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*
Department of Medical Microbiology, Lund University, Lund, Sweden; and
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
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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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Among the virulence factors of S. pyogenes, particular
attention has been paid to the M proteins, which are fibrillar surface
structures that inhibit phagocytosis (3, 4). The M proteins exhibit
antigenic variation and confer protective immunity that is type
specific (5). The structure that is subject to antigenic variation is
the N-terminal region, which protrudes into the environment. This
region encompasses 
50 to 100 amino acid residues and shows extensive
sequence variation between different clinical isolates, but not within
a single strain. There are probably >100 different serologic types of
M protein, each identified by its hypervariable region. The structural
variation within a single type is surprisingly limited, possibly
because the existing types have been selected as the most fit variants
(6, 7).
Although much is known about the genetic mechanisms that give rise to antigenic variation in microbial virulence factors (8, 9), the exact function of the variable structure is rarely known. However, it can be assumed that the variable structure plays an important role during infection, since it apparently is a major target for the immune system and is not lost by mutation. Here, we describe studies aimed at defining the function of the hypervariable region in M proteins.
A clue to the role of the hypervariable region in M proteins was provided by studies with the human complement inhibitor C4BP3 (10, 11, 12). Several hypervariable regions were found to bind C4BP with high affinity, although the binding regions showed no or little amino acid residue identity. Importantly, bacteria-bound C4BP was found to retain its inhibitory function, suggesting that C4BP contributes to virulence (10). However, many other M proteins, including the extensively studied M5 and M6 proteins, do not bind C4BP (10, 11). Thus, the binding of C4BP is not a general property of hypervariable regions, and the role of this region has remained unclear.
Although the M5 and M6 proteins do not bind C4BP, they bind another human complement inhibitor, the plasma protein factor H (FH) (13, 14, 15, 16). However, this property cannot explain the function of the hypervariable region of these M proteins, since FH was reported to bind in the conserved part of the M protein (15, 17). On the other hand, the M5 and M6 proteins were recently found to bind yet another complement inhibitor, the plasma protein FHL-1 (factor H-like protein 1), a naturally occurring splice variant of FH (16, 18). We now report that FHL-1 binds to the hypervariable region of the M5 protein and probably also to the corresponding region of M6. This finding was surprising, but fits with other data indicating that FHL-1 has properties not shared by FH (19). Bacteria-bound FHL-1 was found to retain its complement-inhibitory function, indicating that it is important in pathogenesis. Thus, the studies with FHL-1 and the previous studies of C4BP now make it possible to attribute a specific function to the hypervariable region of many M proteins: the binding of a human complement inhibitor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. pyogenes strain M5 Manfredo (20) was from Dr. M.
Kehoe (University of Newcastle-upon-Tyne, U.K.). The S.
pyogenes M6 strain JRS4 and its isogenic derivatives, JRS145 and
JRS251, were from Dr. June R. Scott (Emory University, Atlanta, GA).
Strain JRS4 is referred to here as M6. In strain JRS145, the gene
encoding protein M6 is deleted; this strain is referred to here as
M6. Strain JRS251, herein referred to as M6C, expresses an M6
deletion mutant lacking the entire central C repeat region; however,
this protein is expressed on the bacterial surface, since the wall
attachment region is intact (21). Escherichia coli LE392 was
the host for construction of plasmids and for purification of
streptococcal proteins. pKEJ1 is a pBR322 derivative carrying the
emm5 gene of strain M5 Manfredo (11, 20). pSIR2202 is a
pK19-derivative carrying the emm22(sir22) gene,
which encodes the Emm22(Sir22) protein (22). The E. coli-S.
pyogenes shuttle vector pLZ12Spec has been described (23). Shuttle
vectors were electroporated into streptococci as described (24).
S. pyogenes strains were grown in Todd-Hewitt (TH) broth
(Difco, Detroit, MI) in 5% CO2 at 37°C. Strains
harboring derivatives of pLZ12Spec were grown in the presence of
spectinomycin at 100 µg/ml for streptococci or 20 µg/ml for
E. coli. The nomenclature for streptococcal genes and
proteins used here follows that of Whatmore et al. and Thern et al. (6, 12).
