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Department of Medical Microbiology, Lund University, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strains of S. pyogenes can also be divided into two major lineages, OF+ and OF-, based on their ability to produce opacity factor (OF),3 a secreted protein with apoproteinase activity (5, 6). The role of OF in S. pyogenes infections is not clear, but the ability, or lack of ability, to produce this protein appears to reflect a fundamental difference between strains and is correlated to several other properties, such as M protein structure and expression of Fc receptors (7, 8, 9, 10). With regard to molecular mechanisms of disease, it is therefore of considerable interest to compare the properties of OF+ and OF- strains.
For many years, it was assumed that strains of S. pyogenes express a single antiphagocytic member of the M protein family. However, many strains (both OF+ and OF-) are now known to express more than one M-like protein, the biologic role of which is unclear (11). In particular, all strains of the OF+ lineage have three linked genes encoding members of the M protein family: the mrp, emm, and enn genes (10, 11, 12, 13). This triplet of genes has also been found in some OF- strains (12, 13), but these OF- strains are of rare serotypes (14). Thus, the presence of a triplet of genes encoding the Mrp, Emm, and Enn proteins can be considered to be a characteristic property of OF+ strains.
Little is known about the biologic function of the Mrp, Emm, and Enn proteins encoded by OF+ strains, and the contribution of these proteins to phagocytosis resistance is unclear. The Enn protein has not been shown to be expressed on the surface of OF+ bacteria, and the enn gene is transcribed at a very low level, if at all, making it unlikely that Enn makes any contribution to phagocytosis resistance in these strains (15, 16, 17). In contrast, the Mrp and Emm proteins are known to be expressed on the bacterial cell surface (18), suggesting that they may have antiphagocytic function. Interestingly, Mrp and Emm show considerable structural differences (19, 20) and do not cross-react immunologically (21). The ligand-binding properties of Mrp and Emm are also different: Mrp binds fibrinogen (18, 20) and Emm binds the complement inhibitor C4BP (22), ligands that may be of importance for phagocytosis resistance (23, 24, 25). In addition, both Mrp and Emm bind Ig-Fc, a property that might contribute to virulence (18, 19, 26, 27).
Surprisingly, recent studies have suggested that the contribution of Mrp and Emm to phagocytosis resistance may vary between different strains (28, 29). However, these studies employed strains that had been passed extensively through mice or in the laboratory, procedures that are known to alter the expression of S. pyogenes surface proteins and virulence (3, 30, 31). In fact, some of the strains that were analyzed grew poorly in human blood, unlike fresh clinical isolates of S. pyogenes (3, 32). This situation made it desirable to study phagocytosis resistance in an ordinary OF+ clinical isolate of S. pyogenes.
Here, we report a study of a clinical isolate of serotype M22, one of the most common S. pyogenes serotypes in the Western world (14, 33). Like other OF+ isolates of S. pyogenes, this strain expresses a fibrinogen-binding Mrp protein and a C4BP-binding Emm protein, and it grows rapidly in blood. The construction of a double mutant lacking both Mrp and Emm, and comparison of this strain with strains deficient in only one of the proteins, allowed us to directly assess the contribution of these two proteins to phagocytosis resistance. The results indicate that each of the Mrp and Emm proteins is sufficient to confer phagocytosis resistance in whole blood, i.e., they are antiphagocytic M proteins according to the classical definition (2, 3).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For historical reasons, the proteins in the M protein family expressed by OF+ strains have received many different names. Here, we follow the simplified nomenclature proposed by Whatmore et al. (34), and use the designations mrp, emm and enn for the three genes present in OF+ strains. Following standard rules, the corresponding three proteins are designated Mrp, Emm, and Enn. It follows that the protein that was previously referred to as Sir22 (18, 35) is referred to here as Emm22, etc. However, the term "M protein" is reserved for a protein that has been shown to have antiphagocytic function (2, 3).
