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Infection and Immunity, August 2008, p. 3399-3404, Vol. 76, No. 8
0019-9567/08/$08.00+0     doi:10.1128/IAI.01392-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cathelicidin LL-37 in Severe Streptococcus pyogenes Soft Tissue Infections in Humans{triangledown}

Linda Johansson,1,{dagger} Pontus Thulin,1,{dagger} Parham Sendi,1 Erika Hertzén,1 Adam Linder,2 Per Åkesson,2 Donald E. Low,3 Birgitta Agerberth,4 and Anna Norrby-Teglund1*

Center for Infectious Medicine, Karolinska Institutet, Department of Medicine—F59, Karolinska University Hospital, Huddinge, SE-141 86 Stockholm,1 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm,4 Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden,2 Department of Microbiology, Mount Sinai Hospital, and the University of Toronto, Toronto, Ontario, M5G 1x5, Canada3

Received 17 October 2007/ Returned for modification 16 December 2007/ Accepted 11 May 2008


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ABSTRACT
 
Severe soft tissue infections, such as necrotizing fasciitis and severe cellulitis, caused by group A streptococci (GAS) are rapidly progressing life-threatening infections characterized by massive bacterial loads in the tissue even late after the onset of infection. Antimicrobial peptides are important components of the innate host defense, and cathelicidins have been shown to protect against murine necrotic skin infections caused by GAS. However, it has been demonstrated that the streptococcal cysteine protease SpeB proteolytically inactivates the human cathelicidin LL-37 in vitro. Here we have investigated the expression of LL-37 and its interaction with GAS and SpeB during acute severe soft tissue infections by analyses of patient tissue biopsy specimens. The results showed large amounts of LL-37, both the proform (hCAP18) and the mature peptide, in the tissue. Confocal microscopy identified neutrophils as the main source of the peptide. A distinct colocalization between the bacteria and LL-37 could be noted, and bacterial loads showed positive correlation to the LL-37 levels. Areas with high LL-37 levels coincided with areas with large amounts of SpeB. Confocal microscopy confirmed strong colocalization of GAS, SpeB, and LL-37 at the bacterial surface. Taken together, the findings of this study provide in vivo support of the hypothesis that SpeB-mediated inactivation of LL-37 at the streptococcal surface represents a bacterial resistance mechanism at the infected tissue site in patients with severe GAS tissue infections.


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INTRODUCTION
 
Since the late 1980s, a resurgence of severe invasive group A streptococcus (GAS) infections associated with high morbidity and mortality, including streptococcal toxic shock syndrome and necrotizing fasciitis, has been reported worldwide (11). GAS cause disease primarily by activating and modulating host immune responses. The immunomodulatory properties of the bacteria include, among others, avoidance and modulation of host innate defense mechanisms. A central component of the first line of defense is the antimicrobial peptides expressed in epithelia and several of the innate immune cells (3). Cathelicidins are cationic antimicrobial peptides, referred to as LL-37 and CRAMP in humans and mice, respectively, that exert their antimicrobial activity through disruption of bacterial membranes, resulting in cell lysis (8, 16). The mature and active LL-37 is enzymatically derived from a larger protein, the human cationic antimicrobial peptide of 18 kDa (hCAP-18). hCAP18 is produced in many cell types and tissues, including skin and intestinal epithelia, sweat glands, neutrophils, monocytes, NK cells, and mast cells. Cathelicidins were ascribed a central role in the control of GAS infections, since CRAMP-deficient mice were significantly more prone to succumb to invasive GAS infections than their wild-type littermates (13).

Proteolytic inactivation of host antimicrobial peptides is a resistance mechanism shared by several significant human pathogens. GAS secrete at least two factors capable of inactivating the antimicrobial peptide LL-37 in vitro, namely, the cysteine protease SpeB (18) and streptococcal inhibitor of complement (SIC) (7). The proteolytic activity of SpeB is tightly regulated at the bacterial surface through expression of the surface-attached GRAB (G-related {alpha}2-macroglobulin-binding) protein, which binds {alpha}2-macroglobulin, a major protease inhibitor of human plasma (17). However, entrapment of SpeB in the GRAB-{alpha}2-macroglobulin complex has been shown to result in enhanced LL-37 inactivation in vitro, as SpeB is retained at the bacterial surface, where it remains proteolytically active against small peptides, such as LL-37, that can penetrate the complex (15).