Deletion of the emm5 gene in strain M5 Manfredo
The temperature-sensitive shuttle vector pJRS233 (25) was used
to delete the entire emm5 gene. Two PCR-amplified fragments
were prepared first. One of these fragments corresponded to the 3' part
of the mga5 gene (located upstream) and part of the
intergenic region. The other PCR fragment corresponded to the 5' part
of the scpA5 gene (located downstream) and part of the
intergenic region. These PCR fragments were ligated into pJRS233, and
the kanamycin resistance cassette 
Km2 (26) was introduced between
the two PCR fragments. The resulting plasmid was used to isolate a
mutant of strain M5 Manfredo, in which the emm5 gene had
been replaced with the Km2 cassette, as described (12, 26). The
structure of the mutant was verified by PCR analysis. The
mga5 gene, which positively regulates emm5, was
shown to be intact in the mutant, as demonstrated by expression of M5
after introduction of the emm5 gene on a plasmid. The M5
protein binds fibrinogen (4), and the mutant was found to completely
lack fibrinogen-binding ability. Moreover, the strain was shown to lack
reactivity with anti-M5 antiserum. This strain is designated herein
as M5.
Construction of streptococcal strains expressing chimeric proteins
Isogenic streptococcal strains expressing M5 or Emm22, or one of
the chimeric proteins M5-Emm22 or Emm22-M5, were constructed by
transforming strain M5 with derivatives of plasmid pLZ12Spec
carrying the corresponding four genes. The emm5 gene was
introduced into pLZ12Spec as described (16). The
emm22(sir22) gene was recovered as a 2.2-kbp
HindIII fragment of pSIR2202 and ligated into pLZ12Spec
digested with HindIII. The chimeric gene
emm5-emm22 was derived from pLZ12Spec carrying
the emm5 gene, by exchanging the 3' end of the
emm5 gene, located downstream of the BglII site
(11), with a BglII fragment of pSIR2202 encoding the 3' part
of the sir22 gene. The chimeric gene
emm22-emm5, which includes the region of
emm5 located downstream of the BglII site, was
constructed as described (11), recovered on an
EcoR/SphI fragment, and ligated into pLZ12Spec.
The structure of the chimeric genes was verified by DNA sequencing.
Surface expression of the four proteins in the M5 strain was
verified by analyzing the ability of the transformants to bind various
ligands for which the binding sites in the streptococcal proteins are
known. As expected, the strain expressing M5 bound fibrinogen (4); the
strain expressing Emm22 bound C4BP and IgG (11, 22); the strain
expressing M5-Emm22 bound IgG; and the strain expressing Emm22-M5 bound
C4BP and fibrinogen.
Purified proteins and antisera
Streptococcal proteins were purified after expression of the corresponding genes in E. coli carrying the appropriate plasmids. The Emm22 and Emm22-M5 proteins were purified from osmotic shock lysates, as described (11, 22). (Note that the Emm22 protein was previously referred to as Sir22 (12, 22)). The M5 and M5-Emm22 proteins were purified from whole cell lysates, using affinity chromatography on immobilized fibrinogen or IgG, respectively (11, 22). Small amounts of contaminating proteins in the latter two preparations were removed by Sephacryl S-300 HR gel filtration (Pharmacia, Uppsala, Sweden). Recombinant FHL-1, fitted with a histidine tag, was expressed in the baculovirus system and purified as described (27). Purified FH, factor I, and C3b used for cofactor assays were purchased from Advanced Research Technologies (San Diego, CA). Two other preparations of highly purified FH were the kind gifts of Drs. Andreas Hillarp and Lennart Truedsson (Lund University). Human fibrinogen was from IMCO (Stockholm, Sweden). Sheep anti-human FH serum was from The Binding Site (Birmingham, U.K.).
Absorption of plasma with streptococci and elution of bound proteins
Bacteria in an overnight culture were harvested, washed, and
suspended in PBS to 1010 bacteria/ml. Bacterial samples of
increasing size, pelleted in microfuge tubes, were mixed with 300 µl
of pooled human plasma diluted 4x in PBS, and the mixtures were
incubated for 1 h at room temperature. The samples had been
supplemented with EDTA (0.34 M) to prevent complement activation. After
centrifugation, supernatants were collected, and the presence of
material reacting with anti-FH serum was analyzed by rocket
electrophoresis (28). In elution experiments, incubations were
performed essentially as described above. However, for technical
reasons elutions could not be performed with very small samples of
bacteria, unlike the absorption tests. Therefore, the elution analysis
was performed in such a way that several bacterial samples of the same
size were incubated with varying amounts of plasma. The ratios (µl
plasma)/(number of bacteria) in the three samples analyzed in the
elution experiment covered the same range as in the absorption analysis
(see Fig. 1
). For the elution analysis, three samples of pelleted
bacteria (100 µl, corresponding to 1010 bacteria) were
mixed with different volumes of EDTA-plasma, diluted 2xx in PBS. After
incubation for 1 h at room temperature, the samples were
centrifuged and the pellets were washed with 1.5 ml of PBSAT (PBS with
0.02% sodium azide and 0.05% Tween-20). Bound proteins were eluted
with 300 µl of 0.1 M glycine-HCl, pH 1.8, for 20 min at room
temperature, and the eluates were neutralized.