Bacterial strains, bacteriophage, plasmids, and media
AL168 is a nonmucoid M22 strain of S. pyogenes, isolated in 1989 from a Swedish patient with acute pharyngitis (18). Following primary isolation, the strain was immediately frozen at -80°C, and bacteria from the original ampoules were used for this study. A streptomycin-resistant mutant of AL168 (AL168str-r) was isolated and shown to have the same properties as the parental strain with regard to binding of different ligands and ability to grow in human blood. Strain JRS4 and its derivative JRS145, which has a deletion in the structural gene for the M6 protein (36), were obtained from Dr. J. R. Scott (Emory University, Atlanta, GA). A derivative of JRS145 expressing the Emm22 (Sir22) protein was constructed by transformation with plasmid pSIR2202 (35). The Enterococcus faecalis strain CG110, carrying Tn916 (37), was provided by Dr. D. Clewell (University of Michigan, Ann Arbor, MI). M13 was used as the vector for the sequencing of the mrp22 gene and was propagated in Escherichia coli JM101. Plasmid pJRS233 (38) was provided by Dr. J. R. Scott. Strains of S. pyogenes were grown in Todd-Hewitt broth (TH) (Difco, Detroit, MI), which was supplemented with 0.2% yeast extract (THY) for experiments involving transposon mutagenesis, electroporation, and phagocytosis. Sheep blood agar plates and THY plates were used for growth on solid phase media. Strains of E. coli were grown in LB broth or in 2x YT (39). Antibiotics were used in the following concentrations: 100 µg/ml ampicillin, 200 µg/ml erythromycin, and 50 µg/ml kanamycin for work with E. coli; and 5 µg/ml erythromycin, 200 µg/ml kanamycin, 1 mg/ml streptomycin, and 10 µg/ml tetracyclin for S. pyogenes.
DNA preparation, cloning techniques, and sequencing
Plasmid DNA was prepared by the Qiagen tip-100 purification system (Qiagen, Hilden, Germany). Standard procedures were used for work with phage M13 and restriction enzymes (39). The mrp22 gene was amplified by PCR from AL168 chromosomal DNA, using primers derived from the surrounding mga and emm genes (35, 40), restriction enzyme digestion of the PCR fragments, and subcloning into M13 mp18/19. Chain termination ssDNA sequencing was performed with Sequenase (United States Biochemical, Cleveland, OH) according to the instructions of the manufacturer. PCR products obtained in three independent reactions were sequenced.
Proteins
Fibrinogen was from IMCO (Stockholm, Sweden). C4BP, purified from human plasma (41), was the kind gift of Dr. Björn Dahlbäck, Malmö General Hospital, Malmö, Sweden. Polyclonal IgG was from Pharmacia (Uppsala, Sweden) and polyclonal serum IgA was from Cappel-Organon Teknika (Turnhout, Belgium). All proteins were of human origin.
Tn916 mutagenesis: isolation of mrp22 andmga22 mutants
Mutagenesis with Tn916 was used in an experiment designed to
isolate mutants lacking expression of mga22,
mrp22, or emm22. These mutants were expected to
have different ligand-binding properties, allowing the use of
appropriate screening procedures. For mutants affected in mga22,
mrp22, or emm22, the expected phenotype was lack of
ability to bind both fibrinogen and C4BP, only fibrinogen, or only
C4BP, respectively (cf. Fig. 3
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Among 
10,000 Tn916 mutants analyzed, 6 were found to be deficient
for fibrinogen binding. PCR analysis of 1 mutant demonstrated that
Tn916 was located in or close to the mrp22 gene, since PCR
products were obtained when primers derived from the ends of Tn916 were
combined with primers derived from the 3' end of mga22 and
from the intergenomic region between the mrp22 gene and the
emm22 gene (Fig. 1,
lanes 3 and 4). The insertion site was
estimated to be located in the promotor region of mrp22
based on the length of the PCR fragments. Several putative
mga22 mutants were also found among the Tn916 mutants, and
PCR analysis showed that one of them had Tn916 inserted in the
mga22 gene (Fig. 1, lanes 1 and
2). The insertion site was estimated to be located in
the middle of the mga22 gene. Southern blot analysis showed
that the Tn916 mutants affected in mrp22 and
mga22 had only one copy of the transposon inserted into the
chromosome (data not shown).
