FASEB J. Integrated DNA Technologies
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.07-105643.
 (The FASEB Journal. 2008;22:2715-2722.)
© 2008 FASEB
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
fj.07-105643v1
fj.07-105643v2
22/8/2715    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sanderson-Smith, M. L.
Right arrow Articles by Walker, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sanderson-Smith, M. L.
Right arrow Articles by Walker, M. J.

M protein-mediated plasminogen binding is essential for the virulence of an invasive Streptococcus pyogenes isolate

M. L. Sanderson-Smith*,{dagger}, K. Dinkla{dagger}, J. N. Cole*, A. J. Cork*, P. G. Maamary*, J. D. McArthur*, G. S. Chhatwal{dagger} and M. J. Walker*,1

* School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia; and

{dagger} Department of Microbial Pathogenesis, Helmholtz Centre for Infection Research, Braunschweig, Germany

1Correspondence: School of Biological Sciences, University of Wollongong, Wollongong, NSW, 2522, Australia. E-mail: mwalker{at}uow.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human protease plasmin plays a crucial role in the capacity of the group A streptococcus (GAS; Streptococus pyogenes) to initiate invasive disease. The GAS strain NS88.2 was isolated from a case of bacteremia from the Northern Territory of Australia, a region with high rates of GAS invasive disease. Mutagenesis of the NS88.2 plasminogen binding M protein Prp was undertaken to examine the contribution of plasminogen binding and cell surface plasmin acquisition to virulence. The isogenic mutant NS88.2prp was engineered whereby four amino acid residues critical for plasminogen binding were converted to alanine codons in the GAS genome sequence. The mutated residues were reverse complemented to the wild-type sequence to construct GAS strain NS88.2prpRC. In comparison to NS88.2 and NS88.2prpRC, the NS88.2prp mutant exhibited significantly reduced ability to bind human plasminogen and accumulate cell surface plasmin activity during growth in human plasma. Utilizing a humanized plasminogen mouse model of invasive infection, we demonstrate that the capacity to bind plasminogen and accumulate surface plasmin activity plays an essential role in GAS virulence.—Sanderson-Smith, M. L., Dinkla, K., Cole, J. N., Cork, A. J., Maamary, P. G., McArthur, J. D., Chhatwal, G. S., Walker, M. J. M protein-mediated plasminogen binding is essential for the virulence of an invasive Streptococcus pyogenes isolate.


Key Words: plasmin • group A streptococcus • innate immunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
STREPTOCOCCUS PYOGENES [group A streptococcus (GAS)] is the major etiological agent of a variety of human infections. These include simple pharyngitis (>600 million cases per year) and impetigo (>100 million cases per year), but also life-threatening invasive infections such as necrotizing fasciitis, bacteremia, and toxic shock syndrome (>600,000 cases and 163,000 deaths per year) (1) . Over the past two decades, there have been numerous reports of a resurgence in GAS invasive disease of increasing severity in Western countries (2 , 3) . In addition, in developing nations, the epidemiology of GAS disease is less well described, and GAS infection remains endemic in many areas (4) . In the Northern Territory of Australia, the incidence of invasive GAS disease in indigenous communities is extremely high, with the rate of bacteremia reported to be 5 times that seen in the nonindigenous population (4) . Similar to the epidemiology seen in developing countries, diverse emm types are found to be associated with both invasive and endemic infections in the Northern Territory, and invasive disease is generally secondary to endemic skin infection (1 , 4 5 6) .

A mounting body of clinical, epidemiological, and experimental evidence suggests an important role for plasminogen activation in GAS virulence (7 , 8) . Plasminogen is a single-chain glycoprotein found in plasma and extracellular fluids at concentrations of ~2 µM (9) . Cleavage of plasminogen at a single site (Arg560Val561) by specific plasminogen activators results in the formation of the two-chain plasmin molecule, which contains a serine protease active site in the C-terminal region (10) . Human plasminogen can also be activated to plasmin by the GAS protein streptokinase, as part of a highly species-specific plasminogen/streptokinase activator complex (11) . Plasmin has the ability to degrade fibrin clots, connective tissue, and the extracellular matrix (9 , 10) . Thus, activation of this proteolytic system by GAS may have significant pathological consequences for the host. A subset of GAS strains expresses M proteins that bind plasminogen and plasmin directly and with high affinity. The first of these M proteins to be identified was PAM, initially identified in M53 serotype GAS (12) . Plasminogen-binding motifs in M proteins have been identified in other GAS serotypes associated with both invasive and noninvasive disease (13) . Plasminogen-binding M proteins have been well characterized in vitro (12 , 14 15 16 17 18 19 20) ; however, the direct contribution of these proteins to GAS virulence has not been defined. Although the deletion of the PAM gene results in a loss of virulence (7 , 14) , this may be due to a loss of other functions such as protection from phagocytosis or binding of other host molecules attributed to M proteins (21) .

