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J. Biol. Chem., Vol. 279, Issue 51, 52820-52823, December 17, 2004

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₂-Macroglobulin-Proteinase Complexes Protect Streptococcus pyogenes from Killing by the Antimicrobial Peptide LL-37^*

Patrik Nyberg, Magnus Rasmussen, and Lars Björck

From the Department of Cell and Molecular Biology, Section for Clinical and Experimental Infectious Medicine, Lund University, S-22184 Lund, Sweden

Received for publication, October 13, 2004 , and in revised form, October 29, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The significant human bacterial pathogen Streptococcus pyogenes expresses GRAB, a surface protein that binds ₂-macroglobulin (₂M), a major proteinase inhibitor of human plasma. ₂M inhibits proteolysis by trapping the proteinase, which, however, still remains proteolytically active against smaller peptides that can penetrate the ₂M-proteinase complex. Here we report that SpeB, a cysteine proteinase secreted by S. pyogenes, is trapped by ₂M bound to protein GRAB. As a consequence, SpeB is retained at the bacterial surface and protects S. pyogenes against killing by the antibacterial peptide LL-37.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Streptococcus pyogenes is a common and clinically important human bacterial pathogen causing a wide range of invasive and non-invasive disease, as well as non-suppurative sequelae (for review, see Ref. 1). Work in many laboratories has underlined the significance of proteolysis in S. pyogenes virulence (for review, see Ref. 2). These Gram-positive bacteria secrete proteinases and they inhibit/activate proteolytic cascades of the human host, including the complement, contact, coagulation, and fibrinolytic systems. As S. pyogenes penetrates into normally sterile sites, this will attract neutrophils, which, when activated, will release proteinases. Proteinases also leak from damaged cells and tissues into the infectious nidus. Combined this generates massive proteolytic activity, which has been suggested to promote S. pyogenes virulence by enhancing tissue spread and dissemination. However, to exploit this activity efficiently, the bacteria should also be able to regulate and modify the proteolysis they induce. It was therefore interesting to find that S. pyogenes expresses a surface protein called GRAB, which binds the proteinase inhibitor ₂-macroglobulin (₂ M)¹with high specificity and affinity (3). ₂M bound to the bacterial surface via GRAB entraps and inhibits host and S. pyogenes proteinases and thereby protects bacterial surface proteins and virulence determinants from degradation (3). Among these proteinases is SpeB, a classical enzyme of S. pyogenes (4).

SpeB is a cysteine proteinase that is secreted in large quantities by S. pyogenes. It has broad proteolytic activity and cleaves a large number of human proteins as well as surface proteins of S. pyogenes (for review, see 2). Like several other secreted bacterial proteinases, SpeB degrades and inactivates LL-37 (5), one of the major human antibacterial peptides (for review, see Ref. 6). LL-37 is small enough (4.5 kDa) to penetrate into the cage that ₂M forms around the trapped proteinase, which commonly remains proteolytically active in the cage. The hypothesis that active SpeB could be retained at the bacterial surface in complex with ₂M and protect S. pyogenes against killing by LL-37 initiated the present investigation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Proteins and Peptides—The streptococcal cysteine proteinase was purified as described previously (7). The activity of the purified protein was examined by active site titration as described (8). The SpeB preparation used in this paper was 46% active. Throughout the paper the amount of active SpeB is given. To activate SpeB, the proteinase was incubated in activation buffer (1 mM EDTA, and 1 mM DTT in 0.1 M NaAc-HAc, pH 5.0) for 30 min at 37 °C, and activated SpeB was used throughout this investigation. Since high concentrations of DTT were found to reduce the inhibitory capacity of ₂M, the amount of DTT used in the assays was reduced compared with previous investigations (7, 8). The final concentration of DTT in all assays was 0.1 mM, which did not affect SpeB activity toward azocasein. To block SpeB activity, E64 (Sigma) was added to the proteinase to a final concentration of 10 µM. ₂M was purified from human plasma as described (9), or purchased from Sigma. For some experiments ₂M was pretreated with 0.5 M methyl amine for 20 min at 37 °C to convert ₂M to the fast-migrating form (10). Plasmin from human placenta and bovine trypsin were from Sigma. The SpeB was radiolabeled using the Bolton and Hunter reagent (Amersham Biosciences, Amersham, UK) according to the manufacturer's instructions. Labeled proteins were separated from free ¹²⁵I on a PD10 column (Amersham Biosciences, Uppsala, Sweden). The cleavage site of SpeB in ₂M was determined by Edman degradation of fragments obtained after incubation of ₂M with activated SpeB for 15 min. LL-37 and Texas Red-labeled LL-37 were synthesized by Innovagen AB, Lund, Sweden.