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Isolation of an emm22 deletion mutant and amrp22-emm22 double mutant
A deletion mutant of AL168str-r was constructed in which the 5'
half of the emm22 gene was replaced with a kanamycin
resistance cassette. The procedure was based on the use of the shuttle
vector pJRS233, in which replication is temperature sensitive in
S. pyogenes, allowing efficient selection of recombinants
arising through homologous recombination (38). A derivative of pJRS233
was constructed, as described below, carrying a region of streptococcal
DNA corresponding to the
mrp22-emm22-enn22 region, but with the
5' half of the emm22 gene replaced by a kanamycin resistance
cassette (Fig. 2). This plasmid,
designated pJRS233 mks, was transformed into AL168str-r (43).
Transformants (isolated on kanamycin plates incubated for 48 h at
30°C anaerobically) were employed to isolate emm22
deletion mutants as described (38), with the following minor
modifications. The transformants were reisolated once at 30°C on
blood agar plates without kanamycin and then reisolated once more at
30°C on the selective medium. (The kanamycin was omitted from the
first reisolation plates, since preliminary experiments demonstrated
better recovery of transformants with this procedure.) Single colonies
were used to start 10-ml overnight liquid cultures (in TH +
kanamycin), which were incubated at 37°C for elimination of free
plasmid. From these cultures, 103 CFU were transferred
to the same medium (10 ml) and incubated again at 37°C. This
procedure was repeated once, and the cultures were plated for single
colonies at 37°C on plates with kanamycin (200 µg/ml). Sheep blood
agar plates were used throughout. Recombinants derived from two
crossovers in mrp22 and in the emm22-enn22
region, respectively, were selected by analyzing for resistance to
erythromycin and kanamycin. The desired double crossover was expected
to result in a recombinant resistant only to kanamycin, while a single
crossover would cause insertion of the whole plasmid encoding both
kanamycin and erythromycin resistance. Among colonies picked after the
selection procedure, 40% were resistant only to kanamycin,
indicating that they had the desired structure. PCR analysis with
primers from the kanamycin cassette and the surrounding streptococcal
genes gave products of the expected sizes (see Fig. 1
, lanes
5 and 6).
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), used for the selection described
above, was constructed in two steps. The insert was first constructed
in pUC18. A 1304-bp XbaI-HindIII fragment,
covering the 3' end of the emm22
(sir22) gene and 5' part of the
enn22 gene, was recovered from plasmid pSIR2202 (35) and
cloned into pUC18 cleaved with the same enzymes, resulting in plasmid
pUC18s. This plasmid was subsequently cleaved with BamHI,
upstream of the XbaI site, and ligated to
BamHI-cleaved 
Km2 element (44), yielding plasmid pUC18ks.
Finally, the entire mrp22 gene was amplified from
chromosomal DNA with primers 3 and 5 (Fig. 3; Table I), cleaved with EcoRI and
KpnI, and cloned into pUC18ks cleaved with the same enzymes,
using sites located upstream of the BamHI site, resulting in
plasmid pUC18 mks. This plasmid was cleaved with EcoRI,
blunted and cleaved with BglII, at a site located 83 bp
upstream the HindIII site in the enn22 gene, to
obtain the fragment mks that was ligated to pJRS233, cleaved with
SmaI and BamHI, generating pJRS233 mks (Fig. 2).
The host for the construction work with pUC18 and pJRS233 was E.
coli DH5
.
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, lanes 7 and 8). Binding tests with radiolabeled ligands
For binding assays with whole bacteria, the bacteria were grown overnight in TH broth, washed twice in PBSAT (0.03 M phosphate, 0.12 M NaCl, 0.02% NaN3 and 0.05% Tween-20, pH 7.2) and resuspended to a concentration of 109 bacteria/ml. Serial dilutions of the bacteria were made in suspensions of the M-negative nonbinding strain JRS145. Duplicate samples of 125I-labeled proteins (about 15,000 cpm) were mixed with 200 µl of bacterial suspension in plastic tubes and incubated at room temperature for 90 min. The bacteria were washed twice with 2 ml PBSAT, and the radioactivity in the pellets was measured. The quantity bound is expressed as a percentage of added radioactivity. Nonspecific uptake (<5%) was determined with the nonbinding strain JRS145.