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The ability of whole streptococcal cells to bind
125I-FHL-1 was determined as described (16). Binding of
125I-FHL-1 to purified streptococcal proteins was analyzed
after immobilization of the bacterial proteins in microtiter wells. The
wells were coated overnight at 4°C with 50 µl of a protein solution
(1 µg/ml in PBS). After washing and blocking with cold PBSAT, 50 µl
of 125I-FHL-1 (16,000 cpm) was added to each well, and
the plates were incubated for 2 h at 4°C. After washing with
PBSAT, the radioactivity associated with each well was determined.
Unspecific binding (<0.6%) was determined in control wells and has
been subtracted. The purified streptococcal proteins had a similar
ability to bind to the plastic wells, as shown by control experiments
(data not shown).
Cofactor activity of FHL-1 after binding to bacteria
A pellet containing 2 µl of washed bacteria (2 x
108 bacteria) was resuspended with 18 µl of TBS (50 mM
Tris, 150 mM NaCl, pH 7.3) containing 2 µg of pure FHL-1. After
incubation for 90 min at room temperature, unbound FHL-1 was removed by
washing with 450 µl of TBST (TBS with 0.05% Tween-20). To determine
the cofactor activity of bound FHL-1, 125I-labeled C3b
(350,000 cpm, corresponding to 9 ng) and factor I (44 ng) were added
in a total volume of 20 µl. After incubation for 2 h at 37°C,
the tubes were centrifuged and the supernatants were recovered. For
analysis of C3b degradation, samples (30,000 cpm) were subjected to
SDS-PAGE under reducing conditions, and the gels were dried and
autoradiographed. As a positive control, pure FHL-1 without bacteria
was used as the cofactor, using the same amount of FHL-1 as that added
to the bacteria.
Competitive inhibition experiments
The procedures were similar to those previously described (16).
The wells of microtiter plates were coated by incubation with a
solution of M5 (2 µg/ml) or FH (10 µg/ml). After blocking,
radiolabeled ligand (FHL-1 or M5; 16,000 cpm) was added together
with the purified protein indicated. The wells were incubated for
2 h at 4°C and then washed, and the radioactivity associated
with each well was determined.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human complement regulators FH and FHL-1 are plasma proteins
that cause degradation of C3b by acting as cofactors for the protease
factor I (27, 29, 30, 31, 32). In addition, both FH and FHL-1 reduce the
formation of C3b by causing decay of the C3 convertase of the
alternative pathway (29, 31, 33). The 150-kDa FH protein is composed of
20 short consensus repeat (SCR) modules (Fig.
1A) and has a plasma
concentration of 400 µg/ml, while the 42-kDa FHL-1 protein, which
is composed of the first 7 SCRs of FH, has a molar concentration that
is 2 to 10 times lower (19). Previous work has shown that both of these
plasma proteins bind to the streptococcal M5 and M6 proteins (13, 14, 15, 16, 17).
To study the ability of FH/FHL-1 to bind to surface-exposed M proteins
in a physiologic setting, samples of plasma were absorbed with
increasing amounts of bacteria expressing the M5 or M6 protein (Fig. 1
B). The plasma was supplemented with EDTA to prevent
complement activation. After absorption, the plasma samples were
analyzed for remaining FH and FHL-1 (detected together as material
reacting with anti-FH antiserum). The M5 and M6 strains efficiently
bound plasma proteins reacting with anti-FH, but isogenic strains
without M protein lacked this ability.
Since the absorption analysis did not distinguish between binding of FH
and FHL-1, proteins that had bound to M5 bacteria were eluted, and the
eluates were analyzed for presence of FH and FHL-1 by Western blot. The
samples that were analyzed corresponded to arrows I through III in
Figure 1B and represent samples with very different ratios
of bacteria to plasma. For technical reasons (see Materials and
Methods), the elution analysis was performed with samples
containing fixed numbers of bacteria, which were incubated with
different amounts of plasma (Fig. 1C). The analysis showed
that FHL-1 was present in eluate I, while the other two eluates
contained little or no FHL-1 (Fig. 1C, left). The absence of
FHL-1 from eluates II and III is most likely due to the small amounts
of plasma used, allowing too little FHL-1 to bind in these samples. As
expected, the binding of FHL-1 in sample I required expression of the
M5 protein, as shown by a control experiment with the mutant lacking M
protein (Fig. 1C, right).