The PAM-related protein Prp is associated with an S. pyogenes strain isolated from a severe invasive infection in the Northern Territory. Prp has been shown to interact with plasminogen via the same mechanism, and with a similar affinity to other plasminogen binding M proteins. It has also been demonstrated that the plasminogen binding ability of Prp can be abrogated without resulting in a loss of protein structure (18) . Thus, Prp provides an ideal candidate for examining the contribution of plasminogen-binding M proteins to streptococcal virulence. Here, we report the construction of an isogenic GAS Prp mutant (NS88.2prp), which is attenuated for plasminogen binding and surface plasmin accumulation. Reverse complementation of the prp gene to wild type (NS88.2prpRC) restored both cell surface plasminogen binding and activation. NS88.2, NS88.2prp and NS88.2prpRC expressed equivalent levels of hyaluronic acid capsule, streptokinase, {alpha}-enolase, and GAPDH, and showed no difference in the ability to bind fibrinogen, survive in whole blood, or interact with human polymorphonuclear leukocytes. The virulence of the isogenic mutant NS88.2prp was, however, significantly attenuated in a humanized plasminogen mouse model of invasive infection, underpinning the central role of plasminogen binding and surface plasmin accumulation in GAS virulence.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains and culture methods
GAS strains were routinely cultured in Todd-Hewitt broth containing 1% yeast (THBY) or grown on horse blood agar (HBA) plates at 37°C. GAS strain NS88.2 has been described previously (13) . Allelic exchange was used to create NS88.2prp via precise replacement of the wild-type prp gene in GAS strain NS88.2 with the previously described prpA96A101A107A108gene, which encodes a protein that is completely attenuated for plasminogen binding (18) . The mutation was subsequently reverse complemented by the replacement of prpA96A101A107A108 with the wild-type prp gene to create NS88.2prpRC. Escherichia coli MC1061 was propagated using Luria Bertani (LB) broth or agar. For selection of the plasmid pHY304 in either GAS or E. coli, erythromycin was used at a concentration of either 4 µg/ml (GAS) or 500 µg/ml (E coli), respectively, and bacteria were cultured at 30°C.

Construction of prp mutant GAS strains
GAS mutants were constructed essentially as described previously (22 , 23) . Both the wild-type prp gene and the prpA96A101A107A108 gene were previously cloned into the expression vector pGEX2T (18) . To facilitate allelic replacement, these genes were subcloned into the temperature-sensitive vector pHY304, which contains the gene for erythromycin resistance, using BamHI/EcoRI restriction enzyme digestion and ligation with T4 DNA ligase. Plasmids were transformed into E. coli MC1061 using standard electroporation procedures, and the recombinant plasmids screened by DNA sequence analysis using primers PAMN1 and PAMN2 (12) to confirm the presence of either the mutated or the wild-type gene. The resulting plasmids (pHYprp and pHYprpRC) were transformed into NS88.2 or NS88.2prp, respectively, by electroporation (24) . Integration into the chromosome was achieved by 2 h incubation at the permissive temperature for plasmid replication (30°C). Following subculture at 37°C, single crossover insertions were selected using erythromycin resistance screening. Chromosomal DNA was also screened for the presence of the erythromycin resistance gene by polymerase chain reaction (PCR) using primers pHYermF (5'-GAAGGAGTGATTACATGAAC-3') and pHYermR (5'-CATAGAATTATTTCCTCCCG-3'), to avoid the selection of spontaneously resistant mutants. Serial passage at 30°C and the removal of antibiotic selection was used to achieve double crossover. The resulting isogenic mutants NS88.2prp and NS88.2prpRC containing the prpA96A101A107A108 and prp genes were confirmed by DNA sequence analysis using primers M1 and M2, respectively (12) . The growth rates of the wild-type and isogenic mutant strains in THBY were found to be identical (data not shown).

Vir typing
The Vir regulon was amplified from GAS chromosomal DNA, and the resulting amplification product was subjected to restriction enzyme digestion using HaeIII, as described previously (5) .

Capsule assay
Overnight GAS cultures were used to inoculate fresh THBY. Cultures were grown to an OD600 of 0.5–0.6. Capsule was extracted and assayed using the Stains-All method, as described previously (25) .

Determination of M protein, streptokinase, GAPDH, and {alpha}-enolase expression
Mutanolysin extracts and GAS culture supernatants from overnight cultures were prepared as described previously (26) . Following SDS-PAGE of the protein samples according to the method of Laemmli (27) , proteins were transferred to nitrocellulose membrane using a Bio-Rad Trans-Blot apparatus (Bio-Rad, Hercules, CA, USA). For the determination of M protein expression, mutanolysin extracts were subjected to Western blot analysis using rabbit polyclonal antisera raised against PAM. Specific rabbit antisera were also used to examine expression of the plasminogen receptors GAPDH and {alpha}-enolase. TCA-precipitated proteins from overnight GAS culture supernatants were assayed for the presence of streptokinase using rabbit streptokinase antiserum. The antisera used and the conditions for Western blot analysis have been described in detail elsewhere (19 , 26) .

Cell surface plasmin acquisition
Cell surface plasmin activation assays were conducted following incubation of GAS in human plasma, essentially as described previously (26) . Briefly, overnight GAS cultures were adjusted to OD600 0.5 and incubated with human plasma for 3 h at 37°C. Following washing twice with PBS, 0.01% gelatin, and 0.01 M EDTA, bacteria were resuspended in PBS and 0.01% gelatin, and the plasmin activity of this resuspension was determined using the chromogenic substrate Spectrozyme PL.

Binding of human plasminogen and fibrinogen
Bacteria were collected from overnight GAS cultures by centrifugation, and resuspended to 10% transmission at OD600 in PBS and 0.01% TWEEN-20. A 250-µl sample of this cell suspension was used for 125I-plasminogen and 125I-fibrinogen binding analysis as described previously (13) . For analysis of plasminogen and fibrinogen capture from human plasma, 400 µl of bacterial cell suspension was pelleted by centrifugation and resuspended in 100 µl of human plasma. Following 1 h incubation at 37°C, bacteria were washed 3 times with 1 ml PBS, and bound proteins were eluted by incubation in 50 µl 100 mM Glycine-HCl (pH 2.0) for 15 min at room temperature. Following centrifugation, the supernatant was collected, and 4 µl of 1.5 M Tris-HCl was added to 40 µl of protein solution. Following SDS-PAGE and Western blot transfer as described above, membranes were probed with polyclonal goat anti-human plasminogen or polyclonal rabbit anti-human fibrinogen antibodies diluted 1:3000. Goat anti-rabbit or rabbit anti-goat horseradish peroxidase-conjugated secondary antibodies diluted 1:3000 were used for detection of primary antibody binding. Membranes were developed by enhanced chemiluminescence.