Phagocytosis assays
Resistance to phagocytosis in heparinized human blood was
analyzed in the bactericidal test (45). An overnight culture of
bacteria in THY was diluted 1:50 in THY and grown without agitation to
A620 = 0.15 at 37°C in a CO2 box with
5% CO2. The bacteria were diluted x 104
in THY, and 50 µl of this suspension, containing 50 CFU, was added
to 1 ml of fresh heparinized blood in acid-cleaned glass tubes
(100 x 10 mm). The tubes were incubated with rotation at 37°C
for 3 h. The number of CFU in the inoculum and after growth in
blood was analyzed by the pour plate method. Blood for these
experiments was drawn in 7-ml Vacutainer tubes (Becton Dickinson,
Meylan, France) containing 143 U of heparin.
When hirudin was used to prevent coagulation, some modifications were
made to the phagocytosis assay due to the expensiveness of this
anticoagulant. Polypropylene tubes (2.2 ml) were used instead of glass
tubes, and 250 µl of fresh hirudinized blood was mixed with 50 CFU
of log phase bacteria in THY. Hirudin (Sigma Chemical Co., St. Louis,
MO; H7380) was used at a final concentration of 100U/ml. The hirudin
solution was stored at 4°C, since freezing was found to destroy the
activity of the protein.
Electron microscopy
Bacteria were grown in 10 ml THY to OD620 = 0.6, chilled on ice, and spun down at 2200 x g. The supernatant was poured off and the pellet, with remaining liquid, transferred to a 1.5-ml polypropylene tube and briefly centrifuged to assemble a new pellet. Excess medium was removed, and the sample was fixed for 1 h in 1% glutaraldehyde, phosphate buffer, pH 7.0, postfixed in 1% OsO4 in the same buffer, and dehydrated in a graded series of ethanol. The sample was subsequently embedded in epoxy resin. Thin sections were collected on formvar-coated copper grids, stained in uranyl acetate and lead citrate, and observed in a Philips electron microscope CM10 (Eindhoven, The Netherlands).
Other methods
SDS-PAGE was conducted according to standard methods. Radiolabeling was done with the chloramine-T method (46). OF testing was performed according to Maxted et al. (47).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clinical isolate studied here, the M22 strain AL168, has a
triplet of genes encoding members of the M protein family: the Mrp22,
Emm22, and Enn22 proteins (Fig. 3) (35).
As in other OF+ strains, the regulatory gene
mga is located upstream of the mrp gene. The
Mrp22 and Emm22 proteins bind human fibrinogen and C4BP, respectively
(18, 20, 22, 24). Moreover, Mrp22 and Emm22 are both Ig-binding
proteins: Mrp22 binds IgG and Emm22 binds IgA as well as IgG (18, 19, 20, 35). As mentioned above, expression of the Enn protein has not been
demonstrated in any OF+ strain. Therefore, this study was
focused on the function of the Mrp22 and Emm22 proteins expressed by
AL168. The sequence of Emm22 has been reported previously (35), and the
sequence of Mrp22 is described below.
Strain AL168 is nonmucoid, like most clinical isolates of S. pyogenes. Therefore, it does not produce the hyaluronic acid capsule that is an important virulence factor in some isolates of S. pyogenes (48) and that may contribute to phagocytosis resistance in such strains (49).
Sequence of the Mrp22 protein
The Mrp22 protein (Fig. 4) has an
organization similar to that of previously sequenced Mrp proteins,
including a putative 41-residue signal peptide, a central region with
three A repeats, and a hydrophobic COOH-terminal region preceded by the
wall-anchoring LPXTG motif (19, 20, 55).
