SIC, a Secreted Protein of Streptococcus pyogenes That Inactivates Antibacterial Peptides*

Inga-Maria FrickDagger §, Per Åkesson, Magnus RasmussenDagger , Artur Schmidtchen, and Lars BjörckDagger

From the Dagger  Section for Molecular Pathogenesis, Department of Cell and Molecular Biology, Lund University, and the  Department of Medical Microbiology, Dermatology, and Infection, Lund University Hospital, S-221 85 Lund, Sweden

Received for publication, August 30, 2002, and in revised form, February 25, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Some isolates of the significant human pathogen Streptococcus pyogenes, including virulent strains of the M1 serotype, secrete protein SIC. This molecule, secreted in large quantities, interferes with complement function. As a result of natural selection, SIC shows a high degree of variation. Here we provide a plausible explanation for this variation and the fact that strains of the M1 serotype are the most frequent cause of severe invasive S. pyogenes infections. Thus, protein SIC was found to inactivate human neutrophil alpha -defensin and LL-37, two major antibacterial peptides involved in bacterial clearance. This inactivation protected S. pyogenes against the antibacterial effect of the peptides. Moreover, SIC isolated from S. pyogenes of the M1 serotype was more powerful in this respect than SIC variants from strains of M serotypes 12 and 55, serotypes rarely connected with invasive infections.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Streptococcus pyogenes is one of the most common and important human bacterial pathogens. It causes relatively mild infections such as pharyngitis (strep throat) and impetigo but also serious clinical conditions like rheumatic fever, poststreptococcal glomerulonephritis, necrotizing fasciitis, septicemia, and a toxic-shock syndrome (1, 2). Increases in the number of life-threatening systemic S. pyogenes infections have been reported world-wide since the late 1980s and have attracted considerable attention and concern (3, 4). Based on the highly polymorphic M protein, a surface protein of S. pyogenes (for references see Ref. 5), isolates are divided into more than 100 serological subtypes, and systemic infections are most frequently caused by organisms of the M1 serotype (6).

Protein SIC was originally isolated from the growth medium of an M1 strain (7). All strains of the M1 serotype secrete SIC and so do M57 organisms, whereas strains of 53 other serotypes were found to lack the sic gene (7). Subsequent work has identified distantly related sic variants also in M12 and M55 strains (8). SIC stands for streptococcal inhibitor of complement, as the protein incorporates into the membrane attack complex of complement and inhibits complement-mediated lysis of sensitized erythrocytes (7). This inhibition of membrane attack complex was recently shown to be the result of SIC preventing uptake of C567 onto cell membranes (9). A remarkable property of SIC was reported by Stockbauer et al. (10). They found that the sequences of a large number of sic genes from different strains of the M1 serotype showed a unique degree of variation, which is in striking contrast to the lack of M1 protein variation. Moreover, in a mouse model of infection, Hoe et al. (11) discovered that SIC variants arise rapidly on mucosal surfaces by natural selection. They also reported that the inhibition of complement-mediated lysis by SIC was not affected in the new SIC variants arising from natural selection, suggesting that complement inhibition is not the only function of SIC. Complement belongs to the innate immune system, and antibacterial peptides represent another important part of this defense system. These peptides, originally described in silk worms (12), play important roles in the clearance of bacteria at biological boundaries susceptible for infection (for references see reviews in Refs. 13-16), and the starting point for this investigation was the hypothesis that SIC, secreted in substantial amounts by S. pyogenes (7), could interfere with the activity of antibacterial peptides. Such a mechanism could help explain the variation of SIC and the high frequency of S. pyogenes infections caused by strains of the M1 serotype.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Bacterial Strains and Purification of Protein SIC-- The S. pyogenes strains AP1 (40/58) of serotype M1 and AP12 (1/71) of serotype M12 were from the World Health Organization Collaborating Centre for Reference and Research on Streptococci, Prague, Czech Republic. The S. pyogenes strain W38 (GT 71-154) of serotype M55 was from the late Dr. L. W. Wannamaker, and the S. pyogenes strains U15 and U17 of serotype M1 were kindly provided by Dr. Stig Holm, Umeå University, Umeå, Sweden. Bacteria were grown in Todd-Hewitt broth (TH1; Difco) at 37 °C. Protein SIC was purified from the S. pyogenes strains AP1, U15, U17, AP12, and W38 as described (7) by precipitation of the culture medium with 30% ammonium sulfate, followed by ion-exchange chromatography on Mono Q (Amersham Biosciences). Fractions containing protein SIC were combined, dialyzed against 2 mM NH4HCO3 and freeze-dried. For the antimicrobial assay (see below) protein SIC was dissolved in 10 mM Tris-HCl, pH 7.5, containing 5 mM glucose.