Surprisingly, the presence of FH in the eluates did not parallel that of FHL-1. Thus, FH was not present in eluate I, but was found in increasing amounts in the other two eluates. The lack of binding of FH in sample I can be explained by the presence of fibrinogen, which is found in high concentration in plasma and binds to M protein, thereby blocking the binding of FH, but not of FHL-1, to the M protein (see below). In samples II and III, in which relatively small volumes of plasma were used for the absorption, the bacteria caused depletion of fibrinogen, allowing binding of FH (data not shown).
Elution analysis with the M6 strain and its isogenic M6-deficient mutant gave results similar to those obtained with the M5 system (data not shown). Thus, the elution experiments indicated that the M5 and M6 proteins bind FHL-1, rather than FH, when bacteria are exposed to a large volume of plasma. Since this situation corresponds to the initial stages of a streptococcal infection, when bacteria enter the bloodstream or encounter plasma proteins transudated into the respiratory pathway (34), the emphasis of the following work was placed on studies of FHL-1. Pure FHL-1 and the M5 system were used for this work.
FHL-1 binds to the hypervariable N-terminal region of the M5 protein
In agreement with the results obtained in elution experiments,
pure FHL-1 was found to bind to M5-expressing bacteria, but not to the
isogenic M5-deficient strain (Fig. 2A).
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The chimeric protein M5-Emm22 includes the N-terminal 103 amino acids
of M5, corresponding to the hypervariable region of the protein (3, 37), whereas the reciprocal construct Emm22-M5 includes the 347
C-terminal amino acids of the M5 protein (Fig. 3A). The four proteins shown
in Figure 3A were expressed by transformants of the
M5-deficient strain, and the four transformants were studied for
ability to bind FHL-1 (Fig. 3B). Only strains expressing the
M5 or the M5-Emm22 protein bound FHL-1, indicating that the FHL-1
binding region of M5 is located in the N-terminal hypervariable part of
the protein.
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C). Finally, the ability to bind FHL-1 was analyzed after
immobilization of the four purified proteins in microtiter wells (Fig. 3D). Only the M5 and M5-Emm22 proteins bound FHL-1.
Together, these experiments show that FHL-1 binds to the N-terminal
hypervariable region of M5. Competitive inhibition experiments with different ligands of the M5 protein
The M5 protein not only binds FHL-1, but also binds FH (16). Moreover, M5 binds fibrinogen via the B repeat region (4). Possible effects of FH and fibrinogen on the binding of FHL-1 were studied in inhibition experiments.
The binding of radiolabeled FHL-1 to M5 was inhibited by unlabeled
FHL-1, as expected, but not by fibrinogen (Fig. 4A). Thus, the binding of
fibrinogen to the B repeat region does not block the binding of FHL-1
to the N-terminal region. The binding of FHL-1 to M5 was inhibited very
poorly by FH, suggesting that these two structurally related ligands
may bind to different sites in M5.
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B). This binding was inhibited by FH, as expected, but was
inhibited only weakly, if at all, by FHL-1. However, the binding of FH
to M5 was completely inhibited by fibrinogen, suggesting that FH and
fibrinogen have overlapping binding sites in the B repeat region of M5.
The ability of fibrinogen to inhibit the binding of FH is in agreement
with data reported for the M6 protein (38) and may explain the
surprising result with FH obtained in the elution analysis (Fig. 1C).
Taken together, the inhibition experiments indicate that the major
binding sites for FHL-1 and FH are located in separate regions of M5
and that FH may bind in the B repeat region of M5. This conclusion is
in good agreement with the elution analysis, which gave very different
results for FHL-1 and FH (Fig. 1C). No further attempts were
made to characterize the binding site(s) for FH in M5, since only FHL-1
appeared to interact with the streptococci under physiologic conditions
(see Fig. 1C).
Binding of FHL-1 to the M6 protein: evidence for binding in the N-terminal region
Like M5, the extensively studied M6 protein binds both FH and
FHL-1 and shows selective binding of FHL-1 in human plasma (see above;
and Refs. 13 and 16). It has been reported that FH binds to M6 in the
central C repeat region (17), which is homologous to the central C
repeat region of the M5 protein (cf Fig. 3A). It was
therefore of interest to analyze the role of the C repeat region for
binding of FHL-1. Isogenic mutants of an M6 strain were used for this
analysis (21).