Growth of GAS in whole blood
Survival of GAS strains in whole blood was determined using the Lancefield method (28) . Fold increase in colony forming units (CFU) was determined by dividing the number of CFU present after 3-h incubation in heparanized human blood by the number of CFU present in the original inoculum, as determined by plating on HBA and colony counting. Assays were performed in triplicate, using two different blood donors.

Polymorphonuclear leukocyte phagocytosis assay
Polymorphonuclear leukocytes (2.5x105 cells) were isolated from fresh human blood and incubated with 2.5 x 106 Alexa-488-labeled GAS in the presence of nonimmune human serum, as described previously (29 , 30) . Cells were analyzed using flow cytometry, and the percentage of fluorescent polymorphonuclear leukocytes was used as a measure of phagocytosis.

Humanized plasminogen mouse model of invasive disease
Transgenic humanized plasminogen AlbPLG1 mice heterozygous for the human plasminogen transgene (7) were backcrossed with C57BL/J6 mice. Groups of 10 AlbPLG1 mice were infected subcutaneously with 100 µl each of NS88.2, NS88.2prp, or NS88.2prpRC, and survival was monitored over a 10-day period. The number of CFU used for infection was determined by serial dilution of the inoculum, plating on HBA, and colony counting following overnight incubation at 37°C.

Statistical analyses
Differences in the survival of humanized plasminogen transgenic mice infected with GAS strains were determined by log-rank test. All other data were analyzed via one-way ANOVA with Dunnett’s multiple-comparison test. Data sets were considered significantly different at P < 0.05. All analysis was perfomed using GraphPad Prism 4.00 (GraphPad, San Diego, CA, USA).

Ethics approval
Ethics permission was obtained from the University of Wollongong ethics committee prior to the commencement of animal experiments. Volunteers provided informed consent before blood samples were obtained.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of GAS strains NS88.2, NS88.2prp, and NS88.2prpRC
The mga regulon of NS88.2, a pattern D GAS strain (13) , encodes multiple emm and emm-like genes (31) . As the C terminus of these gene products is highly conserved between different emm and emm-like genes, it was important to ensure that the integrity of NS88.2prp (prp gene replaced with prpA96A101A107A108gene) and NS88.2prpRC (prpA96A101A107A108gene replaced with prp gene) and that illegitimate gene rearrangements had not occurred. The mga regulon from NS88.2, NS88.2prp, and NS88.2prpRC was PCR amplified, and a restriction profile was generated using HaeIII (Fig. 1 A). Amplification of the mga regulon from each strain resulted in amplicons of comparative size (~7 kb), and the restriction pattern generated for each strain was identical, suggesting that integration of modified prp genes into the mga regulon of NS88.2 did not result in unwanted chromosomal rearrangements. The integrity of the recombined prp genes in the chromosomes of NS88.2prp and NS88.2prpRC was confirmed by DNA sequence analysis (results not shown).


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		Figure 1. Characterization of GAS strains NS88.2, NS88.2prp, and NS88.2prpRC. A) Amplification of the mga regulon of NS88.2 (lane 1), NS88.2prp (lane 2), and NS88.2prpRC (lane 3), and HaeIII digestion of mga amplification products for NS88.2 (lane 4), NS88.2prp (lane 5), and NS88.2prpRC (lane 6). Molecular size markers are given in kilobase pairs (kbp). B) Western blot analysis of mutanolysin cell wall extracts from NS88.2 (lane 1), NS88.2prp (lane 2), and NS88.2prpRC (lane 3) using rabbit polyclonal anti-PAM antibodies. Bold arrowhead indicates Prp. Molecular mass markers are given in kilodaltons (kDa). C) NS88.2, NS88.2prp, and NS88.2prpRC produce equivalent levels of hyaluronic acid capsule (mean±SD, n=3; P>0.05). D) Western blot analysis of TCA-precipitated GAS supernatants from NS88.2 (lane 1), NS88.2prp (lane 2), and NS88.2prpRC (lane 3), using rabbit polyclonal anti-streptokinase antibodies. Bold arrowhead indicates streptokinase.

To further characterize these strains, the expression of Prp by NS88.2, NS88.2prp, and NS88.2prpRC was examined. Western blot analysis of mutanolysin cell wall extracts was used to confirm equivalent expression levels of M protein by GAS strains NS88.2, NS88.2prp, and NS88.2prpRC (Fig. 1B ). As expected, mutagenized Prp expressed by NS88.2prp displays a modified electrophoretic mobility on SDS-PAGE (18) . No significant difference in the expression of hyaluronic acid capsule by NS88.2, NS88.2prp, and NS88.2prpRC was evident (P>0.05; Fig. 1C ). Furthermore, each of these strains expressed equivalent amounts of the plasminogen activator streptokinase (Fig. 1D ), as well as the other known plasminogen receptors GAPDH and {alpha}-enolase (results not shown). Thus, the techniques used for the construction of the isogenic NS88.2prp and NS88.2prpRC strains did not result in changes to the expression of other virulence factors involved in the interaction of GAS with human plasminogen.

