of the growth period, RNase B-supplemented cultures were transferred to microcentrifuge tubes with a nominal volume of 1.5 ml and centrifuged at 11,600 × g for 3 min at ambient temperature to pellet bacterial cells. The cell-free culture supernatants were transferred to fresh microcentrifuge tubes and stored at20°C for a period of up to 2 weeks prior to analysis by
MALDI-TOF MS.
Assay for glycosidase activities using synthetic substrates.
Glycosidase activities of bacterial suspensions from FAA plates were
assayed using fluorogenic (4MU-linked) or chromogenic (pNP-linked)
substrates. Fluorogenic assays were set up in 96-well microtiter trays
and each were comprised as follows: 50 µl of 0.2 mM
4MU--D-mannopyranoside,
4MU--D-mannopyranoside, or
4MU--D-N-acetylglucosaminide (Sigma), 50 µl of 0.2 M sodium phosphate buffer (pH 7.0), an appropriate volume
of cell suspension (up to 50 µl), and distilled water to a total
volume of 200 µl. Trays were incubated at 37°C for up to 24 h and
fluorescence values were recorded in a fluorimeter fitted with a
96-well plate reader (Fluoroscan Ascent FL; Labsystems) at excitation
and emission wavelengths of 355 and 460 nm, respectively. Released 4MU
was quantified by comparison with standard curves of authentic 4MU
(Sigma) obtained under the same conditions. Chromogenic assays were set
up in 96-well microtiter trays and each was comprised as follows: 25 µl of 0.2 mM pNP--D-mannopyranoside or
pNP--D-mannopyranoside (Sigma), 25 µl of 0.2 M sodium
phosphate buffer (pH 7.0), an appropriate volume of cell suspension (up
to 25 µl), and distilled water to a total volume of 100 µl. Trays
were incubated at 37°C for up to 24 h, and then the reaction was
terminated and the pH of each assay was elevated by the addition of 100 µl of 0.1 M sodium carbonate buffer (pH 10.2). Absorbances at 450 nm
were recorded by a 96-well plate-reading spectrophotometer (Titertek
Multiscan MCC340), and released pNP was quantified by comparison with
standard curves of authentic pNP (Sigma) obtained under the same
conditions. Whole-cell protein in bacterial suspensions was determined
using the bicinchoninic acid assay described by Smith et al.
(28; reagents were purchased from Sigma) and was
quantified by comparison with bovine serum albumin as a standard. Where
appropriate, specific activities of enzymes are given as
nanomoles/hour/milligram of whole-cell protein. Assays were carried out
on at least two separate occasions, and mean data are presented.
MALDI-TOF-MS analysis of RNase B. RNase B-supplemented culture supernatants were diluted 10-fold with 0.1% trifluoroacetic acid (Sigma), and 0.5 µl of each was applied to a gold-coated target plate. 3,5-Dimethoxy-4-hydroxycinnaminic acid (sinapinic acid; Aldrich) was used as matrix. This was freshly prepared to a concentration of 10 mg/ml in 50% acetonitrile in 0.1% trifluoroacetic acid, and 0.5 µl was applied to each sample. Spectra were acquired using a PerSeptives Voyager MALDI-TOF MS system operating in linear mode with delayed extraction and a nitrogen laser giving 337-nm output. Scans (typically 256) were acquired and averaged, and mass values were calibrated by reference to intact RNase B as an external standard or RNase A, which forms a minor component of all Sigma preparations of RNase B (10) as an internal standard.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Growth on RNase B as a measure of mannosidase production.
There was a linear relationship between the concentration of mannose
supplied to S. oralis ATCC 35037 and Streptococcus
mutans ATCC 25175 and the maximum increase in absorbance of
cultures over the concentration range of 0 to 10 mM (r > 0.9 for both strains; data not shown), demonstrating the
dependence of growth on the presence of a source of fermentable
carbohydrate as the supplement to minimal medium. Both strains grew on
10 mM mannose with a maximum increase in A620 of
0.299 and 0.304, respectively (Fig. 2).
RNase B supported growth of S. oralis cells, with a maximum
increase in A620 of 0.067 (Fig. 2b), but
S. mutans cells did not utilize the glycans of RNase B, with
no significant increase in absorbance of the culture when compared with
unsupplemented minimal media.
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) or
RNase B (
), and growth was measured by monitoring
A620.
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Growth of bacteria on RNase B and mannosidase production monitored
by MALDI-TOF MS.