SpeB Trap Assay—Radiolabeled SpeB was mixed with 2.5 µgof ₂M or 1 µl of heparinized human plasma in 10 µl of PBS and followed by incubation for 15 min at 37 °C. The mixture was subjected to SDS-PAGE using non-reducing conditions, followed by autoradiography using a BAS-III imaging plate. The plate was scanned with a Bio-Imaging Analyzer BAS-2000 (Fuji Photo Films Co. Ltd.), and intensity was calculated using the Image Gauche program. For competition experiments, radiolabeled SpeB and different amounts of proteinase were added to ₂M simultaneously. The reactions were terminated by the addition of SDS-PAGE sample buffer and the samples were put on ice.

Analysis of Proteolysis—SpeB was mixed with 1% (w/v) azocasein (Sigma) in PBS and incubated for 1 h at 37 °C. Non-degraded azocasein was precipitated with 5% trichloroacetic acid, centrifuged at 10 000 x g for 5 min, and A₃₆₆ of the resulting supernatant was determined. Alternatively SpeB was incubated with 40 nM Z-Arg-AFC (Enzyme Systems Products) (11) in PBS for 1 h at 37 °C. Fluorescence (excitation 400 nm, emission 505 nm) was measured using a LS-5B luminescence spectrometer (PerkinElmer Life Sciences). SpeB activity against LL-37 was determined by incubating 1.25 nmol of Texas Red-labeled LL-37 with 10 pmol of SpeB preincubated with PBS or 25 pmol of ₂M. After various time points aliquots were taken, and E64 was added to inactivate SpeB. The samples were separated by SDS-PAGE on a 16.5% Tris-Tricine gel (reducing conditions).

SpeB Binding Assays—S. pyogenes of strain KTL3 or its derivative MR4 (lacking GRAB at the bacterial surface (3)) were grown to mid-log phase (A₆₂₀ = 0.5), washed in PBS, and resuspended in PBS to a final concentration of 5 x 10⁹ cfu/ml. The bacteria were preincubated for 30 min with PBS or ₂M, washed three times in PBS, and resuspended in PBS. Radiolabeled SpeB was activated and preincubated for 15 min with PBS or E64 and added to the bacterial suspensions. After 15 min, the bacteria were washed three times, and the radioactivity of bacterial pellets was determined. Alternatively, incubation proceeded for various time points after the washing steps, and the radioactivity in the pellets was determined as described above.

LL-37 Killing Assays—KTL3 or MR4 bacteria were grown to mid-log phase (A₆₂₀ = 0.5), washed, and resuspended in 0.1 M Tris, pH 7.5, with 10 mM glucose (Tris-glucose). LL-37 (100 pmol) was pretreated for 1 h with different concentrations of SpeB, or SpeB complexed with ₂M, and added to the bacterial suspension. After 2 h, the bacteria were washed in Tris-glucose, and viable counts were performed. Alternatively, for determining the protective effects of the ₂M-SpeB complex bound to the bacterial surface via GRAB, KTL3 or MR4 bacteria were pretreated with ₂M for 30 min. The bacteria were washed three times in Tris-glucose and incubated with activated SpeB for 15 min. After three additional washes, different concentrations of LL-37 were added, and viable counts were performed after 2 h.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Initial experiments were performed to expand previous studies (3) on the relationship between SpeB and ₂M. Proteinase entrapment by ₂M results in inhibition of proteinase activity against larger protein substrates, and when SpeB was incubated with different concentrations of ₂M, a dose-dependent inhibition of SpeB activity against azocasein was observed (Fig. 1A). The SpeB-₂M interaction was further investigated by mixing radiolabeled SpeB with ₂M or plasma, followed by SDS-PAGE and autoradiography. A fraction of the radiolabeled SpeB co-migrated with ₂M, demonstrating the formation of ₂M-proteinase complexes (Fig. 1B). Pretreatment of ₂M or plasma with methylamine, converting ₂M to an inactive state, or pretreatment of radiolabeled SpeB with the cysteine proteinase inhibitor E64, prevented complex formation, as did the addition of a molar excess of unlabeled SpeB (Fig. 1B).