Interaction of NS88.2, NS88.2prp, and NS88.2prpRC with innate immune effectors
A significant factor in GAS virulence is the ability of strains to evade phagocytosis by host immune cells, and the interaction of GAS with fibrinogen has been shown to provide protection against phagocytosis by polymorphonuclear leukocytes (32 , 33) . In addition, some GAS strains are able to interact with the plasminogen activation system indirectly via fibrinogen-binding M proteins (34) . As such, the interaction of wild-type and mutant strains with fibrinogen, whole human blood, and human polymorphonuclear leukocytes was investigated.

Incubation of GAS strains in human plasma and subsequent analysis of the eluted proteins indicated that NS88.2 wild-type and mutant strains of GAS are able to acquire fibrinogen from human plasma (Fig. 2 A). Furthermore, in a quantitative analysis of the binding of 125I-fibrinogen (Fig. 2B ), NS88.2, NS88.2prp, and NS88.2prpRC bound equivalent levels of fibrinogen (P>0.05). Therefore, it can be assumed that fibrinogen-mediated interactions with both host immune cells and the plasminogen activation system will be maintained.


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Figure 2. Interaction of NS88.2, NS88.2prp, and NS88.2prpRC with human fibrinogen. A) Western blot analysis of eluted cell surface proteins following incubation in human plasma, using anti-human fibrinogen polyclonal antibodies. Lane 1, purified fibrinogen; lane 2, NS88.2; lane 3, NS88.2prp; lane 4, NS88. 2prpRC. B) Binding of 125I-fibrinogen by GAS strains NS88.2, NS88.2prp, and NS88.2prpRC (mean±SD, n=6; P>0.05).

This observation is supported by data from whole-blood assays, indicating no significant difference in the ability of NS88.2, NS88.2prp, and NS88.2prpRC to replicate in whole blood from two separate blood donors (P>0.05; Table 1 ). Notably, all strains showed a greater than 32-fold increase in population, which is regarded as the threshold for an intact antiphagocytic capacity (35) . Furthermore, in three independent experiments, no significant difference was seen between the uptake of GAS strains NS88.2, NS88.2prp, or NS88.2prpRC by polymorphonuclear leukocytes (Fig. 3 P>0.05). Thus, it appears that the expression of the mutated prp gene by NS88.2prp does not alter the antiphagocytic properties of NS88.2.


View this table:
[in this window]
[in a new window]

  		Table 1. Fold increase in bacterial colonies compared to inoculum following growth in whole human blood


Figure 3
View larger version (10K):
[in this window]
[in a new window]

  		Figure 3. Flow cytometric analysis of the uptake of NS88.2, NS88.2prp, and NS88.2prpRC by human polymorphonuclear leukocytes. A–C) Representative data are shown from a single experiment. Data have been gated to exclude autofluorescence. Uptake by polymorphonuclear leukocytes, as indicated by the percentage of flourescent polymorphonuclear leukocytes, of GAS strains NS88.2 (A), NS88.2prp (B), and NS88.2prpRC (C). D) Combined data from 3 separate experiments (mean±SD, n=9; P>0.05).

Plasminogen binding analysis
GAS have been found to interact directly with plasminogen via multiple receptors. Of these receptors, the plasminogen-binding M protein has been shown to have the highest affinity for the circulating form of plasminogen. In additon, there appears to be a direct correlation between the presence of a plasminogen-binding M protein gene in the GAS chromosome, and the ability of isolates to bind plasminogen (13) . However, to date, the overall contribution of plasminogen binding M protein to the acquisition of plasminogen by GAS has not been established. Data presented in Fig. 4 clearly indicates that the abrogation of plasminogen binding by the M protein Prp significantly decreases the ability of GAS strain NS88.2 to acquire plasminogen, both from human plasma (Fig. 4A ) and in direct plasminogen-binding assays (Fig. 4B ). The plasminogen-binding ability of NS88.2prp was restored by reverse complementation with the wild-type prp gene. In addition, the expression of the mutant Prp protein by NS88.2 completely prevented the acquisition of cell surface plasmin activity (Fig. 4C ). Thus, it appears that for GAS strains that express plasminogen-binding M proteins, the ability to bind and activate plasminogen at the cell surface is almost exclusively dependent on the expression of these M proteins.


Figure 4
View larger version (6K):
[in this window]
[in a new window]

  		Figure 4. Interaction of NS88.2, NS88.2prp, and NS88.2prpRC with human plasminogen. A) Western blot analysis of eluted cell surface proteins following incubation in human plasma, using anti-human plasminogen polyclonal antibodies. Lane 1, purified plasminogen; lane 2, NS88.2; lane 3, NS88.2prp; lane 4, NS88.2prpRC. B) Binding of 125I-plasminogen by GAS strains NS88.2, NS88.2prp, and NS88.2prpRC (mean±SD, n=6; P<0.05). C) Acquisition of cell surface plasmin activity in human plasma by GAS strains NS88.2, NS88.2prp, and NS88.2prpRC (mean±SD, n=9; P<0.05).

Virulence in the humanized plasminogen mouse infection model
A mounting body of evidence suggests that the ability to localize plasminogen at the cell surface is critical for the virulence of certain GAS isolates. The role of M-protein dependent plasminogen acquisition in virulence was investigated using a humanized plasminogen transgenic mouse infection model. Following subcutaneous injection of AlbPLG1, mice with either NS88.2, NS88.2prp, or NS88.2prpRC, survival was monitored over a 10-day period. Although the wild-type strain was highly virulent, with 80% of infected mice dead by day 5, NS88.2prp was significantly attenuated for virulence in this model of infection (10% mortality; P<0.05; Fig. 5 ). Replacement of the wild-type gene resulted in restoration of virulence equivalent to that of the wild type (90% mortality; P>0.05).