Growth on RNase B was a reliable indicator of the
production of mannosidase activity and thus the ability to degrade the
Man5 glycoform of the glycoprotein, releasing fermentable
carbohydrate (Table 2). For isolates
which lacked this ability, the maximum increase in
A620 for RNase B-supplemented minimal media was
always less than 0.010 when compared with the control cultures without an added carbohydrate source, and no degradation of the RNase B
glycoforms (Man5 to Man9) could be detected
when culture supernatants were analyzed by MALDI-TOF MS. All isolates
grew in glucose-supplemented minimal media (Table 2) and virtually all
strains utilized mannose as efficiently as glucose, as judged by the
maximum increase in A620 over a 24-h period and
the doubling time of the cultures (data not shown). One notable
exception to this was the low rate of growth of the Streptococcus
peroris isolates on mannose, suggesting that mannose was utilized
less efficiently than glucose.
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-(13), -(16), and -(14) mannosidase activities, these isolates also produced N-acetylglucosaminidase activity. Among the mitis group,
Streptococcus sanguinis and S. peroris were the
only species which did not exhibit Man5-degrading activity
(Table 2). For strains degrading the Man5 glycan of RNase
B, there was no evidence for proteolytic cleavage of the RNase B
polypeptide backbone; no new species with an m/z
ratio of less than 13,682 were detected in spent culture supernatants.
Glycosidase activities measured using synthetic substrates.
Measurement of mannosidase activities using chromogenic or fluorogenic
substrates revealed a different pattern of production by viridans group
streptococci, and there was no correlation between the production of
these enzymic activities and the ability to degrade RNase B (Table 2).
None of the isolates investigated degraded
4MU--D-mannopyranoside or
pNP--D-mannopyranoside, all strains yielding specific
activities of less than 0.5 nmol/h/mg of bacterial protein.
4MU--D-mannopyranoside and
pNP--D-mannopyranoside degradation was limited to
isolates of S. gordonii, Streptococcus anginosus,
and S. intermedius, with all strains degrading these substrates. Specific activities for the degradation of
4MU--D-mannopyranoside within these isolates ranged
from 1.1 to 9.8 nmol/h/mg of bacterial protein (data not shown). All
strains which degraded the Man5 glycan produced
N-acetylglucosaminidase activity, which was detectable using
either the respective chromogenic or fluorogenic substrates, with the
notable exception of S. mitis strains, which did not hydrolyze this substrate. All isolates which degraded the
Man5 glycoform grew when N-acetylglucosamine was
provided as the supplement to minimal media (data not shown).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In our earlier studies with S. oralis strain AR3, we
demonstrated the presence of - and -mannosidase activities which
cleaved mannose residues from the core pentasaccharide of the complex N-glycans of human serum 1-acid glycoprotein
(4). Subsequently, we used bovine pancreatic RNase B to
show that of all the high-mannose glycans (Man5 to
Man9), the Man5 glycoform alone was degraded during in vitro growth of S. oralis cells in what appeared
to be a single-step reaction involving these - and -mannosidases (30), and the mannosidase activities were undetectable
with synthetic substrates. We now extend these observations to all currently recognized species of human viridans group streptococci by
using RNase B as a model. The ability to utilize the high-mannose glycans of RNase B as sole carbohydrate sources during in vitro growth
was restricted to the mitis group (S. oralis, S. mitis, Streptococcus parasanguinis, S. gordonii, S. cristatus, S. infantis, and
S. pneumoniae) and one species within the anginosus group, namely S. intermedius. Analysis of the spent culture
supernatants from these organisms by MALDI-TOF MS of RNase B
demonstrated that only the Man5 glycoform was degraded,
yielding a product corresponding to the polypeptide backbone with a
single N-acetylglucosamine residue attached. This
degradation may be accounted for by the production of -(13),
-(16), and -(14) mannosidase activities and a
-N-acetylglucosaminidase activity cleaving the
chitobiose core. The lack of ability to degrade glycoforms higher
than Man5 has been ascribed to the lack of an -(12)
mannosidase in S. oralis (30), and our data
indicate that all viridans group streptococci degrading RNase B do so
via a mechanism similar to that observed for S. oralis. In
addition, the degradation of the Man5 glycoform of RNase B
is brought about without production of a detectable endoglycosidase
activity (30), thus differing from the mechanism by which
these glycans are utilized by Enterococcus faecalis
(23), formerly part of the genus Streptococcus.