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). The NH2-terminal sequences of the processed
proteins vary substantially between the groups, but are at least 92%
identical within each group. In contrast, the COOH-terminal parts,
including the central repeat regions, are virtually identical in all of
these Mrp proteins. These data support previous studies indicating that
the sequence variability among Mrp proteins is less extensive than
among Emm proteins (50, 56). Isolation of bacterial mutants
Mutants of strain AL168 deficient in expression of the
mrp22 and emm22 genes, and a mutant affected in
the regulatory gene mga22, were isolated through Tn916
mutagenesis or through the introduction of a deletion, as described in
Materials and Methods. For all mutants, the structure and
location in the chromosome was analyzed by PCR, giving products of the
expected sizes (Fig. 1). Since the mga, mrp, and
emm genes have been found to be transcribed
monocistronically in different strains (15, 17, 28), an insertion in
one of these genes was not expected to exert a polar effect on
downstream genes.
The mutagenesis with Tn916 was devised to allow the isolation of mutants affected in each of the mga22, mrp22, and emm22 genes, but only mga22 and mrp22 mutants were found. The lack of emm22 mutants after Tn916 mutagenesis may be due to the partial site specificity of Tn916 (57). However, a mutant with a deletion in emm22 could be isolated by homologous recombination, employing a temperature-sensitive shuttle vector.
For phagocytosis experiments, it was essential to have available a double mutant affected in both of the mrp22 and emm22 genes. Such a strain was derived from the Tn916-induced mrp22 mutant, by introducing a deletion in the emm22 gene.
An important property of the strain studied here, AL168, is the ability to produce OF, the synthesis of which is positively controlled by the Mga protein (58). As expected, the mrp22 and emm22 single mutants and the mrp22-emm22 double mutant retained the ability to produce OF, but the mga22 mutant did not (data not shown).
Ligand-binding properties of the mutants
The properties of the mutants were verified by analysis of
their ability to bind human serum proteins (cf. Fig. 3). In the
sensitive assay used for this purpose, the binding of radiolabeled
C4BP, fibrinogen, IgG, or IgA to whole bacteria was measured (Fig. 5).
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A). The mrp22 mutant did not bind
fibrinogen, but bound C4BP, IgA, and IgG at a level comparable with
that of the wild-type strain (Fig. 5B). The
emm22 mutant lacked the ability to bind C4BP and IgA, as
expected, and showed a reduced ability to bind IgG (Fig. 5C). The rather poor IgG-binding capacity of the
emm22 mutant is consistent with previously reported data,
showing that Mrp makes only a limited contribution to the IgG-binding
ability of the strain (18). As expected, the
mrp22-emm22 double mutant failed to bind any of
the four ligands and had binding properties similar to those of the
mga22 mutant (Fig. 5, D and
E). The binding assays described above indicate that the ability of strain AL168 to bind C4BP, fibrinogen, IgA, and IgG can be attributed solely to the Mrp22 and Emm22 proteins. Moreover, the data indicate that the emm22 gene was expressed normally in the mrp22 mutant, confirming that the presence of Tn916 in mrp22 did not have a polar effect on the expression of emm22.
As mentioned above, the enn gene has not been shown to be
expressed in any OF+ strain. However, in all
OF+ strains tested, the enn gene encodes an
IgA-binding protein that can be expressed in E. coli (15, 52, 54, 59), and a characteristic IgA-binding region is found in the
NH2-terminal part of these proteins (59, 60), including
Enn22 (35). If the Enn22 protein were expressed in the strain studied
here, the mrp22-emm22 double mutant would
therefore have been expected to bind IgA. However, this mutant
completely lacked IgA-binding ability (Fig. 5D).
Resistance of mutants to phagocytosis
The role of the Mrp22 and Emm22 proteins in phagocytosis
resistance was analyzed in the bactericidal test (45), in which
streptococcal strains are analyzed for ability to grow in whole human
blood (Fig. 6; Table II). The growth indices of strain AL168,
the mrp22 mutant, the emm22 mutant, the
mrp22-emm22 double mutant, and the
mga22 mutant were compared. The availability of the double
mutant allowed a direct comparison of the relative contribution of the
Mrp22 and Emm22 proteins to phagocytosis resistance. Strain JRS145, an
OF- strain that does not grow in human blood due to a
deletion in the gene for the M6 protein (36), was included as a
negative control. The factor of increase in titer during a 3-h
incubation period is referred to as the multiplication factor
(MF).