Figure 5
View larger version (8K):
[in this window]
[in a new window]

  		Figure 5. Virulence of NS88.2, NS88.2prp, and NS88.2prpRC. Cohorts of 10 humanized plasminogen transgenic mice were subcutaneously infected with 1.13 x 107 CFU NS88.2 ({blacksquare}), 1.3 x 107 CFU NS88.2prp ({blacktriangleup}) or 2.6 x 107 CFU NS88.2prpRC ({blacktriangledown}). Survival was measured over a 10-day period. NS88.2prp was significantly attenuated for virulence when compared to both NS88.2 and NS88.2prpRC (P<0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The human plasminogen system has been shown to significantly contribute to GAS virulence. The introduction of the human plasminogen transgene into C57B/J6 mice resulted in reduced survival of transgenic mice, in comparison to nontransgenic littermate control mice, on GAS subcutaneous infection (7) . In the same study, deletion of the GAS plasminogen activator streptokinase brought about a significant reduction in virulence of isogenic GAS mutants, in comparison to the wild-type parental strain (7) . Here, we have demonstrated that the capacity of the plasminogen-binding M protein Prp to bind plasminogen to the GAS cell surface is also a requirement for full GAS virulence. While displaying intact M protein-mediated innate immune defense and streptokinase expression equivalent to wild type, the isogenic mutant NS88.2prp is reduced in capacity to cause lethal infection in the humanized plasminogen mouse model of invasive infection. These observations indicate the capacity to accumulate human plasmin activity at the bacterial surface is an important additional step in the transition of GAS from asymptomatic or benign infection to life-threatening invasive disease.

Isolated from a bacteremic infection in the Northern Territory, GAS strain NS88.2 has been shown to bind high levels of plasminogen when compared to other GAS isolates from this region (13) . This strain expresses the PAM-related protein Prp, which functions as a high-affinity plasminogen receptor (18) . To investigate the contribution of M protein to plasminogen binding and cell surface plasmin acquisition, mutagenesis of the NS88.2 M protein Prp was undertaken. The isogenic mutant NS88.2prp was engineered, whereby two lysine residues, an arginine and a histidine residue critical for plasminogen binding by Prp, were converted to alanine codons in the GAS genome sequence. Expression of virulence factors {alpha}-enolase, GAPDH, and streptokinase, which have been shown to interact with plasminogen (34 , 36 37 38) , were unaffected in NS88.2prp. The ability of this isogenic mutant to interact with fibrinogen, resist phagocytosis by polymorphonuclear leukocytes and grow in whole blood was unchanged compared to the wild-type parental strain. However, replacement of the wild-type prp gene in NS88.2 with a prp gene encoding a protein attenuated for plasminogen binding, significantly reduced the ability of this strain to accumulate both plasminogen and plasmin at the cell surface.

The lack of either plasminogen acquisition or activation in human serum by NS88.2prp suggests that for GAS-expressing plasminogen binding M proteins (PAM-positive GAS), {alpha}-enolase, and GAPDH, which likely interact with kringle domains 4 and 5 of plasminogen (11 , 39) , do not significantly contribute to plasminogen binding and activation. Plasminogen-binding M proteins interact with kringle domain 2 of plasminogen and are specifically coinherited with the subcluster 2b allele of streptokinase (8) . These observations suggest that, for PAM-positive S. pyogenes strains, the specific interaction between human plasminogen kringle 2 domain, subcluster 2b streptokinase and plasminogen-binding M proteins provide selection pressure for the coinheritance of these GAS virulence factors (8 , 11) . A number of PAM-negative GAS strains, including GAS serotype M1T1, also demonstrate human plasminogen-dependent virulence (7 , 26 , 40) We, therefore, hypothesize that PAM-positive and PAM-negative GAS strains employ differing strategies for surface plasminogen acquisition and activation. For PAM-negative isolates, the formation of a trimolecular complex of streptokinase, plasminogen, and fibrinogen, bound to the GAS cell surface via fibrinogen receptors or the plasminogen receptors {alpha}-enolase and GAPDH may represent an alternative human plasminogen-dependent virulence pathway employed by this strain set (11 , 13 , 34 , 41) .

Previous studies investigating the role of PAM in GAS disease have focused on strain ALAB49. This isolate was associated with uncomplicated impetigo infection (7 , 14) . Studies using the hu-skin-SCID mouse model for streptococcal impetigo found that a PAM-negative isogenic deletion mutant of GAS strain ALAB49 displayed partial attenuation for virulence when compared to the wild-type strain (14) . However, a recently published animal study of GAS infection showed that in mice expressing the human plasminogen transgene, infection with the PAM-positive GAS strain, AP53 resulted in 80% mortality, whereas an isogenic mutant of AP53, where the PAM gene was replaced by an antibiotic resistance cassette, exhibited only minimal virulence (7) . While this observation supports the findings of the present study, the creation of isogenic PAM deletion mutants may also reduce resistance to phagocytosis (14) . Hence, the contribution of plasminogen binding, resistance to phagocytosis (42) , and other potential polar effects cannot be accounted for. The GAS isogenic mutant strains NS88.2prp and NS88.2prpRC used in this study were precisely constructed, were found to have comparable levels of interaction with polymorphonuclear leukocytes, and showed equivalent levels of growth in human blood.