These mannosidase activities were not detected using synthetic
substrates. -Mannosidase activity was detected in only a limited number of species of the viridans group streptococci (S. gordonii, S. intermedius, and S. anginosus)
when assayed using synthetic 4MU-linked or pNP-linked substrates, and
these data were consistent irrespective of the leaving group or the
concentration of substrate presented to bacterial cell suspensions.
This extends the observations of Kilian et al. (20), who
reported the activity in S. gordonii and S. anginosus only when using a chromogenic substrate. In addition, we
have used the corresponding -linked mannosides as substrates and
demonstrated that none of the viridans group streptococci had the
capacity to degrade these substrates. -Mannosidases from yeast or
mammalian sources which cleave -(12) mannose residues from native
glycoproteins or free glycans have previously been shown to lack
activity when pNP--D-mannoside or
4MU--D-mannoside was presented as a substrate
(26) and that synthetic substrates may underrepresent the
incidence of this enzyme activity. Given the failure of synthetic
substrates to detect mannosidases, a number of strategies have been
adopted for the measurement of the these enzymes, including monitoring
the release of mannose from free oligosaccharides and resolving
enzyme-treated glycans using high-performance liquid chromatography
methodologies (26, 34). In the present study, we have
utilized MALDI-TOF MS to monitor RNase B degradation. MALDI-TOF MS is
particularly applicable to the rapid analysis of biological samples due
to its ability to tolerate high concentrations of salts and buffers as
encountered in biological systems and is relatively rapid. This
methodology has recently been shown to be applicable to the detection
of mannosidases acting on RNase B (31) and has been
applied here to investigate the presence of mannosidase activities
within the viridans group streptococci.
The recent availability of genomic sequence data for a number of
bacterial species, including S. pneumoniae
(http://www.tigr.org/tdb, The Institute for Genomic Research) has
facilitated investigations linking genome organization with the
production of functional proteins. Interrogation of these data
using the WIT suite of software (WIT;
http://wit.integratedgenomics.com/IGwit) indicates the
presence of an open reading frame in S. pneumoniae coding for a protein with homology to an
hypothetical -mannosidase present in E. faecalis and
Streptococcus pyogenes (P score = 1.17 × 10
115 and 6.86 × 10111,
respectively). These in silico data are in accordance with our demonstration of functional -mannosidase in this organism and other
closely related species. There is no evidence for a -mannosidase in
this genomic data, but this may form one of a number of genes for which
no function can be ascribed at present. Further biochemical characterization of mannosidase of viridans group streptococci and the
substrate specificity of the enzyme(s) will be required to elucidate
the mechanism by which this degradation is achieved.
The function of mannosidases produced by viridans group streptococci is unclear. High-mannose-type glycans are frequent constituents of host tissues and are found as components of the extracellular matrix, e.g., laminin, being involved in a wide range of cellular functions (11, 25, 27). In addition, the major platelet integrin, GP IIb/IIIa, contains high-mannose-type oligosaccharides, predominantly the Man5 and Man6 glycoforms (33). Degradation of the Man5 glycoform of human glycoproteins in vivo may not only modulate their function but also release carbohydrates and play a role in the persistence of viridans group streptococci at infection sites. We have demonstrated that mannosidase activities are produced by major pathogens within the viridans group of streptococci, including S. oralis and S. mitis, which are associated with infective endocarditis and infections in immunocompromised patients, S. pneumoniae, which is the cause of pneumonia, otitis media, and meningitis, and S. intermedius, which is associated with purulent abscesses of the liver and brain (3, 12, 13, 22, 37). The effect of mannosidase activities on host tissues and high-mannose-type glycoproteins remains to be established. The ability of viridans group streptococci to grow on RNase B, however, may be a useful test to assist in the identification of this complex group of microorganisms.
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ACKNOWLEDGMENTS |
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This work was supported in part by grant PG/97015 from the British Heart Foundation.
Y. Kawamura (Department of Microbiology, Gifu University School of Medicine, Gifu, Japan) is thanked for providing S. infantis and S. peroris strains.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Oral Microbiology, GKT Dental Institute, King's College London, Caldecot Rd., Denmark Hill, London SE5 9RW, United Kingdom. Phone: 44 20 7346 3272. Fax: 44 20 7346 3073. E-mail: karen.a.homer{at}kcl.ac.uk.
Present address: Glaxo Wellcome, Stevenage, United Kingdom.
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Journal of Clinical Microbiology, March 2001, p. 995-1001, Vol. 39, No. 3
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.3.995-1001.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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