In our recent study of GAS pathogenesis at the tissue site of infection (19), we showed that there is a massive bacterial load in the tissue for a prolonged time despite the use of intravenous antibiotics as well as the presence of inflammatory cells. This indicated inefficient eradication of the bacteria at the tissue site of infection despite the fact that cultivation of bacteria from the tissue on blood agar plates revealed the presence of bacterial-growth-inhibitory substances, presumably antibiotic and/or host antimicrobial peptides, in the tissue. To our knowledge, there are no reports available that describe LL-37 responses during severe acute GAS tissue infections. Therefore, in this study, we investigated whether LL-37 is present in infected patient tissue and the degree of its interaction with the bacteria and bacterial factors. We provide in vivo evidence that LL-37 is present at the tissue site but does not contribute to efficient bacterial clearance, likely due to SpeB-mediated inactivation at the bacterial surface.

(This work was presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2007 [20].)


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MATERIALS AND METHODS
 
Patient material. Snap-frozen tissue biopsy specimens (n = 25) were collected from patients with various severe soft tissue infections, including necrotizing fasciitis and cellulitis, caused by GAS of various serotypes (Table 1). These patients were identified through active surveillance in Ontario, Canada, from 1995 to 1997. All patients had received intravenous clindamycin in combination with a β-lactam antibiotic at admission. Biopsy specimens, including subcutaneous tissue, muscle, or fascia, were collected at surgical procedures performed on different days, ranging from 1 to 9 days after diagnosis. Each biopsy specimen received a clinical grade at the time of sampling: grade 1 for distal tissue not visually inflamed (n = 5) and grade 2 for inflamed tissue, including cellulitis, fasciitis, and necrotizing fasciitis (n = 20). The biopsy material has been described in detail previously (14, 19).


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  		TABLE 1. Characteristics of tissue biopsy material

Snap-frozen punch biopsy specimens collected from the epicenter of infection from 12 patients with erysipelas at Lund University Hospital were also included. The Human Subjects Review Committees of the University of Toronto and of Lund University approved the studies, and informed consent was obtained from all patients.

Immunohistochemical staining of tissue biopsy specimens. The biopsy specimens were cryosectioned, fixed, stained, and analyzed by image analysis as previously described (14, 19). The following antibodies were used: anti-CD68 (EBM11), anti-neutrophil elastase (NP57), and anti-human mast cell tryptase (AA1) (all murine immunoglobulin G1 [IgG1]; Dako, Carpinteria, CA). LL-37 was identified by use of either a rabbit polyclonal or a murine monoclonal anti-LL-37 antibody (produced in the laboratory of Birgitta Agerberth). Both LL-37 antisera were raised against the mature peptide and recognize both the proform and the mature peptide but not the cathelin domain. A polyclonal rabbit antibody against GRAB was produced in, and kindly provided by, the laboratory of Lars Björck, Lund University. GAS and SpeB were identified by use of polyclonal rabbit antisera raised against the Lancefield group A carbohydrate (Difco, Detroit, MI) or against native SpeB (Toxin Technology Inc, Sarasota, FL), respectively (14, 19). SpeB was also detected by use of a polyclonal rabbit antiserum raised against the prodomain of the SpeB zymogen (IAS19) (kindly provided by Mattias Collin, Lund University). The specificities of the polyclonal anti-SpeB and anti-LL-37 antisera were verified by preincubation of each antiserum with a 10-fold excess of purified SpeB or synthetic LL-37, respectively, which effectively blocked staining for the antigen in the tissue sections. Irrelevant isotype-specific murine antibodies (Vector Laboratories, Burlingame, CA) and preimmune rabbit sera at appropriate dilutions were used to control for nonspecific staining reactions. Biotinylated secondary antibodies included goat anti-mouse IgG1, goat anti-mouse IgG2a (Caltag Laboratories, San Francisco, CA), and goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA).

Immunohistochemically stained specimens were analyzed by acquired computerized image analysis (ACIA). Despite the sizes of the biopsy specimens, the whole section was included in the analysis, which yielded an analyzed cell area (defined by the blue hematoxylin counterstaining) ranging from 2.1 x 105 to 1.6 x 107 µm2 for the deep tissue specimens and 9.6 x 105 to 7 x 106 µm2 for the erysipelas specimens. The results are presented as ACIA values, calculated as the percentage of the area that is positively stained multiplied by the mean intensity of positive staining. The majority of negative controls were completely negative; however, in case of any background staining, the section was subjected to ACIA analysis, and the resulting value was subtracted from the sample value.