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Results from eight different experiments with heparinized blood, using
three different blood donors, are summarized in Figure 6A
and Table II. The wild-type strain AL168 showed an average MF of 210,
corresponding to a generation time of 23 min. Thus, AL168 grew rapidly
in blood, like other fresh clinical isolates of S. pyogenes
(2, 32). The mrp22-emm22 double mutant grew
poorly, with an average MF of 7.4, i.e., 28-fold less than the
wild-type strain. Similar poor growth was observed for the
mga22 mutant and for JRS145. In contrast, the
mrp22 and emm22 mutants had MFs that were only
2-fold lower than for the wild-type strain. The differences in MF
between the different strains cannot be explained by a general effect
on growth, because all strains grew at similar rates in human plasma.
Since the single mutants mrp22 and emm22 grew
much better than the double mutant, these data indicate that each of
the Mrp22 and Emm22 proteins confers phagocytosis resistance. However,
optimal growth appeared to require the expression of both Mrp22 and
Emm22.
Data from six different experiments with hirudin as the
anticoagulant are summarized in Figure 6B and Table II. The
results are in good agreement with those obtained with heparinized
blood. Thus, the average MF for the double mutant was 1.2, while it was
243 for the mrp22 mutant and 98 for the emm22
mutant. These data confirm that each of the Mrp22 and Emm22 proteins
have antiphagocytic function.
Although the experiments with heparin and hirudin gave similar results, there were some noteworthy differences. First, the wild-type strain AL168 grew somewhat faster in the experiments with hirudin (MF = 750, corresponding to a generation time of 19 min), possibly due to small technical differences between the two types of experiments. More importantly, the difference between AL168 and the mrp22 and emm22 mutants was larger than in heparinized blood. Thus, the MFs of the mrp22 and emm22 mutants were reduced by factors of 3 and 8, respectively, as compared with AL168. These results show that both the Mrp22 protein and the Emm22 protein were required for optimal growth in blood with hirudin, as appeared to be the case also in heparinized blood. Moreover, the data suggest that the Emm protein was more important than the Mrp protein under these experimental conditions.
In the experiments with hirudin, the mga22 mutant unexpectedly had a higher MF than the mrp22-emm22 double mutant. This result was partially due to a MF value of 32, obtained in one experiment. In any case, the mga22 mutant still grew poorly, as compared with the mrp22 and emm22 single mutants. Moreover, the control strain JRS145 did not grow at all, showing that the increased MF value was not due to a general enhancement of growth in blood with hirudin.
Some of the bacterial mutants used here were generated through the insertion of Tn916, encoding tetracycline resistance. However, antibiotics were not present in the bacterial cultures used for phagocytosis experiments. To exclude that growth of a mutant in blood was due to the appearance of revertants, survivors from several experiments were analyzed for their ligand-binding properties. In all cases tested, the survivors had the expected properties.
Electron microscopy
Members of the M protein family form -helical coiled-coils that
extend from the bacterial cell surface and appear as tuftlike or
fibrillar projections in the electron microscope (2, 66). Since the
OF+ strain studied here expresses two different
members in the M protein family, the Mrp22 and Emm22 proteins, it was
of interest to analyze the surface structure of the different mutants
described above. The wild-type strain AL168 had the typical tuftlike
surface structures (Fig. 7, A
and B). As expected from other studies (21, 66, 67),
the mga mutant lacked these structures and had a smooth
surface (Fig. 7F). Interestingly, not only the single
mutants lacking Mrp22 or Emm22, but also the double mutant lacking both
Mrp22 and Emm22, still expressed the characteristic surface structures
(Fig. 7, C–E). However, comparison of
many different bacteria indicated that the double mutant expressed
about twofold less tufts than the single mutants or the wild-type
strain. These results indicate that Mrp22 and Emm22 are not the only
mga-controlled molecules that contribute to the tuftlike
surface structure of strain AL168.