DNA Sequencing-- PCR was used to amplify the sic genes from the S. pyogenes strains U15, U17, AP12, and W38. For serotype M1 strains (U15 and U17), primers were constructed corresponding to the nucleotide sequence starting 72 base pairs upstream (ACCTTTACTAATAATCGTCTTTGTTTTATAATGA) and 179 base pairs downstream (ATCTTTCTCGGACTCAGATAGTCCATAGC) of the coding sequence for sic in the AP1 strain (7). For serotype M12 (AP12) and M55 (W38) strains, a forward primer (CATTAACGAAATAATTTATTAAGGAGAG) corresponding to a region upstream of the coding sequence of sic from AP1 and a reverse primer (CCAATGATAGTCACCAGCAATTCAGG) corresponding to a region downstream of the coding sequence of the distantly related sic from M12 and 55 strains (8). The PCR products were purified with a High Pure PCR purification kit (Roche Applied Science) and used as templates in sequencing reactions using an ABI PRISM® BigDyeTM dideoxy terminator kit (BigDye terminator version 3.0 cycle sequencing Ready Reaction, Applied Biosystems), according to the manufacturer's instructions.

Proteins, Peptides, Antibodies, and radiolabeling-- Recombinant M1 protein was prepared as described previously (17), alpha -defensin (HNP-1), ACYCRIPACIAGERRYGTCIYQGRLWAFCC (Mr 3442) was purchased from Sigma, and LL-37, LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (Mr 4492) was synthesized by Innovagen AB, Lund, Sweden. The peptide GCP28, GCPRDIPTNSPELEETLTHTITKLNAEN, based on a sequence in human kininogen has been described previously (18) and was a kind gift from Dr. Heiko Herwald, Lund University, Lund, Sweden. Mouse monoclonal alpha -defensin antibodies were from Bachem AG, and rabbit polyclonal LL-37 antibodies were from Innovagen AB. Antiserum against protein SIC was raised in rabbits. Protein SIC was labeled with 125I using the chloramine T method (19).

Generation of a sic Mutant in S. pyogenes and Northern Blotting-- To inactivate the gene encoding protein SIC, AP1 bacteria were subjected to an allelic replacement mutagenesis strategy. A fragment of the up-stream sph and of the down-stream IS1562 was amplified from AP1 by PCR as described (20) using synthetic oligonucleotides hybridizing with nucleotides 486-512 and 1559-1533 in (21) and 516-539 and 1614-1641 in (22). The oligonucleotides had sites for restriction enzymes that were used to clone the sph product into multiple cloning site I of plasmid pFW13 (23) and the IS1562 product into multiple cloning site II to generate pFW13sic-. 20 µg of pFW13sic- was electroporated as described (24) into AP1 bacteria. Recombinants were selected on plates containing 150 µg/ml kanamycin. One transformant (SIC-) was obtained, and growth media from this strain was incubated with 6% trichloroacetic acid for 30 min on ice followed by centrifugation at 15,000 × g (4 °C for 20 min). Precipitated material was analyzed for SIC content using polyclonal anti-SIC antibodies in Western blot experiments.