Double and triple immunostaining was achieved with anti-GAS and anti-SpeB antibodies that were biotinylated by using EZ-Link NHS-LC-biotin (Pierce, Rockford, IL) according to the manufacturer's manual. For evaluation, a Leica confocal scanner (model TCS SP II) coupled to a Leica DMR microscope was used.

Western blotting. Two 5-µm-thick biopsy cryosections were resuspended in 12 µl 0.1% trifluoroacetic acid, heated to 95°C for 5 min, and diluted in NuPAGE sample buffer (4x) (Invitrogen, Carlsbad, CA) and dithiothreitol to a final concentration of 0.05 M. Samples were then incubated at 70°C for 10 min. Fifteen microliters of each biopsy solution was loaded onto a 4 to 12% NuPAGE Novex Bis-Tris gel (Invitrogen, Carlsbad, CA). Separated proteins were transferred to a polyvinylidene difluoride membrane (Invitrogen, Carlsbad, CA) by electrophoretic transfer according to the instructions of the manufacturer. The membrane was blocked in phosphate-buffered saline containing 5% fat-free milk. The presence of LL-37 in tissue biopsy specimens was detected by use of an affinity-purified rabbit polyclonal antibody against LL-37, followed by anti-rabbit IgG conjugated with horseradish peroxidase (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Immunoreactive protein bands were visualized with an electrochemiluminescence kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The sensitivity of this technique is 5 pg, according to the manufacturer's specification.

Statistical evaluation. Data were analyzed by GraphPad Prism, version 4.0 for Windows (GraphPad Software, San Diego, CA). Correlations between variables were determined by use of the Spearman test.


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RESULTS AND DISCUSSION
 
Our recent finding that severe GAS soft tissue infections are characterized by massive bacterial loads in the tissue even after prolonged intravenous antibiotic therapy (19) indicated inefficient eradication of the bacterial infection at the tissue site. Since the host's own antimicrobial, LL-37, has been ascribed a central role in the control of GAS tissue infections, it was of interest to assess LL-37 responses at the tissue site during acute infection. For this purpose, 25 tissue biopsy specimens from patients with severe invasive GAS infections were immunohistochemically stained for LL-37 and then evaluated by microscopy and in situ image analysis. The peptide was present in all biopsy specimens, and LL-37 staining demonstrated a pattern consistent with intracellular localization as well as a secreted form (Fig. 1 A). LL-37 is generated by proteolytic processing of hCAP18 upon secretion; hence, these staining patterns indicated the presence of both the proform and the mature peptide in the tissue. Since these forms cannot be distinguished by immunostaining with the antibody used, the tissue biopsy specimens were further analyzed by Western blotting. For all but one patient, the processed mature LL-37 could be identified (Fig. 1B). In the biopsy specimen lacking the mature LL-37 peptide (Fig. 1B, biopsy specimen 2, lane 3), no LL-37 could be detected, correlating well with the image analysis, which revealed low levels of LL-37 in this tissue biopsy specimen. Although the ratio of the precursor to the LL-37 peptide differed between biopsy specimens, no relation to the bacterial load, clinical grade, degree of inflammation, or severity of disease could be noted (data not shown). Unique cathelicidin peptides, some of which demonstrated even greater antimicrobial activity than LL-37, have recently been identified in normal skin (21). In our Western blot analysis of the tissue, no evidence of further processing of mature LL-37 could be seen. Nonetheless, this does not exclude the presence of such processed forms in the tissue but may merely reflect the sensitivity of the Western blot technique in the assessment of complex tissue with high levels of the hCAP-18 precursor and the mature peptide.