View larger version (18K): [in this window] [in a new window]   FIG. 1. SpeB is trapped by 2M. A, M inhibits SpeB activity against azocasein. SpeB (12 pmol) was incubated with different concentrations of 2M for 15 min, and the enzymatic activity against azocasein was measured. Values from a representative experiment are depicted. B, 2M-SpeB association is dependent on 2M configuration and SpeB activity. Radiolabeled SpeB was mixed with purified 2M (left gel) or plasma (right gel) for 15 min, separated by SDS-PAGE under non-reducing conditions, and subjected to autoradiography. The wells were loaded as follows: radiolabeled SpeB only (a), radiolabeled SpeB and 2M/plasma (b), radiolabeled SpeB and 2M/plasma pretreated with methylamine (c), radiolabeled SpeB and 2M/plasma treated with an excess of unlabeled SpeB (d), and radiolabeled SpeB pretreated with E64 and 2M/plasma (e). The high molecular weight bands correspond to SpeB complexed with 2M and the low molecular weight bands to uncomplexed SpeB. C, time course of the SpeB-2M complex formation. Radiolabeled SpeB was mixed with an excess of 2M and allowed to react for different periods of time, followed by SDS-PAGE (reducing conditions) and autoradiography. Values are given as percent of maximum association of SpeB to 2M (n = 3 ± S.D.). D, proteinase competition experiments. Different amounts of unlabeled SpeB (), trypsin (), or plasmin () were added together with radiolabeled SpeB to 0.7 pmol of 2M and allowed to react for 15 min, followed by SDS-PAGE (reducing conditions) and autoradiography. Values from a representative experiment are depicted and given as percent of association of SpeB to 2M in the absence of competitor.

The SpeB trap assay was used to investigate the time dependence of the interaction between radiolabeled SpeB and a molar excess of ₂M. The association between SpeB and ₂M followed a hyperbolic curve reaching 50% association after 1 min (Fig. 1C). Both trypsin and plasmin are efficiently inhibited by ₂M, and unlabeled trypsin, plasmin, and SpeB were used to compete with the binding of radiolabeled SpeB to ₂M (Fig. 1D). SpeB and trypsin had similar competition profiles, whereas plasmin was less efficient in inhibiting ₂M-SpeB complex formation under the conditions used.

These data show that SpeB is inactivated by ₂M when tested against azocasein in solution, and previous work has demonstrated that SpeB cleaves and inactivates the antimicrobial peptide LL-37 (5). Since proteinases in complex with ₂M generally retain their activity against small substrates, we investigated whether SpeB in complex with ₂M was still active against LL-37. SpeB was pretreated with a molar excess of ₂M, followed by incubation with Texas Red-labeled LL-37. Samples were taken at various time points and separated by SDS-PAGE (Fig. 2A). The degradation pattern of LL-37 by SpeB is very similar to that of Pseudomonas aeruginosa elastase (5), which cleaves LL-37 in the region responsible for its antibacterial activity (15). Unexpectedly, LL-37 was cleaved more efficiently when incubated with SpeB-₂M complexes than with only SpeB. ₂M alone did not induce degradation of LL-37 (data not shown). When the activity of SpeB against the small fluorescent substrate Z-Arg-AFC (11) was tested, SpeB in complex with ₂M was found to be three to five times more active than SpeB alone (data not shown). The molecular basis for the enhanced peptide degradation by SpeB in complex with ₂M is unclear, but the observation was further substantiated in experiments with S. pyogenes bacteria. Thus, ₂M-SpeB complexes inhibited LL-37-mediated killing more efficiently than SpeB alone (Fig. 2B). Again, ₂M alone had no effect (Fig. 2B, 0 pmol of SpeB, filled bar).

View larger version (27K): [in this window] [in a new window]   FIG. 2. Trapped SpeB degrades and inactivates LL-37. A, degradation of LL-37 by SpeB or 2M-SpeB complexes. SpeB was incubated with PBS (–) or a molar excess of 2M (+) for 15 min, followed by the addition of Texas Red-labeled LL-37. Aliquots were taken at various time points and subjected to SDS-PAGE (16.5% Tris-Tricine gel, reducing conditions). B, inactivation of LL-37 by SpeB or 2M-SpeB complexes. Different concentrations of SpeB were incubated with PBS (open bars) or a molar excess of 2M (filled bars) for 15 min, followed by the addition of LL-37. After 1 h, the samples were added to 2 x 106 KTL3 bacteria, and following incubation for 2 h, viable counts were performed. Values are the means of three experiments ± S.D.