Total RNA from the AP1 and SIC- strains was isolated at early logarithmic (A620 0.3), late logarithmic (A620 0.6), or early stationary phase (A620 0.8) as described previously (20). Northern blotting was performed as described (20) using a probe generated with primers hybridizing to sic (1-28, 912-889 in Ref. 7).

Animal Experiments-- Female NMRI mice weighing ~25 g were injected subcutaneously with 104 mutant or wild-type bacteria in 100 µl of PBS followed by 900 µl of air. Mice were observed for 10 days. Statistical analysis of survival time was performed with the Wilcoxin rank sum test.

Antimicrobial Assay-- AP1 bacteria were grown to mid-log phase in TH broth, washed, and diluted in 10 mM Tris-HCl, pH 7.5, containing 5 mM glucose. 50 µl of bacteria (2 × 106 colony forming units (cfu)/ml) were incubated together with alpha -defensin or LL-37 at various concentrations for 2 h at 37 °C. In subsequent experiments, bacteria were incubated with alpha -defensin or LL-37 at a concentration of 448 nM together with different concentrations of protein SIC or M1 protein and the reactions were carried out for 2 h (alpha -defensin) or 1 h (LL-37). To quantitate the bactericidal activity serial dilutions of the incubation mixtures were plated on TH agar, incubated overnight at 37 °C, and the number of cfus were determined.

Bacterial Growth Assay-- AP1 and SIC- bacteria were grown to stationary phase in TH broth. 200 µl of TH was inoculated with 5 µl of the bacterial suspension in 96-well plates (Falcon) at 37 °C. At early logarithmic phase various amounts of LL-37 was added, and growth was followed by measuring the absorbance at 490 nm (using a BioRad 550 microplate reader). The amount of protein SIC in the growth medium was estimated by ELISA (see below).

Slot-binding, SDS-PAGE, and Western Blot Analysis-- Peptides were applied to polyvinylidene difluoride (PVDF) membranes (Immobilon, Millipore) using a Milliblot-D system (Millipore). Membranes were blocked in TBS (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl) containing 3% bovine serum albumin, incubated with 125I-labeled protein SIC for 3 h, and washed with TBS containing 0.05% Tween 20. Autoradiography was carried out using Kodak x-Omat AR films and regular intensifying screens. SDS-PAGE was performed as described by Laemmli (25) using a polyacrylamide concentration of 10% or 12% and 3.3% cross-linking. Samples were boiled for 3 min in sample buffer containing 2% SDS and 5% 2-mercaptoethanol. Gels were stained with Coomassie Blue or subjected to Western blot analysis. Separated proteins were transferred to PVDF membranes using a trans-blot semidry transfer cell (Bio-Rad). Membranes were blocked with PBS containing 0.05% Tween 20 (PBST) and 5% dry milk powder (blocking buffer), incubated with antibodies against protein SIC (1:1000) in blocking buffer for 30 min at 37 °C, washed with PBST, and incubated with a horseradish peroxidase-conjugated antibody against rabbit IgG (1:3000) for 30 min at 37 °C. The membranes were washed, and detection of bound antibodies was performed using the chemiluminescence method.