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, G and
H).
To further analyze the structure of the surface proteins expressed by
the OF+ strain AL168, the emm6 deletion mutant
JRS145 was transformed with a plasmid expressing the Emm22 protein. The
resulting strain had the typical tuftlike surface structures (Fig. 7I), confirming that Emm22 contributes to these
structures in strain AL168. A similar experiment with the Mrp22 protein
could not be performed, since cloning of the entire mrp gene
in a shuttle vector was not possible (20).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Mrp and Emm proteins of OF+ strains have previously been characterized with regard to structure and ligand-binding properties (10, 15, 18, 19, 20, 22, 26, 34, 56). These studies showed that the Emm proteins have the greatest structural similarity to the known antiphagocytic M proteins of OF- strains, which could be taken as evidence that the Emm protein has antiphagocytic function. On the other hand, Mrp binds fibrinogen (18, 20), a well-known property of S. pyogenes that has been implicated in phagocytosis resistance (23, 25, 68).
We analyzed bacterial mutants affected in the mrp and emm genes for resistance to phagocytosis in whole human blood, using heparin or hirudin as the anticoagulant. The overall conclusions that could be drawn from the different experiments were similar, regardless of the method used to achieve anticoagulation. Both sets of experiments showed that a double mutant lacking both Mrp and Emm grew at least as poorly as a mutant affected in the regulatory gene mga. The simplest explanation for this finding is that Mrp and Emm are the only mga-controlled proteins that contribute to phagocytosis resistance. Most importantly, the two sets of phagocytosis experiments showed that the single mutants lacking either Mrp or Emm still grew well in blood, unlike the double mutant lacking both proteins. This result implies that each of the Mrp and Emm proteins has antiphagocytic activity, i.e., they are M proteins, according to classical definitions (2, 3, 45). However, the two sets of experiments also indicated that optimal growth required the presence of both Mrp and Emm. Indeed, lack of the Emm protein reduced growth considerably in the experiments with hirudin. Since all available data indicate that hirudin is a more specific anticoagulant than heparin (61, 62, 64, 65), the results obtained with this compound may be more representative of the in vivo situation. The results can therefore be interpreted to mean that Emm is more important than Mrp for prevention of phagocytosis in whole human blood.
The conclusion that both Mrp and Emm have antiphagocytic function implies a functional redundancy. This situation is not unique to S. pyogenes, since many pathogenic microorganisms have been reported to express more than one protein with similar function. For example, Listeria monocytogenes expresses two structurally related surface molecules, members of the internalin family, that promote entry into human hepatocytes. Each of these proteins alone promotes entry, but optimal entry requires both proteins (69). Similarly, the adhesion of nontypable Haemophilus influenzae to human epithelial cells is promoted by two structurally related surface proteins, both of which can mediate attachment (70). It seems possible that the expression of several proteins with similar, but not identical, function may allow a pathogen to adapt to a changing environment in an optimal fashion. With regard to OF+ strains of S. pyogenes, the relative importance of the Mrp and Emm proteins could vary during different stages of the infectious process, possibly reflecting roles other than the ability to inhibit phagocytosis.