Most strains of S. pyogenes express GRAB, an ₂M-binding cell wall-attached protein (3, 16). To determine whether GRAB could mediate binding of SpeB to the bacterial surface via ₂M, radiolabeled SpeB was added to the S. pyogenes strain KTL3 or to MR4, a mutant of KTL3 lacking GRAB (3), that had been preincubated with PBS or ₂M (Fig. 3A). The results demonstrate that SpeB is bound to the surface of GRAB-expressing KTL3 bacteria preincubated with ₂M, whereas only background levels of SpeB were associated with KTL3 bacteria preincubated with PBS, or with MR4 bacteria preincubated with ₂M. Background binding was also obtained when radiolabeled SpeB was inactivated with the cysteine proteinase inhibitor E64 and then added to GRAB-expressing KTL3 bacteria preincubated with ₂M (Fig. 3A, bar c). Following binding to ₂M at the bacterial surface, the amount of surface-associated SpeB remained approximately the same for at least 6 h (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   FIG. 3. 2M-SpeB complexes bound to the bacterial surface via GRAB protect S. pyogenes from LL-37-mediated killing. A, binding of radiolabeled SpeB to the surface of S. pyogenes bacteria. KTL3 or MR4 bacteria were mixed with radiolabeled SpeB, incubated for 15 min, washed, and pelleted by centrifugation. Finally, the radioactivity of the pellets was determined. Pretreatments were as follows: KTL3 bacteria were pretreated with PBS (a), KTL3 bacteria were pretreated with 2M (b), KTL3 bacteria were pretreated with 2M and then mixed with radiolabeled SpeB inactivated with E64 (c),and MR4 bacteria were pretreated with 2M (d). Values are the means of three experiments ± S.D. B, time-dependent binding of SpeB to the surface of S. pyogenes bacteria. KTL3 () or MR4 () bacteria were incubated with 2M and washed, and radiolabeled SpeB was added. After 15 min, the bacteria were washed and resuspended in PBS. Aliquots taken at different time points were pelleted by centrifugation, and the radioactivity of the pellets was determined. Data are from a representative experiment. C, effect of surface bound 2M-SpeB complexes on LL-37-mediated killing. KTL3 () or MR4 () bacteria were incubated with 2M and washed.SpeB was added followed by incubation (15 min) and washing.Different concentrations of LL-37 were added. The incubation proceeded for 2 h, and the number of cfus was determined. The values are the means from three experiments ± S.D.

Fig. 4 summarizes the observations of this study, demonstrating that ₂M-SpeB complexes can be formed at the surface of S. pyogenes through protein GRAB and that these complexes protect the bacteria from killing by LL-37. Previously, other mechanisms for S. pyogenes defense against antimicrobial peptides have been described. SIC, another secreted protein of S. pyogenes (17), inactivates both LL-37 and the neutrophil-derived antimicrobial peptide -defensin (18). Moreover, apart from cleaving LL-37, SpeB degrades proteoglycans, thereby releasing dermatan sulfate that binds to and inactivates -defensin (19). Thus, S. pyogenes has developed different defense strategies against antimicrobial peptides. This apparent redundancy could be explained by the temporal aspects of an S. pyogenes infection. It has been proposed that the early phase of S. pyogenes infection is characterized by inhibition of proteolytic activity, including inhibition of complement activation, at the bacterial surface, while later phases are characterized by massive proteolysis due to the release of SpeB and host proteinases (2). In this context, SIC would be an important early defense against antimicrobial peptides, being produced in the early growth phase (17). As the infection proceeds, SpeB production will start and soluble and surface bound ₂M-SpeB complexes are formed. Thus, at this stage bacterial surface proteins are protected, and SpeBs activity is directed against smaller substrates, like antimicrobial peptides. When SpeB production further increases, the protective mechanism is overridden and bacterial surface proteins responsible for bacterial attachment to host structures will be degraded, probably facilitating bacterial spread (2, 8). When the local ₂M pool is depleted, SpeB will degrade proteoglycans, thus generating free dermatan sulfate that inactivates -defensin. At this late phase of infection, activation of potent host proinflammatory systems by SpeB (20, 21) will induce clinical symptoms of S. pyogenes disease.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Schematic model for the generation of protective 2M-proteinase complexes at the surface of S. pyogenes. Box 1, GRAB is expressed at the bacterial surface during early growth phase. As the infection proceeds, the inflammatory response induces vascular leakage and 2M diffuses into the infectious site. Box 2, 2M binds to GRAB and secreted SpeB is trapped by 2M. Box 3, antimicrobial peptides (AMPs), such as LL-37, are cleaved and inactivated by SpeB retained at the bacterial surface in complex with 2M.

	FOOTNOTES

 
^* This work was supported by grants from the Swedish Research Council (Projects 7480 and 14379), the Medical Faculty, Lund University, the Foundations of Kock and Österlund, and Hansa Medical AB. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Section for Clinical and Experimental Infectious Medicine, Dept. of Cell and Molecular Biology, Lund University, BMC, B14, Tornavägen 10, S-22184 Lund, Sweden. Tel.: 46-46-2224489; Fax: 46-46-157756; E-mail: Patrik.Nyberg{at}medkem.lu.se.

¹ The abbreviations used are: ₂M, ₂-macroglobulin; DTT, dithiothreitol; PBS, phosphate-buffered saline; Z, benzyloxycarbonyl; AFC, 7-amino-4-trifluoromethylcoumarin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; cfu, colony-forming unit.

	ACKNOWLEDGMENTS

 
We thank Gustaf Grände for excellent technical assistance and Hans-Peter Müller for important technical and intellectual support.

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