ELISA-- Indirect ELISA was performed by coating microtiter plates (Maxisorb, NUNC) overnight with protein SIC or M1 protein at a concentration of 2.9 nM. The plates were washed in PBST, blocked in PBST containing 2% bovine serum albumin for 30 min and incubated with 58 nM alpha -defensin or LL-37 for 1 h. Bound antibacterial peptides were detected with specific antibodies against alpha -defensin (1:2000) or LL-37 (1:5000), and binding was visualized by a peroxidase-conjugated secondary antibody against mouse or rabbit IgG (1:3000). All incubations were performed at 37 °C for 1 h followed by a washing step. Substrate solution, 0.1% (w/v) diammonium-2,2-azino-bis-(3-ethyl-2,3-dihydrobenzthiazoline)-6-sulfonate, 0.012% (v/v) H2O2 in 100 mM NaH2PO4, pH 4.5, was added, and the change in absorbance at 405 nm was determined after 5 min. To determine the concentration of SIC in growth medium plates coated with AP1 or SIC- growth medium were incubated with antibodies against protein SIC (1:1000) followed by a secondary antibody against rabbit IgG (1:3000). Visualization of binding was detected as above, and absorbance was determined after 15 min.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Two major and well characterized human antibacterial peptides, alpha -defensin (HNP-1) and LL-37, were used in this investigation. These peptides have broad antibacterial activity against both Gram-positive and Gram-negative bacteria. alpha -defensin (HNP-1) is found in the azurophilic granules of human neutrophils, but analogues to neutrophil alpha -defensins are produced also by intestinal Paneth cells (26, 27). LL-37 is produced by neutrophils and epithelial cells. alpha -defensin and LL-37 are both found in extracellular fluids, including wound fluid, and the two peptides act synergistically on target bacteria (28). In S. pyogenes, the sic gene is part of the so-called mga regulon, and like the other genes of this regulon sic is expressed at an early growth phase (7), suggesting that SIC will be secreted as soon as S. pyogenes bacteria carrying the sic gene start to grow on an epithelial surface. To investigate whether alpha -defensin and LL-37 have affinity for SIC, these peptides and a control peptide (GCP28) based on a sequence in H-kininogen (18), were applied to Immobilon filters that were probed with 125I-labeled SIC (if not indicated otherwise SIC is from the M1 strain AP1). Fig. 1A shows that SIC interacts with the antibacterial peptides, an observation that was confirmed also by experiments where SIC and M1 protein were applied to plastic wells, followed by the addition of alpha -defensin or LL-37 and antibodies to the peptides. M1 protein was chosen as a control. It was isolated from the same strain of S. pyogenes (AP1) as SIC (7), and although the protein is predominantly associated with the cell wall, it is also released from the bacterial surface by proteolytic cleavage (29). In the experiments summarized in Fig. 1B, alpha -defensin and LL-37 showed affinity for SIC, whereas the interaction with M1 protein was at background level.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   SIC interacts with antibacterial peptides. A, various amounts of the antibacterial peptides alpha -defensin and LL-37 and the peptide GCP28 derived from human H-kininogen were applied to a PVDF membrane. The membrane was incubated with radiolabeled protein SIC (2 × 105 cpm/ml) for 3 h and autoradiographed for 3 days. B, microtiter plates were coated with protein SIC or M1 protein at 2.9 nM, followed by incubation with alpha -defensin or LL-37 (58 nM). Binding was detected with specific antibodies against alpha -defensin and LL-37, respectively. The bars represent the mean ± S.E. of at least three experiments.

Next we investigated the antibacterial effect of alpha -defensin and LL-37 on the AP1 strain. In these experiments the bacteria were washed and resuspended in buffer prior to the addition of the peptides to exclude that SIC was present during the incubation period. As shown in the left panel of Fig. 2, the peptides killed the bacteria. The concentration required for 100% killing was ~0.4 µM for both peptides. The inhibitory effect of SIC was then tested at a bactericidal concentration (0.448 µM) of the peptides and the results (see right panel, Fig. 2) show that SIC blocks the antibacterial activity of alpha -defensin and LL-37. The inhibition curves indicate that SIC is about 10 times more efficient in blocking LL-37 than alpha -defensin. M1 protein was also tested (at a maximum of 0.72 µM) but showed no inhibitory activity.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   SIC protects S. pyogenes against antibacterial peptides. S. pyogenes strain AP1 (2 × 106 cfu/ml) was incubated with the antibacterial peptides alpha -defensin (open circle ) or LL-37 (triangle ) at indicated concentrations for 2 h at 37 °C in buffer, and cfus were determined (left panel). The bactericidal effect of alpha -defensin (open circle ) or LL-37 (triangle ), at a concentration of 448 nM, was inhibited with various concentrations of protein SIC (right panel). Experiments were repeated at least three times, and representative experiments are shown.

When grown to stationary phase, the growth medium of S. pyogenes of the M1 serotype contains large quantities (10-15 µg/ml) of protein SIC (7). To inactivate the gene encoding protein SIC, AP1 bacteria were subjected to an allelic replacement mutagenesis strategy. One mutant, SIC-, was obtained, and it secreted minute amounts of protein SIC to the growth medium as compared with wild-type AP1 bacteria (Fig. 3A). The expression of sic from the AP1 and SIC- strains was investigated at different stages of growth using Northern blotting. As shown in Fig. 3B, the expression of sic reaches its maximum at late logarithmic growth phase, and no expression was detected at early stationary phase. The same filter was hybridized with a 16 S probe, showing that the same amount of RNA was applied to each well (data not shown). In experiments where the bacteria were washed before the addition of the antibacterial peptide LL-37, the mutant strain SIC- was found to be as sensitive to LL-37 as the wild-type strain AP1 (see Fig. 2, left panel). In a series of experiments we then investigated whether SIC produced by growing M1 bacteria could protect the organisms against LL-37 (these experiments required substantial amounts of antibacterial peptide, and only LL-37 was tested). The wild-type AP1 strain and the mutant strain SIC- were grown to early logarithmic growth phase where the concentration of SIC produced by AP1 bacteria was 0.85 µg/ml growth medium as determined by ELISA (Fig. 3C). The secretion of protein SIC by the mutant SIC- was below the detection level (Fig. 3C). At this point different amounts of LL-37 were added. As shown in Fig. 3D, SIC- bacteria were more sensitive to LL-37 as compared with the SIC-producing strain AP1. The concentration of LL-37 required to kill 50% of SIC- bacteria in these experiments was 22.3 µM, while at this concentration only 20% of the wild-type bacteria were killed. Lower concentrations of LL-37 killed 20% of SIC- bacteria, but the AP1 strain was unaffected. The amount of protein SIC produced was determined at different time points during growth. While the concentration of SIC produced by AP1 bacteria in this experimental system, reached its maximum (4.75 µg/ml) at early stationary phase (6 h), the amount of protein SIC in the growth medium of the mutant strain SIC- was below the level of detection (Fig. 3C). The results suggest that SIC secretion provides protection against antibacterial peptides already at an initial stage of infection.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 3.   Secretion of protein SIC during growth of S. pyogenes inhibits the antibacterial activity of LL-37. A, growth media from S. pyogenes strain AP1 and the SIC- mutant were precipitated with trichloroacetic acid. The precipitated proteins were subjected to SDS-PAGE analysis. One gel was stained with Coomassie Blue (Stain), and one gel was blotted to a PVDF membrane and probed with a polyclonal anti-SIC antiserum (Blot). B, total RNA samples from the AP1 and SIC- strains were isolated from bacteria in early logarithmic phase (EL), late logarithmic phase (LL), or early stationary phase (ES), subjected to Northern blotting, and probed with a probe hybridizing with sic. C, AP1 bacteria (SIC+) and the mutant strain SIC- were grown in microtiter plates, and the concentration of protein SIC was determined at different time points by ELISA. D, bacteria were grown in microtiter plates and various amounts of LL-37 were added to AP1 (SIC+) and SIC- bacteria at early logarithmic phase (indicated with an arrow). Bacterial growth was determined by measuring the absorbance at 490 nm at different time points. Growth medium alone (); 5.55 µM LL-37 (diamond ); 11.1 µM LL-37 (open circle ); 22.3 µM LL-37 (triangle ). Experiments were repeated at least three times, and a representative experiment is shown.