Two previous studies have addressed the role of the Mrp and Emm proteins in resistance of S. pyogenes to phagocytosis. One of these studies (28) analyzed the ability of single mutants affected in the mrp or emm gene to grow in heparinized blood, and it was concluded that the role of Mrp and Emm varies between strains. One of the strains included in that study, an OF- strain designated 64/14 expresses surface proteins (including an Enn-related protein) with very atypical binding properties (50, 71), making it difficult to compare this strain with other isolates of S. pyogenes, particularly since the strain had been extensively passed through mice with the purpose of changing the expression of surface proteins (72). Three other strains analyzed in that study (28) were Lancefield reference strains that had been passed extensively through mice or in the laboratory. This situation may have contributed to the variable results obtained. However, the general conclusion that Mrp contributes to phagocytosis resistance (28) is supported by the results reported here. Another study (29) analyzed the role of the Emm protein for phagocytosis resistance by introducing the emm4 gene, cloned from an OF+ strain, into the M-deficient mutant JRS145 (derived from an OF- strain of serotype M6). Although the Emm4 (Arp4) protein was expressed on the bacterial cell surface, it did not confer phagocytosis resistance, implying that expression of this Emm protein was not sufficient to prevent phagocytosis. This result may appear to be at odds with the data reported here. However, recent studies in our laboratory have shown that not even the M5 protein, a known antiphagocytic M protein (4), confers phagocytosis resistance on the M-negative mutant JRS145 (H. Kotarsky, G. Lindahl, and U. Sjöbring, manuscript in preparation). This type of problem was avoided in the experiments reported here, which were performed with mutants derived from a single OF+ strain.
As mentioned in the introduction, some strains in the OF- lineage express more than one member of the M protein family, as is the case in OF+ strains. For example, some strains of serotype 1 express two M-like proteins, and a similar situation has been described in some strains of serotype 5 (73, 74). It remains to be analyzed whether each of the two proteins expressed by these OF- strains has an antiphagocytic function. In this context, it is interesting to note that a report published more than 35 years ago (75) demonstrated the presence of two antiphagocytic M proteins in some OF- strains of the rare serotype 14. At the time, these strains were considered to be unusual exceptions, but the properties of OF+ strains now suggest that the expression of two antiphagocytic M proteins is very common.
The mechanism by which M proteins prevent phagocytosis is not known. However, the available evidence suggests that one important function of these proteins is to protect against complement attack (76, 77). Interestingly, the mechanism used may be to bind human complement inhibitors. Several OF- strains have been shown to bind the complement inhibitor factor H, and direct binding of factor H to purified M6 protein has been demonstrated (77). We have analyzed several OF+ strains and found that they bind factor H poorly, if at all, but the Emm proteins of these strains bind another complement inhibitor, C4BP (22, 24) (K. Berggård and G. Lindahl, manuscript in preparation). Thus, it seems possible that OF+ and OF- strains use similar mechanisms to prevent complement attack, although they exploit different host proteins to achieve this result. However, the resistance of OF+ strains to phagocytosis cannot be completely explained by the ability of Emm proteins to bind C4BP, since the Mrp protein also has antiphagocytic function, as shown here. Possibly, the antiphagocytic function of Mrp is due to its ability to bind fibrinogen (23, 25, 68). In any case, it is interesting to note that neither fibrinogen-binding nor C4BP-binding is absolutely required for phagocytosis resistance, as demonstrated by the ability of mrp and emm single mutants to grow in blood.
From electron microscope analysis, it is well known that M proteins form fibrillar or tuftlike surface structures (2, 66). Our analysis of an OF+ strain confirmed that molecules in the M protein family are an important part of these structures, but studies of the double mutant mrp-emm- showed, surprisingly, that this mutant still expressed the typical surface structures, unlike the mga- mutant. These data indicate that molecules other than those in the M protein family contribute to the characteristic surface structure of OF+ S. pyogenes strains. Moreover, the data imply that the presence of tuftlike surface structures is not directly correlated to phagocytosis resistance in S. pyogenes.
In summary, we have presented evidence that OF+ strains of S. pyogenes express two different antiphagocytic M proteins, Mrp and Emm, both of which are required for optimal resistance to phagocytosis. These data provide a molecular basis for further analysis of pathogenetic mechanisms in OF+ strains. In particular, it will now be of interest to analyze how the different ligands that bind to the Mrp and Emm proteins influence their biologic properties.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gunnar Lindahl, Department of Medical Microbiology, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden. E-mail address:
3 Abbreviations used in this paper: OF, opacity factor; C4BP, C4b-binding protein; MF, multiplication factor; THY, Todd-Hewitt broth with 0.2% yeast extract; VBS, veronal-buffered saline.
Received for publication June 11, 1997. Accepted for publication September 25, 1997.
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