Our results suggest that the plasminogen-binding M protein Prp is a critical requirement for virulence in GAS strain NS88.2. Nonetheless, PAM-negative GAS are capable of virulent infection. This apparent dichotemy is not unprecedented. In two separate studies, isogenic mutagenesis of the fibronectin and fibrinogen-binding protein serum opacity factor (SOF) resulted in a loss of GAS virulence in an animal model of infection (43 , 44) . Yet, sof is associated with only 40–50% of GAS isolates (45 , 46) . Similarly, targeted mutagenesis of the fibronectin binding protein FbaA, which is associated with ~70–85% of GAS isolates (47 48 49) , resulted in a significant increase in survival when compared to wild-type GAS in a murine model of skin infection (47) . These examples provide further evidence for the concept of divergent sets of genotypically distinct GAS isolates exhibiting different virulence strategies in the face of numerous host defenses.

The requirement for GAS surface plasminogen binding and activation for lethal murine infection described in this work suggests an important role for the human plasminogen activation system in the transition from benign to invasive disease. Rather than employing a strategy whereby diffuse activation of plasminogen by streptokinase occurs at or near the site of infection, the formation of active plasmin on the S. pyogenes surface may allow the bacterium to specifically penetrate innate immune barriers leading to the breakout of human plasmin-decorated GAS cells systemically. An obvious innate immune defense, which would be susceptible to this form of bacterial offensive strategy, would be the fibrinogen layer deposited around the site of local GAS infection by the host (7) .

Recent data indicate that more 660,000 cases of invasive GAS infection occur worldwide each year. Of these, almost one quarter are fatal (1) . To establish invasive infection, S. pyogenes must overcome barriers posed by the host innate immune response. Our data support the contention that the human plasminogen activation system plays a central role in this process. The expression of functional Prp at the GAS cell surface is essential for the virulence of strain NS88.2, suggesting that other plasminogen-binding M proteins of both GAS (13 , 16 , 50 , 51) , and different streptococcal species (52) may play an important role in bacterial dissemination within the host.


   ACKNOWLEDGMENTS
 
This work was funded in part by an Australian National Health and Medical Research Council grant (459103). M.L.S.-S. is the recipient of an Alexander von Humboldt Research Fellowship. A.J.C. is the recipient of a University of Wollongong postgraduate award. The authors thank René Bergmann for assistance with the radiolabeling of proteins.