Figure 1
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  		FIG. 1. LL-37 responses during severe soft tissue infections caused by GAS. Snap-frozen tissue biopsy specimens (n = 25) from patients with deep tissue GAS infections were immunohistochemically stained for LL-37, GAS, and cell markers. The stains were evaluated by microscopy and ACIA; for details, see Materials and Methods. (A) Staining for LL-37 in the tissue sections revealed the presence of both diffuse, presumably secreted staining (white arrows) and distinct intracellular staining (black arrows). Magnification, x400. A representative tissue biopsy specimen is shown. (B) Tissue sections were analyzed for LL-37 by Western blotting, and a representative blot is shown. Lane 1, mature LL-37 (1 ng); lane 2, biopsy specimen 1 (LL-37 ACIA value, 30); lane 3, biopsy specimen 2 (LL-37 ACIA value, 6); lane 4, biopsy specimen 15 (LL-37 ACIA value, 45); lane 5, biopsy specimen 35 (LL-37 ACIA value, 54). The upper band represents the hCAP18 precursor, with the expected molecular mass of 17 kDa, and the lower band of 4.5 kDa represents the mature LL-37 peptide. (C and D) Staining for LL-37 revealed that areas of high LL-37 staining intensity (C) coincided with areas of high GAS staining intensity (D). Magnification, x400. (E) Expression of LL-37 in biopsy specimens of clinical grade 2 (n = 20) in relation to the bacterial load, i.e., the GAS ACIA value. (F and G) Dual immunofluorescence staining of the tissue sections for LL-37 (green) and cell markers (magenta), i.e., macrophages identified by CD68 positivity (F) or neutrophils stained for neutrophil elastase (G). A representative tissue biopsy specimen is shown. (H) Expression of LL-37 in relation to neutrophil infiltration was assessed by image analysis of immunohistochemical staining of the respective marker in the biopsy specimens (n = 25). Significant correlations, as determined by the Spearman test, are indicated by P and r values in panels E and H.

Detection of GAS in the tissue revealed that areas with high bacterial loads coincided with high LL-37/hCAP18 staining intensities (Fig. 1C and D). This finding was further supported by quantification of LL-37 and bacteria in severely involved biopsy specimens by use of in situ imaging. The analyses revealed a positive correlation between LL-37 and GAS ACIA results (P = 0.042; r = 0.46) (Fig. 1E), and the correlation was strongest early in the course of infection, i.e., during the first 2 days after the onset of symptoms (n = 13; P = 0.035; r = 0.59). Although upregulation of LL-37 in response to GAS infections is expected (4, 13), such a positive correlation to bacterial loads, together with the fact that there are viable bacteria in the tissue for a prolonged time, strongly implies that LL-37 in the infected tissue does not contribute efficiently to bacterial killing.

A lack of antimicrobial activity by the peptide could be due to several factors that are not mutually exclusive, including peptide variants resulting from differential cleavage, the local milieu, and bacterial counterstrategies. Since a variety of cells have been reported to produce LL-37, it was of interest to determine the main source of LL-37 at the site of local tissue infection. In accordance with our previous report (19), macrophages and neutrophils were dominant cell populations at the local site of infection and thus represented likely contributors to the observed LL-37. Dual immunofluorescence staining of LL-37 combined with antibodies against various cell markers identified neutrophils as the predominant source of LL-37 at the epicenter of invasive GAS infections, whereas very few macrophages stained positive for LL-37 (Fig. 1F and G). LL-37 expression could also be detected in other cell types, including mast cells, but these were few in number and not likely to contribute significantly to the overall LL-37 amount (data not shown). Consistent with the confocal microscopy data implicating neutrophils as the main source of LL-37, the degrees of neutrophil infiltration and LL-37 expression revealed a significant positive correlation (P = 0.0001; r = 0.8) (Fig. 1H).

In order to relate LL-37 expression in severe soft tissue infections to responses during a milder and more confined streptococcal infection, biopsy specimens from erysipelas patients, in which bacteria can only rarely be cultured (6), were analyzed. The LL-37 response in erysipelas was highly similar to the response observed in deep tissue infections, with LL-37 present in all tissues, both secreted in the tissue and within cells, predominantly in neutrophils (data not shown). As in the deep tissue infections, LL-37 expression was found in the same areas as the bacteria, and LL-37 expression correlated positively with the bacterial load (P = 0.00015; r = 0.88) (Fig. 2A). There was no statistically significant difference between the level of LL-37 expressed in erysipelas and that in severe streptococcal tissue infections, although somewhat higher levels of LL-37 were found in severely inflamed tissue, i.e., in clinical grade 2 biopsy specimens (Fig. 2B). Hence, the impaired antimicrobial activity of LL-37 in the tissues of patients with severe acute infections could not be explained by the concentration, the cellular source, or the expression pattern of LL-37 in the tissue.


Figure 2
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  		FIG. 2. LL-37 responses in erysipelas biopsy specimens. Snap-frozen biopsy specimens from patients with erysipelas lesions (n = 12) were stained for LL-37. The stained specimens were evaluated by ACIA; for details, see Materials and Methods. (A) Correlation between LL-37 in biopsy specimens from erysipelas lesions in relation to the bacterial load, as determined by staining for GAS by use of a polyclonal antiserum against Lancefield group A antigen. A significant correlation, as determined by the Spearman test, is indicated by the P and r values. (B) Comparison between the expression of LL-37 in tissues from patients with deep tissue GAS infections and in tissues from patients with erysipelas. Shown is a box-and-whiskers plot of LL37 expression in biopsy specimens from patients with erysipelas and from patients with deep tissue infections classified according to clinical grade 1 (distal tissue) or 2 (epicenter tissue).

Bacterial proteases have been shown to represent an important bacterial counterstrategy against host antimicrobial peptides, because they can degrade and inactivate the peptides. In GAS, both the streptococcal cysteine protease SpeB (15, 18) and SIC (7) have been reported to render LL-37 inactive. SIC is expressed by only a few serotypes of GAS (2), whereas SpeB is present in the vast majority of GAS strains regardless of serotype. Since our tissue material includes tissues from patients infected with GAS strains of various serotypes, many of which have been reported to lack the gene encoding SIC, it was reasonable to focus on a potential effect of SpeB. In particular, we were interested in determining whether the mechanism for inactivation of LL-37 by SpeB at the bacterial surface (15) contributed to the resistance to LL-37 noted at the tissue site of infection. This model involves the following steps: (i) a bacterial surface-bound protein, GRAB, binds the potent proteinase inhibitor {alpha}2-macroglobulin; (ii) SpeB is entrapped in the GRAB-{alpha}2-macroglobulin complex and is thereby retained at the bacterial surface; (iii) SpeB degrades small peptides, such as LL-37, that can penetrate the cage, thereby creating a protective zone around the bacteria. Our recent report demonstrated high expression of SpeB at the tissue site, and in biopsy specimens with large amounts of bacteria and inflammatory cells, SpeB could be observed localized to the bacterial surface (19). Consequently, SpeB-mediated inactivation of LL-37 at the bacterial surface could likely occur at the site of tissue infection. To address this, SpeB expression was first confirmed by immunohistochemical staining, and all biopsy specimens were found to be positive (data not shown). Biopsy specimens in which SpeB was associated with the bacterial surface (Fig. 3A) were subsequently stained with an antibody for the prodomain of the SpeB zymogen. This resulted in a loss of coccus-associated SpeB staining (Fig. 3B), indicating that the SpeB localized to the bacterial surface is proteolytically active SpeB. In biopsy specimens with secreted SpeB, the prodomain antibody resulted in positive staining with only a minor reduction in intensity from that with the antiserum recognizing SpeB (data not shown). These data are in line with the {alpha}2-macroglobulin model, since only the active SpeB protease can be entrapped by the proteinase inhibitor. Importantly, coccus-like structures positive for both GRAB and SpeB were observed in the tissue, providing additional support for this model (Fig. 3C). The localization of LL-37 in relation to SpeB in the tissue was further assessed by confocal microscopy. As shown in Fig. 3D, strong colocalization of the streptococcal SpeB and the host's LL-37 could be seen in large areas of the tissue. Triple staining, including identification of GAS, revealed GAS surrounded by SpeB in areas where LL-37 was present (Fig. 3E). These findings provide in vivo support for the notion that the LL-37-SpeB interaction occurs at the bacterial surface, likely through interaction with GRAB-{alpha}2-macroglobulin complexes, resulting in the accumulation of SpeB around the bacteria, where the biological significance of inactivation will be greatest.


Figure 3
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  		FIG. 3. Interaction between LL-37 and the streptococcal cysteine protease SpeB. (A and B) Tissue biopsy specimens of patients with deep tissue infections were immunohistochemically stained for SpeB by use of a polyclonal rabbit serum raised against native SpeB (A) or a rabbit antiserum raised against the prodomain of SpeB, which recognizes only the inactive zymogen (B). Note that in panel B, the bacterial cocci are no longer stained brown. (C) Double staining of GRAB (green) and SpeB (red) revealed colocalization of these bacterial factors on coccus-like structures whose size corresponds to that of streptococci (white arrows). (D and E) To assess the colocalization of SpeB, LL-37, and bacteria, the biopsy specimens were immunofluorescently stained for SpeB by using a biotinylated anti-SpeB rabbit antibody (magenta), for LL-37 by using a monoclonal anti-LL-37 antibody (green), and for GAS by using a biotinylated polyclonal antiserum against Lancefield group A antigen (red). Cellular nuclei are stained blue by 4',6'-diamidino-2-phenylindole (DAPI). The stained specimens were analyzed by confocal microscopy. (D) Dual staining for SpeB and LL-37. Note the marked colocalization in yellow evident in the overlay image (lower right panel). Each marker is shown separately (cellular nuclei, blue; GAS, red; SpeB, magenta; LL-37, green), and all markers are shown together in an overlay image (lower-right panel). (E) Triple staining for LL-37, GAS and SpeB. Areas in which all three factors colocalize are indicated by arrows. Note the different magnifications in panels D and E, as indicated by the scale bars. The panels show representative stained biopsy specimens.

In recent years, it has become increasingly clear that the role of SpeB in GAS infections is starkly different in blood and at the site of infection. In blood there is an inverse correlation between SpeB expression and the severity of infection (10), whereas at the local site of infection, SpeB has been found to contribute to GAS survival, because it promotes intracellular persistence in tissue macrophages (19). The findings of this study point to yet another mechanism by which SpeB promotes bacterial survival at the tissue site.

Neutrophils were identified as the major source of LL-37 in both severe deep tissue infections and erysipelas. Other studies have shown that neutrophils are absent in GAS-infected tissue due to impaired chemotactic signals, which result in the failure of neutrophils to migrate into the surrounding infected tissue (5, 9). However, in our human biopsy material from patients with severe GAS soft tissue infections, neutrophil aggregates could be observed in several blood vessels, but large amounts of neutrophils were also found in affected tissue areas with high bacterial loads (19). Overall, the data on snap-frozen patient tissue biopsy specimens revealed a significant correlation between neutrophil infiltration and severely involved tissue characterized by high inflammation. It is becoming increasingly evident that many of the antimicrobial peptides act not only as antimicrobial agents but also as significant mediators of other biological effects, including immunomodulatory and chemotactic activities (3, 12). Mature LL-37 has been shown to have chemotactic activity for neutrophils and CD4 T cells (1, 22). It is noteworthy that our previous studies have demonstrated heavy infiltration not only of neutrophils but also of CD4 T cells at the site of tissue infection (14, 19). Whether this is partially attributable to the chemotactic activities of LL-37 remains to be seen, but the scenario is plausible.


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ACKNOWLEDGMENTS
 
We gratefully acknowledge the excellent technical assistance of Anette Hofmann and Monica Lindh. The generous gift of the IAS19 antibody by Mattias Collin at Lund University is highly appreciated.

This work was financially supported by grants from the Swedish Foundation for Strategic Research, Torsten and Ragnar Söderberg's Foundation, The Swedish Research Council, AFA Sjukförsäkring, the Magnus Bergvalls Foundation, the Åke Wibergs Foundation, the Anders Otto Swärds Foundation, the Lars Hiertas Foundation, the Stiftelsen Längmanska Kulturfonden, the Swedish Society of Medicine, The Swiss National Science Foundation (PBBSB—113145, to P.S.), The Margareta and Walter Lichtensteinstiftung (to P.S.), The Karolinska University Hospital, and the Karolinska Institutet.


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FOOTNOTES
 
* Corresponding author. Mailing address: Center for Infectious Medicine—F59, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden. Phone: 46-8-585 872 96. Fax: 46-8-746 7637. E-mail: Anna.Norrby-Teglund{at}ki.se Back

{triangledown} Published ahead of print on 19 May 2008. Back

Editor: A. Camilli

{dagger} L.J. and P.T. contributed equally to this work. Back


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Infection and Immunity, August 2008, p. 3399-3404, Vol. 76, No. 8
0019-9567/08/$08.00+0     doi:10.1128/IAI.01392-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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