As mentioned, SIC variants are produced by S. pyogenes strains of M serotypes 1, 12, 55, and 57 (7, 8). Using the same isolation protocol as for SIC from M1 bacteria (7), SIC was purified from the growth medium of M12 and M55 organisms. Analogous to the M1 strain (7), the M12 and M55 strains produced 10-15 µg of SIC/ml of growth medium when grown to stationary phase. Protein SIC was also purified from two additional strains of the M1 serotype, U15 and U17. The purified variants shown in Fig. 4A were tested for their ability to interfere with the antibacterial activity of alpha -defensin and LL-37. As shown in Fig. 4B, all SIC variants, but not M1 protein, blocked the activity of the peptides. The inhibition curves show that SICM1 is 5-100 times more potent in this respect than the protein SIC variants from the M12 and M55 strains. To investigate a possible molecular basis for this difference in inhibitory activity, sequencing of the genes encoding the SIC variants was performed. As mentioned, there is a high degree of variation among sic genes from different strains of the M1 serotype (10), and nearly 300 alleles are known. The amino acid sequences derived from the obtained sic sequences were compared with that of protein SIC from the M1 strain AP1 (GenBankTM accession number X92968 (7)). A deletion of a region of 29 amino acid residues in SIC from U15 and some shorter insertions and single amino acid substitutions were observed within the M1 variants (Fig. 5A). However, the sic genes of the M12 and M55 strains are clearly more different, as compared with the sic genes of the M1 strains (not shown). The sequence of SICM12 was found to be identical to a recently published sequence (GenBank accession number AJ300679 (30)), and as demonstrated in Fig. 5B a high degree of homology was found when the derived amino acid sequence of SICM55 was compared with that of SICM12. Analysis of the amino acid composition of the SIC variants revealed differences in charged residues, where the M1 variants have a higher negative net charge compared with SICM12 and SICM55. This could explain the reduced capacity of SICM12 and SICM55 in blocking the activity of alpha -defensin and LL-37. It is noteworthy that the four SIC-producing M serotypes (1, 12, 55, and 57) are all known to be associated with poststreptococcal glomerulonephritis, suggesting a role for SIC in this condition (7, 8). Moreover, the fact that M1 strains dominate in cases of invasive disease (6) and that SICM1 is the most potent inhibitor of alpha -defensin and LL-37, support the notion that the interference of SIC with antibacterial peptides could facilitate S. pyogenes invasion through mucosal and skin barriers.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   Inhibitory effect of different SIC variants on the antibacterial activity of alpha -defensin and LL-37. A, different variants of protein SIC (1 µg) were subjected to SDS-PAGE (10% gel) and stained with Coomassie Blue. Protein SICM1 was purified from S. pyogenes strains U15, U17, or AP1. The distantly related variants SICM12 and SICM55 were purified from S. pyogenes strains of M serotypes 12 and 55, respectively. B, S. pyogenes strain AP1 was incubated with antibacterial peptides (448 nM) for 2 h (alpha -defensin) or 1 h (LL-37) in the presence of various amounts of proteins SICM1AP1 (), SICM1U15 (black-square), SICM1U17 (black-triangle), SICM12 (open circle ), SICM55 (), or M1 protein (triangle ). Experiments were repeated at least three times, and representative experiments are shown.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 5.   Structure of the investigated SIC variants. A, insertions, deletions, and amino acid substitutions in the SICM1U15 and SICM1U17 amino acid sequences are shown as compared with the SICM1AP1 sequence (GenBank accession number X92968 (7)). Unbroken lines represent homologous sequences, vertical bars and single-letter amino acid abbreviations represent substitutions indicated at their position in the SICM1AP1 sequence. B, amino acid differences between the SICM55 and the SICM12 sequences are shown by single-letter amino acid abbreviations and indicated at their position in the SICM12 sequence.