Received for publication January 8, 2008. Accepted for publication April 10, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Carapetis, J. R., Steer, A. C., Mulholland, E. K., Weber, M. (2005) The global burden of group A streptococcal diseases. Lancet Infect. Dis. 5,685-694[CrossRef][Medline]
  2. Cunningham, M. W. (2000) Pathogenesis of group A streptococcal infections. Clin. Microbiol. Rev. 13,470-511[Abstract/Free Full Text]
  3. Tart, A. H., Walker, M. J., Musser, J. M. (2007) New understanding of the group A Streptococcus pathogenesis cycle. Trends Microbiol. 15,318-325[CrossRef][Medline]
  4. Carapetis, J. R., Walker, A. M., Hibble, M., Sriprakash, K. S., Currie, B. J. (1999) Clinical and epidemiological features of group A streptococcal bacteraemia in a region with hyperendemic superficial streptococcal infection. Epidemiol. Infect. 122,59-65[CrossRef][Medline]
  5. Gardiner, D. L., Sriprakash, K. S. (1996) Molecular epidemiology of impetiginous group A streptococcal infections in aboriginal communities of northern Australia. J. Clin. Microbiol. 34,1448-1452[Abstract/Free Full Text]
  6. Gardiner, D., Hartas, J., Hibble, M., Goodfellow, A., Currie, B., Sriprakash, K. S. (1997) Molecular epidemiology of group A streptococcal infection in the Northern Territory of Australia. Adv. Exp. Med. Biol. 418,317-321[Medline]
  7. Sun, H., Ringdahl, U., Homeister, J. W., Fay, W. P., Engleberg, N. C., Yang, A. Y., Rozek, L. S., Wang, X., Sjöbring, U., Ginsburg, D. (2004) Plasminogen is a critical host pathogenicity factor for group A streptococcal infection. Science 305,1283-1286[Abstract/Free Full Text]
  8. Kalia, A., Bessen, D. E. (2004) Natural selection and evolution of streptococcal virulence genes involved in tissue-specific adaptations. J. Bacteriol. 186,110-121[Abstract/Free Full Text]
  9. Danø, K., Andreasen, P. A., Grøndahl-Hansen, J., Kristensen, P., Nielsen, L. S., Skriver, L. (1985) Plasminogen activators, tissue degradation, and cancer Adv. Cancer Res. 44,139-266[CrossRef]
  10. Ponting, C. P., Marshall, J. M., Cederholm-Williams, S. A. (1992) Plasminogen: a structural review. Blood Coagul. Fibrinolysis 3,605-614[Medline]
  11. Walker, M. J., McArthur, J. D., McKay, F., Ranson, M. (2005) Is plasminogen deployed as a Streptococcus pyogenes virulence factor?. Trends Microbiol. 13,308-313[CrossRef][Medline]
  12. Berge, A., Sjöbring, U. (1993) PAM, a novel plasminogen-binding protein from Streptococcus pyogenes. J. Biol. Chem. 268,25417-25424[Abstract/Free Full Text]
  13. McKay, F. C., McArthur, J. D., Sanderson-Smith, M. L., Gardam, S., Currie, B. J., Sriprakash, K. S., Fagan, P. K., Towers, R. J., Batzloff, M. R., Chhatwal, G. S., Ranson, M., Walker, M. J. (2004) Plasminogen binding by group A streptococcal isolates from a region of hyperendemicity for streptococcal skin infection and a high incidence of invasive infection. Infect. Immun. 72,364-370[Abstract/Free Full Text]
  14. Svensson, M. D., Sjöbring, U., Luo, F., Bessen, D. E. (2002) Roles of the plasminogen activator streptokinase and the plasminogen-associated M protein in an experimental model for streptococcal impetigo. Microbiology 148,3933-3945[Abstract/Free Full Text]
  15. Wistedt, A. C., Kotarsky, H., Marti, D., Ringdahl, U., Castellino, F. J., Schaller, J., Sjöbring, U. (1998) Kringle 2 mediates high affinity binding of plasminogen to an internal sequence in streptococcal surface protein PAM. J. Biol. Chem. 273,24420-24424[Abstract/Free Full Text]
  16. Wistedt, A. C., Ringdahl, U., Müller-Esterl, W., Sjöbring, U. (1995) Identification of a plasminogen-binding motif in PAM, a bacterial surface protein. Mol. Microbiol. 18,569-578[CrossRef][Medline]
  17. Sanderson-Smith, M., Batzloff, M., Sriprakash, K. S., Dowton, M., Ranson, M., Walker, M. J. (2006) Divergence in the plasminogen-binding group a streptococcal M protein family: functional conservation of binding site and potential role for immune selection of variants. J. Biol. Chem. 281,3217-3226[Abstract/Free Full Text]
  18. Sanderson-Smith, M. L., Dowton, M., Ranson, M., Walker, M. J. (2007) The plasminogen-binding group A streptococcal M protein-related protein Prp binds plasminogen via arginine and histidine residues. J. Bacteriol. 189,1435-1440[Abstract/Free Full Text]
  19. Sanderson-Smith, M. L., Walker, M. J., Ranson, M. (2006) The maintenance of high-affinity plasminogen binding by group A streptococcal plasminogen-binding M-like protein (PAM) is mediated by arginine and histidine residues within the a1 and a2 repeat domains. J. Biol. Chem. 281,25965-25971[Abstract/Free Full Text]
  20. Ringdahl, U., Svensson, M., Wistedt, A. C., Renne, T., Kellner, R., Müller-Esterl, W., Sjöbring, U. (1998) Molecular co-operation between protein PAM and streptokinase for plasmin acquisition by Streptococcus pyogenes. J. Biol. Chem. 273,6424-6430[Abstract/Free Full Text]
  21. McArthur, J. D., Walker, M. J. (2006) Domains of group A streptococcal M protein that confer resistance to phagocytosis, opsonization and protection: implications for vaccine development. Mol. Microbiol. 59,1-4[CrossRef][Medline]
  22. Chaffin, D. O., Beres, S. B., Yim, H. H., Rubens, C. E. (2000) The serotype of type Ia and III group B streptococci is determined by the polymerase gene within the polycistronic capsule operon. J. Bacteriol. 182,4466-4477[Abstract/Free Full Text]
  23. Walker, M. J., Hollands, A., Sanderson-Smith, M. L., Cole, J. N., Kirk, J. K., Henningham, A., McArthur, J. D., Dinkla, K., Aziz, R. K., Kansal, R. G., Simpson, A. J., Buchanan, J. T., Chhatwal, G. S., Kotb, M., Nizet, V. (2007) DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection. Nat. Med. 13,981-985[CrossRef][Medline]
  24. Simon, D., Ferretti, J. J. (1991) Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA. FEMS Microbiol. Lett. 66,219-224[Medline]
  25. Ashbaugh, C. D., Wessels, M. R. (2001) Absence of a cysteine protease effect on bacterial virulence in two murine models of human invasive group A streptococcal infection. Infect. Immun. 69,6683-6688[Abstract/Free Full Text]
  26. Cole, J. N., McArthur, J. D., McKay, F. C., Sanderson-Smith, M. L., Cork, A. J., Ranson, M., Rohde, M., Itzek, A., Sun, H., Ginsburg, D., Kotb, M., Nizet, V., Chhatwal, G. S., Walker, M. J. (2006) Trigger for group A streptococcal M1T1 invasive disease. FASEB J. 20,1745-1747[Abstract/Free Full Text]
  27. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685[CrossRef][Medline]