FHL-1 bound equally well to the wild-type M6 strain and to a mutant
expressing M6 protein without C repeats, but an isogenic strain lacking
expression of M6 did not bind FHL-1 (Fig. 2B). Thus, the
central C repeat region is not required for the binding of FHL-1 to M6.
Moreover, the binding of FHL-1 to M6 was not inhibited by fibrinogen, a
ligand that most likely binds to the B repeats of the M6 protein, which
are almost identical to the fibrinogen-binding B repeats of M5 (data
not shown; 4 . Since the most C-terminal part of the M6 protein is
hidden in the bacterial cell wall (3), these data indicate that the
binding site for FHL-1 in M6 is located in the N-terminal part of the
protein, as found to be the case for the M5 protein.
FHL-1 retains its cofactor activity when bound to streptococci
The cofactor activity of streptococcus-bound FHL-1 was analyzed in
a C3b degradation assay (Fig. 5).
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'- and
ß-chains (Fig. 5, lane A). As expected, incubation of C3b
with FHL-1 and factor I caused cleavage of the '-chain, and
appearance of a 43-kDa fragment (lane B). When FHL-1
was replaced with M5-expressing streptococci that had been preincubated
with FHL-1, degradation of the '-chain was also observed
(lane C). In contrast, degradation of the '-chain
did not occur with M5-deficient streptococci that had been preincubated
with FHL-1 (lane E). The degradation of C3b was not
due to a protease activity in the M5-expressing bacteria, as shown by
incubation of C3b and M5 bacteria in the absence of FHL-1
(lane D). Together, these results indicate that FHL-1
retains its cofactor activity when bound to streptococci. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we studied several members of the M protein family and showed that the N-terminal hypervariable region of these proteins binds the human complement inhibitor C4BP, a high m.w. plasma protein (11). When these studies were performed, the C4BP-binding proteins were known to be Ig-Fc-binding members of the structurally defined M protein family (10, 44), but it was not known whether they have the antiphagocytic function that characterizes "classical" M proteins such as M5 and M6. However, a recent study indicates that this is indeed the case (12). Thus, many antiphagocytic M proteins bind either of the two human complement inhibitors, FHL-1 or C4BP, and the binding sites for these ligands are located in the hypervariable region of the M proteins. This situation suggests that FHL-1 and C4BP may have similar functions in streptococcal pathogenesis, although they have been assigned different functions in complement regulation. C4BP is generally considered an inhibitor of the C3 convertase of the classical pathway (45), while FHL-1 has been identified as an inhibitor of the C3 convertase of the alternative pathway (18, 32). However, several lines of evidence now indicate that the functions of C4BP and FHL-1 overlap (46, 47, 48), which suggests that they may be used for the same purpose by S. pyogenes.
Previous work has shown that FH binds to the M6 protein (13, 14), and a binding site for FH was localized to the conserved C repeat region of this M protein (17). The finding that FHL-1 binds to the hypervariable region of M5 and M6 may therefore seem surprising. However, the inhibition experiments reported here support the conclusion that the major binding sites for FHL-1 and FH are located in separate regions of the M5 protein. A possible explanation for this situation is provided by data showing that the FH molecule can fold back on itself (49), suggesting that ligand-binding regions may be hidden in the native conformation of FH, but exposed in FHL-1. Indeed, FHL-1, but not FH, has been found to promote the attachment of mammalian cells (19). The complexity of the interaction between FH and M protein is underlined by the work of Sharma and Pangburn (14), who studied M6-expressing bacteria and found evidence for a second FH-binding site in M6, in addition to the binding site in the C repeat region. It has been suggested that this second FH-binding site in M6 is located close to the N-terminal part of the C repeat region (15). However, it also seems possible that FH binds to the B repeat region, since the binding of FH to M6 to a large extent can be inhibited by fibrinogen, which most likely binds to the B repeats (4, 38). The binding of FH to M proteins was not analyzed further in this study, since FHL-1 appears to be a more important ligand under physiologic conditions.
The finding that Streptococcus-bound FHL-1 retains its cofactor activity was not unexpected, since the cofactor activity of FHL-1 has been localized to SCR1–4, while the M protein binding site was located to SCR7 (15, 16, 27, 50). However, it cannot be excluded that some of the cofactor activity in our assay was due to FHL-1 that had dissociated from the bacteria during the experiment. In a streptococcal infection, such dissociation may be an advantage, since an increased concentration of free FHL-1 in the bacterial microenvironment could contribute to the protection against complement attack.
Since the well-known M5 and M6 proteins use the hypervariable region to bind FHL-1, it seems likely that many M proteins have this property. This raises the question of the structural basis for the interaction with FHL-1. We propose that regions with very different sequences have similar structures, allowing them to bind FHL-1. Support for this notion comes from studies of the Trypanosoma brucei variant surface glycoprotein (VSG), a protein that shows extensive antigenic variation (8, 9). Studies of two VSG variable domains with little sequence identity showed that they have similar structures (51), suggesting that they may also have similar function. However, the function of the variable domain in VSGs is not known. Our identification of a ligand for the hypervariable region of the M5 and M6 proteins implies that structural analysis of these regions may be particularly rewarding for the analysis of protein-protein interactions and for studies of sequence constraints in antigenic variation. The same argument applies to the interaction between certain hypervariable regions and C4BP.
FHL-1 is a member of the RCA (regulator of complement activation) family of proteins, a complex system of complement inhibitors present in plasma and on cell membranes (31). Accumulated evidence indicates that these proteins not only act as human complement inhibitors but are also major targets for pathogenic microorganisms. Early studies in this field showed that EBV attaches to the RCA protein CR2 on B cells (52) and that S. pyogenes binds the plasma protein FH (13). More recent studies have shown that the RCA proteins CD46 and CD55 act as receptors for measles virus, echoviruses, Neisseria, S. pyogenes, and/or some strains of E. coli (53, 54, 55, 56, 57, 58, 59). The RCA protein CR1 may be a receptor for proteins expressed on the surface of Plasmodium falciparum-infected erythrocytes (60). The plasma protein C4BP not only binds to many strains of S. pyogenes but also binds to Bordetella pertussis (61). Moreover, FH contributes to the serum resistance of Neisseria gonorrhoeae by binding to sialic acid residues on the bacterial surface (62). These examples, and the data reported here, indicate that interactions with RCA proteins are of general importance in microbial pathogenesis.
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Acknowledgments |
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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: C4BP, complement factor 4b-binding protein; FH, complement factor H; FHL-1, complement factor H-like protein 1; SCR, short consensus repeat; PBSAT, PBS with 0.02% sodium azide and 0.05% Tween-20; VSG, variant surface glycoprotein; RCA, regulator of complement activation; 125I-FHL-1, 125I-labeled FHL-1.
Received for publication April 9, 1998. Accepted for publication June 25, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. Poltermann, A. Kunert, M. von der Heide, R. Eck, A. Hartmann, and P. F. Zipfel Gpm1p Is a Factor H-, FHL-1-, and Plasminogen-binding Surface Protein of Candida albicans J. Biol. Chem., December 28, 2007; 282(52): 37537 - 37544. [Abstract] [Full Text] [PDF]
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 A. Kunert, J. Losse, C. Gruszin, M. Huhn, K. Kaendler, S. Mikkat, D. Volke, R. Hoffmann, T. S. Jokiranta, H. Seeberger, et al. Immune Evasion of the Human Pathogen Pseudomonas aeruginosa: Elongation Factor Tuf Is a Factor H and Plasminogen Binding Protein J. Immunol., September 1, 2007; 179(5): 2979 - 2988. [Abstract] [Full Text] [PDF]
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T. Meri, S. J. Cutler, A. M. Blom, S. Meri, and T. S. Jokiranta Relapsing Fever Spirochetes Borrelia recurrentis and B. duttonii Acquire Complement Regulators C4b-Binding Protein and Factor H Infect. Immun., July 1, 2006; 74(7): 4157 - 4163. [Abstract] [Full Text] [PDF]
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 J. Waldemarsson, T. Areschoug, G. Lindahl, and E. Johnsson The Streptococcal Blr and Slr Proteins Define a Family of Surface Proteins with Leucine-Rich Repeats: Camouflaging by Other Surface Structures J. Bacteriol., January 15, 2006; 188(2): 378 - 388. [Abstract] [Full Text] [PDF]
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K. von Lackum, J. C. Miller, T. Bykowski, S. P. Riley, M. E. Woodman, V. Brade, P. Kraiczy, B. Stevenson, and R. Wallich Borrelia burgdorferi Regulates Expression of Complement Regulator-Acquiring Surface Protein 1 during the Mammal-Tick Infection Cycle Infect. Immun., November 1, 2005; 73(11): 7398 - 7405. [Abstract] [Full Text] [PDF]
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J. D. Price, J. Schaumburg, C. Sandin, J. P. Atkinson, G. Lindahl, and C. Kemper Induction of a Regulatory Phenotype in Human CD4+ T Cells by Streptococcal M Protein J. Immunol., July 15, 2005; 175(2): 677 - 684. [Abstract] [Full Text] [PDF]
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L. Wei, V. Pandiripally, E. Gregory, M. Clymer, and D. Cue Impact of the SpeB Protease on Binding of the Complement Regulatory Proteins Factor H and Factor H-Like Protein 1 by Streptococcus pyogenes Infect. Immun., April 1, 2005; 73(4): 2040 - 2050. [Abstract] [Full Text] [PDF]
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R. Wallich, J. Pattathu, V. Kitiratschky, C. Brenner, P. F. Zipfel, V. Brade, M. M. Simon, and P. Kraiczy Identification and Functional Characterization of Complement Regulator-Acquiring Surface Protein 1 of the Lyme Disease Spirochetes Borrelia afzelii and Borrelia garinii Infect. Immun., April 1, 2005; 73(4): 2351 - 2359. [Abstract] [Full Text] [PDF]
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J. V. McDowell, J. Wolfgang, L. Senty, C. M. Sundy, M. J. Noto, and R. T. Marconi Demonstration of the Involvement of Outer Surface Protein E Coiled Coil Structural Domains and Higher Order Structural Elements in the Binding of Infection-Induced Antibody and the Complement-Regulatory Protein, Factor H J. Immunol., December 15, 2004; 173(12): 7471 - 7480. [Abstract] [Full Text] [PDF]
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D. Perez-Caballero, I. Garcia-Laorden, G. Cortes, M. R. Wessels, S. R. de Cordoba, and S. Alberti Interaction between Complement Regulators and Streptococcus pyogenes: Binding of C4b-Binding Protein and Factor H/Factor H-Like Protein 1 to M18 Strains Involves Two Different Cell Surface Molecules J. Immunol., December 1, 2004; 173(11): 6899 - 6904. [Abstract] [Full Text] [PDF]
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M. Weineisen, U. Sjobring, M. Fallman, and T. Andersson Streptococcal M5 Protein Prevents Neutrophil Phagocytosis by Interfering with CD11b/CD18 Receptor-Mediated Association and Signaling J. Immunol., March 15, 2004; 172(6): 3798 - 3807. [Abstract] [Full Text] [PDF]
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P. Kraiczy, J. Hellwage, C. Skerka, H. Becker, M. Kirschfink, M. M. Simon, V. Brade, P. F. Zipfel, and R. Wallich Complement Resistance of Borrelia burgdorferi Correlates with the Expression of BbCRASP-1, a Novel Linear Plasmid-encoded Surface Protein That Interacts with Human Factor H and FHL-1 and Is Unrelated to Erp Proteins J. Biol. Chem., January 23, 2004; 279(4): 2421 - 2429. [Abstract] [Full Text] [PDF]
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M. C. Fontaine, J. J. Lee, and M. A. Kehoe Combined Contributions of Streptolysin O and Streptolysin S to Virulence of Serotype M5 Streptococcus pyogenes Strain Manfredo Infect. Immun., July 1, 2003; 71(7): 3857 - 3865. [Abstract] [Full Text] [PDF]
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A. Eriksson and M. Norgren Cleavage of Antigen-Bound Immunoglobulin G by SpeB Contributes to Streptococcal Persistence in Opsonizing Blood Infect. Immun., January 1, 2003; 71(1): 211 - 217. [Abstract] [Full Text] [PDF]
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 U. Sjobring, U. Ringdahl, and Z. M. Ruggeri Induction of platelet thrombi by bacteria and antibodies Blood, December 15, 2002; 100(13): 4470 - 4477. [Abstract] [Full Text] [PDF]
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V. Pandiripally, E. Gregory, and D. Cue Acquisition of Regulators of Complement Activation by Streptococcus pyogenes Serotype M1 Infect. Immun., November 1, 2002; 70(11): 6206 - 6214. [Abstract] [Full Text] [PDF]
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T. Meri, A. Hartmann, D. Lenk, R. Eck, R. Wurzner, J. Hellwage, S. Meri, and P. F. Zipfel The Yeast Candida albicans Binds Complement Regulators Factor H and FHL-1 Infect. Immun., September 1, 2002; 70(9): 5185 - 5192. [Abstract] [Full Text] [PDF]
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E. Giannakis, T. S. Jokiranta, R. J. Ormsby, T. G. Duthy, D. A. Male, D. Christiansen, V. A. Fischetti, C. Bagley, B. E. Loveland, and D. L. Gordon Identification of the Streptococcal M Protein Binding Site on Membrane Cofactor Protein (CD46) J. Immunol., May 1, 2002; 168(9): 4585 - 4592. [Abstract] [Full Text] [PDF]
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T. Areschoug, M. Stalhammar-Carlemalm, I. Karlsson, and G. Lindahl Streptococcal beta Protein Has Separate Binding Sites for Human Factor H and IgA-Fc J. Biol. Chem., April 5, 2002; 277(15): 12642 - 12648. [Abstract] [Full Text] [PDF]
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P. Kraiczy, C. Skerka, V. Brade, and P. F. Zipfel Further Characterization of Complement Regulator-Acquiring Surface Proteins of Borrelia burgdorferi Infect. Immun., December 1, 2001; 69(12): 7800 - 7809. [Abstract] [Full Text] [PDF]
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S. Ram, M. Cullinane, A. M. Blom, S. Gulati, D. P. McQuillen, B. G. Monks, C. O'Connell, R. Boden, C. Elkins, M. K. Pangburn, et al. Binding of C4b-Binding Protein to Porin: A Molecular Mechanism of Serum Resistance of Neisseria gonorrhoeae J. Exp. Med., February 5, 2001; 193(3): 281 - 296. [Abstract] [Full Text] [PDF]
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A. M. Whatmore Streptococcus pyogenes sclB encodes a putative hypervariable surface protein with a collagen-like repetitive structure Microbiology, February 1, 2001; 147(2): 419 - 429. [Abstract] [Full Text]
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M. Rasmussen, A. Eden, and L. Bjorck SclA, a Novel Collagen-Like Surface Protein of Streptococcus pyogenes Infect. Immun., November 1, 2000; 68(11): 6370 - 6377. [Abstract] [Full Text] [PDF]
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J.-S. Chia, C.-Y. Yeh, and J.-Y. Chen Identification of a Fibronectin Binding Protein from Streptococcus mutans Infect. Immun., April 1, 2000; 68(4): 1864 - 1870. [Abstract] [Full Text] [PDF]
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H. Kotarsky, A. Thern, G. Lindahl, and U. Sjobring Strain-Specific Restriction of the Antiphagocytic Property of Group A Streptococcal M Proteins Infect. Immun., January 1, 2000; 68(1): 107 - 112. [Abstract] [Full Text] [PDF]
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E. Johnsson, T. Areschoug, J. Mestecky, and G. Lindahl An IgA-binding Peptide Derived from a Streptococcal Surface Protein J. Biol. Chem., May 21, 1999; 274(21): 14521 - 14524. [Abstract] [Full Text] [PDF]
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B.-M. Kihlberg, M. Collin, A. Olsen, and L. Bjorck Protein H, an Antiphagocytic Surface Protein in Streptococcus pyogenes Infect. Immun., April 1, 1999; 67(4): 1708 - 1714. [Abstract] [Full Text] [PDF]
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 W. W. Navarre and O. Schneewind Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope Microbiol. Mol. Biol. Rev., March 1, 1999; 63(1): 174 - 229. [Abstract] [Full Text] [PDF]
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R. Janulczyk, F. Iannelli, A. G. Sjoholm, G. Pozzi, and L. Bjorck Hic, a Novel Surface Protein of Streptococcus pneumoniae That Interferes with Complement Function J. Biol. Chem., November 17, 2000; 275(47): 37257 - 37263. [Abstract] [Full Text] [PDF]
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J. Hellwage, T. Meri, T. Heikkila, A. Alitalo, J. Panelius, P. Lahdenne, I. J. T. Seppala, and S. Meri The Complement Regulator Factor H Binds to the Surface Protein OspE of Borrelia burgdorferi J. Biol. Chem., March 9, 2001; 276(11): 8427 - 8435. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), Emm22 (
), M5-Emm22 (
), or
Emm22-M5 (
). C, Western blot analysis.
Highly purified preparations of the four proteins shown in
A were subjected to SDS-PAGE under reducing conditions.
The blotting membrane was incubated with FHL-1 (1 µg/ml), followed by
sheep anti-human FH serum (diluted x1000). After washing and
incubation with 125I-protein G, bound Abs were detected by
autoradiography. No reaction was observed if the incubation step with
FHL-1 was omitted. Molecular mass markers are in kilodaltons.
D, Highly purified preparations of the four proteins
shown in A were used to coat microtiter wells. The
immobilized streptococcal proteins were analyzed for ability to bind
125I-FHL-1. Each of the experiments shown in
B, C, and D was performed
three times with similar results.