Over the past 10-20 years research in many laboratories has firmly established the fundamental role played by antibacterial peptides in the initial clearance of pathogenic bacteria. The demonstration that SIC not only interferes with complement function but also inactivates antibacterial peptides further underlines the significance of the innate immune system. The data of the present study also emphasize the highly complex molecular interplay between S. pyogenes and its human host. Although pathogenicity and virulence are polygenic properties, the results indicate that SIC could represent an important virulence determinant. A previous investigation (31) showed that an isogenic M1 mutant strain in which the sic gene had been inactivated, was significantly less efficient in colonizing the throat of mice as compared with the wild-type strain. Here we find, using a mouse model of subcutaneous infection, that the mutant strain SIC- is significantly attenuated in virulence compared with the wild-type AP1 strain (Table I). Combined these data suggest that SIC promotes early stages of infection by inactivating antibacterial peptides. The results of the present work may also explain the unique variability of the sic gene and the fact that the M1 serotype is the serotype most frequently connected with invasive S. pyogenes infection.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Subcutaneous challenge of mice with S. pyogenes strains AP1 and SIC-
NMRI mice were injected subcutaneously with 104 AP1 or SIC- bacteria, and mice were followed for 10 days.


    ACKNOWLEDGEMENTS

Ingbritt Gustafsson and Ulla Johannesson are acknowledged for excellent technical assistance.

    Addendum

In relation to the present work it is noteworthy that protein SIC also inhibits the antibacterial activities of lysozyme and secretory leukocyte proteinase inhibitor, proteins that are part of the mucosal innate immune system. These data were reported by Fernie-King et al. (32) after the submission of this manuscript.

    FOOTNOTES

* This work was supported by grants from the Swedish Research Council (Projects 7480, 13471, and 14379), the Foundations of Crafoord, Bergvall, and Österlund, and the Royal Physiographic Society.The costs of publication of this article were defrayed in part by the payment of page charges. The 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: Dept. of Cell and Molecular Biology, Lund University, BMC, B14, Tornavägen 10, S-221 84 Lund, Sweden. Tel.: 46-46-222-8569; Fax: 46-46-157756; E-mail: Inga-Maria.Frick@medkem.lu.se.

Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M301995200

    ABBREVIATIONS

The abbreviations used are: TH, Todd Hewitt broth; PBS, phosphate-buffered saline; cfu, colony forming units; PBST, PBS Tween; ELISA, enzyme-linked immunosorbent assay; PVDF, polyvinylidene difluoride.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Bisno, A. L., and Stevens, D. L. (1996) New Engl. J. Med. 334, 240-245[Free Full Text]
2. Cunningham, M. W. (2000) Clin. Microbiol. Rev. 13, 470-511[Abstract/Free Full Text]
3. Nowak, R. (1994) Science 264, 1665[Medline] [Order article via Infotrieve]
4. Stevens, D. L. (2000) Annu. Rev. Med. 51, 271-288[CrossRef][Medline] [Order article via Infotrieve]
5. Fischetti, V. A. (1989) Clin. Microbiol. Rev. 2, 285-314[Medline] [Order article via Infotrieve]
6. Musser, J. M., and Krause, R. M. (1998) in Emerging Infections (Krause, R. M., ed) , pp. 185-218, Academic Press, New York
7. Åkesson, P., Sjöholm, A. G., and Björck, L. (1996) J. Biol. Chem. 271, 1081-1088[Abstract/Free Full Text]
8. Hartas, J., and Sriprakash, K. S. (1999) Microb. Pathog. 26, 25-33[CrossRef][Medline] [Order article via Infotrieve]
9. Fernie-King, B. A., Seilly, D. J., Willers, C., Wurzner, R., Davies, A., and Lachmann, P. J. (2001) Immunology 103, 390-398[CrossRef][Medline] [Order article via Infotrieve]
10. Stockbauer, K. E., Grigsby, D., Pan, X., Fu, Y. X., Mejia, L. M., Cravioto, A., and Musser, J. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3128-3133[Abstract/Free Full Text]
11. Hoe, N. P., Nakashima, K., Lukomski, S., Grigsby, D., Liu, M., Kordari, P., Dou, S. J., Pan, X., Vuopio-Varkila, J., Salmelinna, S., McGeer, A., Low, D. E., Schwartz, B., Schuchat, A., Naidich, S., De Lorenzo, D., Fu, Y. X., and Musser, J. M. (1999) Nat. Med. 5, 924-929[CrossRef][Medline] [Order article via Infotrieve]
12. Steiner, H., Hultmark, D., Engstrom, A., Bennich, H., and Boman, H. G. (1981) Nature 292, 246-248[Medline] [Order article via Infotrieve]
13. Boman, H. G. (2000) Immunol. Rev. 173, 5-16[CrossRef][Medline] [Order article via Infotrieve]
14. Lehrer, R. I., and Ganz, T. (1999) Curr. Opin. Immunol. 11, 23-27[CrossRef][Medline] [Order article via Infotrieve]
15. Schröder, J.-M., and Harder, J. (1999) Int. J. Biochem. Cell Biol. 31, 645-651[CrossRef][Medline] [Order article via Infotrieve]
16. Selsted, M. E., and Ouellette, A. J. (1995) Trends Cell Biol. 5, 114-119[CrossRef][Medline] [Order article via Infotrieve]
17. Åkesson, P., Schmidt, K.-H., Cooney, J., and Björck, L. (1994) Biochem. J. 300, 877-886[Medline] [Order article via Infotrieve]
18. Herwald, H., Hasan, A. A. K., Godovac-Zimmermann, J., Schmaier, A. H., and Müller-Esterl, W. (1995) J. Biol. Chem. 270, 14634-14642[Medline] [Order article via Infotrieve]
19. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963) Biochem. J. 89, 114-123
20. Rasmussen, M., Müller, H.-P., and Björck, L. (1999) J. Biol. Chem. 274, 15336-15344[Abstract/Free Full Text]
21. Gomi, H., Hozumi, T., Hattori, S., Tagawa, C., Kishimoto, F., and Björck, L. (1990) J. Immunol. 144, 4046-4052[Abstract/Free Full Text]
22. Berge, A., Rasmussen, M., and Björck, L. (1998) Infect. Immun. 66, 3449-3453[Abstract/Free Full Text]
23. Podbielski, A., Spellerberg, B., Woischnik, M., Pohl, B., and Lüttiken, R. (1996) Gene 177, 137-147[CrossRef][Medline] [Order article via Infotrieve]
24. Hanski, E., Fogg, G., Tovi, A., Okada, N., Burstein, I., and Caparon, M. (1995) Methods Enzymol. 253, 269-305[Medline] [Order article via Infotrieve]
25. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
26. Ouellette, A. J., and Selsted, M. E. (1996) FASEB J. 10, 1280-1289[Abstract/Free Full Text]
27. Ayabe, T., Satchell, D. P., Pesendorfer, P., Tanabe, H., Wilson, C. L., Hagen, S. J., and Ouellette, A. J. (2002) J. Biol. Chem. 277, 5219-5228[Abstract/Free Full Text]
28. Nagaoka, I., Hirota, S., Yomogida, S., Ohwada, A., and Hirata, M. (2000) Inflamm. Res. 49, 73-79[CrossRef][Medline] [Order article via Infotrieve]
29. Berge, A., and Björck, L. (1995) J. Biol. Chem. 270, 9862-9867[Abstract/Free Full Text]
30. Brandt, C. M., Allerberger, F., Spellerberg, B., Holland, R., Lutticken, R., and Haase, G. (2001) J. Infect. Dis. 183, 670-674[CrossRef][Medline] [Order article via Infotrieve]
31. Lukomski, S., Hoe, N. P., Abdi, I., Rurangirwa, J., Kordari, P., Liu, M., Dou, S. J., Adams, G. G., and Musser, J. M. (2000) Infect. Immun. 68, 535-542[Abstract/Free Full Text]
32. Fernie-King, B. A., Seilly, D. J., Davies, A., and Lachmann, P. J. (2002) Infect. Immun. 70, 4908-4916[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.