  28. Lancefield, R. C. (1962) Current knowledge of type-specific M antigens of group A streptococci. J. Immunol. 89,307-313[Abstract/Free Full Text]
  29. Dinkla, K., Sastalla, I., Gödehart, A. W., Janze, N., Chhatwal, G. S., Röhde, M., Medina, E. (2007) Upregulation of capsule enables Streptococcus pyogenes to evade immune recognition by antigen-specific antibodies directed to the G-related alpha2-macroglobulin-binding protein GRAB located on the bacterial surface. Microbes Infect. 9,922-931[CrossRef][Medline]
  30. Staali, L., Morgelin, M., Björck, L., Tapper, H. (2003) Streptococcus pyogenes expressing M and M-like surface proteins are phagocytosed but survive inside human neutrophils. Cell. Microbiol. 5,253-265[CrossRef][Medline]
  31. Hollingshead, S. K., Readdy, T. L., Yung, D. L., Bessen, D. E. (1993) Structural heterogeneity of the emm gene cluster in group A streptococci. Mol. Microbiol. 8,707-717[Medline]
  32. Courtney, H., Hasty, D., Dale, J. (2006) Anti-phagocytic mechanisms of Streptococcus pyogenes: binding of fibrinogen to M-related protein. Mol. Microbiol. 59,936-947[CrossRef][Medline]
  33. Carlsson, F., Sandin, C., Lindahl, G. (2005) Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway. Mol. Microbiol. 56,28-39[CrossRef][Medline]
  34. Wang, H., Lottenberg, R., Boyle, M. D. (1995) Analysis of the interaction of group A streptococci with fibrinogen, streptokinase and plasminogen. Microb. Pathog. 18,153-166[CrossRef][Medline]
  35. Johnson, D., Kaplan, E., Sramek, J., Bicova, R., Havlicek, J., Havlickova, H., Motlova, J., Kriz, P. (1996) Laboratory Diagnosis of Group A Streptococcal Infections World Health Organisation Geneva.
  36. Pancholi, V., Fischetti, V. A. (1998) Alpha-enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J. Biol. Chem. 273,14503-14515[Abstract/Free Full Text]
  37. Pancholi, V., Fischetti, V. A. (1992) A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity. J. Exp. Med. 176,415-426[Abstract/Free Full Text]
  38. Bajaj, A. P., Castellino, F. J. (1977) Activation of human plasminogen by equimolar levels of streptokinase. J. Biol. Chem. 252,492-498[Abstract/Free Full Text]
  39. Ranson, M., Andronicos, N. M. (2003) Plasminogen binding and cancer: promises and pitfalls. Front. Biosci. 8,s294-s304[Medline]
  40. Khil, J., Im, M., Heath, A., Ringdahl, U., Mundada, L., Cary Engleberg, N., Fay, W. P. (2003) Plasminogen enhances virulence of group A streptococci by streptokinase-dependent and streptokinase-independent mechanisms. J. Infect. Dis. 188,497-505[CrossRef][Medline]
  41. Wang, H., Lottenberg, R., Boyle, M. D. (1995) A role for fibrinogen in the streptokinase-dependent acquisition of plasmin(ogen) by group A streptococci. J. Infect. Dis. 171,85-92[Medline]
  42. Fischetti, V. A. (1989) Streptococcal M protein: molecular design and biological behavior. Clin. Microbiol. Rev. 2,285-314[Abstract/Free Full Text]
  43. Courtney, H. S., Hasty, D. L., Li, Y., Chiang, H. C., Thacker, J. L., Dale, J. B. (1999) Serum opacity factor is a major fibronectin-binding protein and a virulence determinant of M type 2 Streptococcus pyogenes. Mol. Microbiol. 32,89-98[CrossRef][Medline]
  44. Timmer, A. M., Kristian, S. A., Datta, V., Jeng, A., Gillen, C. M., Walker, M. J., Beall, B., Nizet, V. (2006) Serum opacity factor promotes group A streptococcal epithelial cell invasion and virulence. Mol. Microbiol. 62,15-25[CrossRef][Medline]
  45. Kreikemeyer, B., Talay, S. R., Chhatwal, G. S. (1995) Characterization of a novel fibronectin-binding surface protein in group A streptococci. Mol. Microbiol. 17,137-145[Medline]
  46. Delvecchio, A., Currie, B. J., McArthur, J. D., Walker, M. J., Sriprakash, K. S. (2002) Streptococcus pyogenes prtFII, but not sfbI, sfbII or fbp54, is represented more frequently among invasive-disease isolates of tropical Australia. Epidemiol. Infect. 128,391-396[CrossRef][Medline]
  47. Terao, Y., Kawabata, S., Kunitomo, E., Murakami, J., Nakagawa, I., Hamada, S. (2001) Fba, a novel fibronectin-binding protein from Streptococcus pyogenes, promotes bacterial entry into epithelial cells, and the fba gene is positively transcribed under the Mga regulator. Mol. Microbiol. 42,75-86[CrossRef][Medline]
  48. Baldassarri, L., Creti, R., Imperi, M., Recchia, S., Pataracchia, M., Orefici, G. (2007) Detection of genes encoding internalization-associated proteins in Streptococcus pyogenes isolates from patients with invasive diseases and asymptomatic carriers. J. Clin. Microbiol. 45,1284-1287[Abstract/Free Full Text]
  49. Ramachandran, V., McArthur, J. D., Behm, C., Gutzeit, C., Dowton, M., Fagan, P., Towers, R., Currie, B., Sriprakash, K., Walker, M. J. (2004) Two distinct genotypes of prtF2, encoding a fibronectin binding protein, and evolution of the gene family in Streptococcus pyogenes. J. Bacteriol. 186,7601-7609[Abstract/Free Full Text]
  50. Svensson, M. D., Sjöbring, U., Bessen, D. E. (1999) Selective distribution of a high-affinity plasminogen-binding site among group A streptococci associated with impetigo. Infect. Immun. 67,3915-3920[Abstract/Free Full Text]
  51. Ringdahl, U., Sjöbring, U. (2000) Analysis of plasminogen-binding M proteins of Streptococcus pyogenes. Methods 21,143-150[CrossRef][Medline]
  52. Ben Nasr, A., Wistedt, A., Ringdahl, U., Sjöbring, U. (1994) Streptokinase activates plasminogen bound to human group C and G streptococci through M-like proteins. Eur. J. Biochem. 222,267-276[Medline]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
M. L. Vieira, S. A. Vasconcellos, A. P. Goncales, Z. M. de Morais, and A. L. T. O. Nascimento
Plasminogen Acquisition and Activation at the Surface of Leptospira Species Lead to Fibronectin Degradation
Infect. Immun., September 1, 2009; 77(9): 4092 - 4101.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. J. Cork, S. Jergic, S. Hammerschmidt, B. Kobe, V. Pancholi, J. L. P. Benesch, C. V. Robinson, N. E. Dixon, J. A. Aquilina, and M. J. Walker
Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase
J. Biol. Chem., June 19, 2009; 284(25): 17129 - 17137.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
fj.07-105643v1
fj.07-105643v2
22/8/2715    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sanderson-Smith, M. L.
Right arrow Articles by Walker, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sanderson-Smith, M. L.
Right arrow Articles by Walker, M. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS