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Infection and Immunity, September 2007, p. 4541-4551, Vol. 75, No. 9
0019-9567/07/$08.00+0     doi:10.1128/IAI.00518-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

SalY of the Streptococcus pyogenes Lantibiotic Locus Is Required for Full Virulence and Intracellular Survival in Macrophages{triangledown}

Hilary A. Phelps and Melody N. Neely*

Wayne State University School of Medicine, Detroit, Michigan 48201

Received 11 April 2007/ Returned for modification 20 May 2007/ Accepted 10 June 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptococcus pyogenes utilizes numerous mechanisms for evading the host immune response but has only recently been found to survive in the intracellular environment. In this study, we demonstrate the requirement of a putative ABC transporter permease for intracellular survival in macrophages. The highly attenuated S. pyogenes mutant, SalY, was identified from a transposon mutagenesis screen, with over 200-fold attenuation in virulence in a zebrafish invasive-disease model. Sequencing of the region surrounding the insertion identified a locus that is highly conserved in other S. pyogenes genomes and is homologous to an operon involved in lantibiotic production. In vitro analysis demonstrated that the SalY mutant is deficient in intracellular survival in murine macrophages, a phenotype also observed in zebrafish macrophages in vivo. Macrophage crude cell lysates added to bacterial cultures resulted in the death of the SalY mutant but only growth inhibition of the wild-type strain. Specific depletion of zebrafish macrophages in vivo restored the ability of the SalY mutant to cause disease to wild-type levels. The SalY-infected, macrophage-depleted zebrafish exhibit large lesions and invasive dissemination at a rate and level similar to those of the wild type. In contrast, an M protein mutant with a degree of attenuation similar to that of the SalY mutant did not regain full virulence by in vivo depletion of macrophages. The putative SalY ABC transporter may be an example of the ability of S. pyogenes to adapt and evolve new survival strategies that allow dissemination and growth in previously uninhabitable sites.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Invasive Streptococcus pyogenes infections were once an especially rare occurrence; however, since the 1980s a dramatic increase has been observed (22, 35). Although the rates of invasive disease have now stabilized in the United States, invasive S. pyogenes infections still remain a significant health care issue due to the difficulty of diagnosis and management (3) (http://www.cdc.gov/ncidod/dbmd/diseaseinfo/groupastreptococcal_t.htm). Alarming mortality rates of up to 25% for patients afflicted with necrotizing fasciitis and up to 50% for toxic shock syndrome patients are still reported (6, 35). In many cases, these invasive diseases progress rapidly out of control despite antibiotic treatment and surgical debridement (35). The recent increase in invasive S. pyogenes disease emphasizes the ability of this organism to continue to adapt and evolve over time by acquiring new survival strategies that allow dissemination and growth in previously uninhabitable sites.

In an effort to determine the factors involved in S. pyogenes invasive disease, a previously described zebrafish infectious disease model was used for analysis of the necrotizing fasciitis disease state (33). In humans, this disease is characterized as a rapidly progressing infection of the subcutaneous tissue resulting in the extensive destruction of fascia and adipose tissue (3). Bakleh et al. (1a) formulated a novel classification scheme for characterization of clinical cases of necrotizing fasciitis. Patients with stage 3 disease, which was characterized by little to no neutrophil response but a heavy bacterial load in the infected tissue, were found to have a higher mortality rate than patients with stage 1 or 2 disease. Similar observations have been made in both human and animal model studies (8, 17, 50). Hidalgo-Grass et al. (17) profiled two case studies in which one patient was afflicted with necrotizing fasciitis and the other patient suffered from myonecrosis. Both patients were infected with S. pyogenes serotype M14 strains. Histological analysis of the necrotic tissue biopsy specimens from each patient showed high bacterial loads but no neutrophil infiltration. The zebrafish model for necrotizing fasciitis also exhibits little or no inflammatory cell infiltrate and a high bacterial load with an M14 serotype strain, S. pyogenes HSC5 (33). Zebrafish have monocytes and macrophages that are functionally and morphologically similar to those of humans and other mammals (25), and these cells have been shown to phagocytose and clear bacteria in vivo (16). Because of the immunological similarities of the zebrafish model to mammalian systems (see reference 38 and the references within), we can address questions directly related to host-pathogen interactions and the influence on infection. Therefore, the observations of S. pyogenes infection in zebrafish are directly relevant to human disease.

Two mutant strains with insertions in two separate genes of the salivaricin A lantibiotic locus (sal locus) were identified using transposon mutagenesis with the S. pyogenes HSC5 strain. Salivaricin A was first identified and isolated from Streptococcus salivarius (42). The function of this locus in S. salivarius is to produce and process a peptide, which is secreted to act as a signal for a two-component regulatory system for transcriptional regulation of the operon, as well as a lantibiotic to kill other gram-positive bacteria. Genetic analysis revealed that 85 of 87 strains (encompassing 53 M serotypes) of S. pyogenes were found to have the structural gene for the lantibiotic and additional regions of the sal locus; however, various mutations or deletions in genes encoding the SalM lantibiotic-processing protein and/or the SalT transporter protein were observed in S. pyogenes (54). Even though most elements in this locus have been conserved, nearly all S. pyogenes strains tested have been found to be sensitive to the inhibitory effects of salivaricin A produced by S. salivarius (42, 44), suggesting that immunity to the lantibiotic and the ability to produce an active lantibiotic have been lost. Loss of these traits has occurred in spite of the fact that the sequenced genomes of S. pyogenes all appear to have retained intact sequences of the salA gene and the downstream salXYKR genes (putative immunity and two-component regulator genes).

The data presented here demonstrate that the SalY protein of S. pyogenes HSC5 has a significant role in virulence. The loss of SalY results in an inability to survive within macrophages, both in vivo and in vitro. Moreover, depletion of macrophages in the host results in a reestablishment of virulence by the mutant to wild-type levels. The SalY protein has homology to ABC transporter permeases and is proposed to play a role in immunity in S. salivarius by protecting the bacterium from the effects of the secreted lantibiotic (52). Our results are consistent with the hypothesis that the SalY protein performs an immunity function, but the protection conferred is no longer to the lantibiotic but rather to an innate immune function of the host. Multiple amino acid differences, which resulted in a change in charge, were found in the S. pyogenes SalY protein compared to the sequence of S. salivarius SalY. We propose that these changes resulted in an evolutionary adaptation, thereby allowing survival of S. pyogenes in a new environment. The overall conservation of key components of the sal locus in S. pyogenes, in the absence of an active lantibiotic, demonstrates that a selective pressure is present in the natural environment and that these factors contribute to a successful infection.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains, media, and growth conditions. All plasmids were maintained in either Escherichia coli TOP10 cells (Invitrogen) or E. coli DH5{alpha}. E. coli strains were cultured in Luria-Bertani broth (BBL) supplemented with the appropriate antibiotics, at concentrations of 50 µg/ml kanamycin or 15 µg/ml chloramphenicol. E. coli cultures were grown at 37°C under aerobic conditions. The wild-type S. pyogenes strain used in this study was HSC5, which is a serotype M14 strain (15, 30). S. pyogenes strains were cultured in Todd-Hewitt medium (BBL) supplemented with 0.2% yeast extract (THYB) or in Todd-Hewitt medium supplemented with 0.2% yeast extract and 2% proteose peptone (TP; BBL). S. pyogenes cultures were grown in TP overnight and diluted 1:50 in TP the next day to bring the cultures to mid-log phase. When necessary, antibiotics were added to S. pyogenes cultures at concentrations of 500 µg/ml kanamycin or 3 µg/ml chloramphenicol (Sigma). Solid medium was prepared by adding 1.4% agar (BBL). S. pyogenes broth cultures were grown at 37°C in 15-ml screw-cap conical tubes without shaking. Solid media were incubated in an anaerobic gas chamber with GasPak (BBL) envelopes at 37°C.

Construction of transposon mutants. To identify secreted proteins involved in virulence, a suicide vector, pCMG8, which carries an alkaline phosphatase gene, phoZ (cloned from Enterococcus faecalis), and transposon Tn4001, was employed (10). The phoZ gene lacks the first 18 codons of its own signal sequence and acts as a reporter gene in this assay. The alkaline phosphatase activity is dependent on the export of the protein to the cell surface. Within the vector, the phoZ gene is adjacent to the left inverted repeat of the insertion sequence of the transposon. The vector also carries a kanamycin resistance gene for selection. When the transposon inserts into a gene that encodes a designated export protein, the PhoZ protein is trafficked to the cell surface. Colonies are purified on TP plates supplemented with 500 µg/ml kanamycin and then assayed for alkaline phosphatase activity.

Alkaline phosphatase activity. To determine alkaline phosphatase activity, a colorimetric filter lift assay was used as described previously (10). Colonies that were alkaline phosphatase positive were stored as glycerol stocks at –80°C until screening in the zebrafish model.

Sequencing of the sal locus. The sequence of the HSC5 salA operon was determined by sequencing overlapping fragments of the locus, using Platinum high-fidelity Taq polymerase (Invitrogen) for PCR amplification. Primers were designed by referring to the completed genome sequence of the S. pyogenes serotype M3 strain MGAS315 (Table 1). Sequencing was conducted at the Wayne State University genomics core facility. Sequences were evaluated and aligned using the Genomics Computer Group (GCG; University of Wisconsin) program. Each fragment was sequenced using both the 5' and 3' primers.


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  		TABLE 1. Primers used in the sequencing of the HSC5 sal locus

 
Deferred-antagonism assay. The method of deferred antagonism described by Tagg and Bannister (49) was used with minimal modifications. Briefly, S. salivarius was streaked on a Columbia agar plate with 5% sheep blood using a cotton swab and then incubated at 32°C for at least 18 h. The growth was removed by scraping the surface of the agar with the edge of a glass coverslip and then inverting the plate over chloroform-soaked filter paper in the lid of a petri dish for 30 min. Following chloroform exposure, the plate was exposed to air for 15 min. Overnight cultures of the strains to be tested were grown in THYB, cross-streaked at a right angle to the original S. salivarius streak, and then incubated overnight. Growth inhibition was determined by observing zones of interference in the region originally occupied by the S. salivarius strain. The strains used were a laboratory strain of Micrococcus luteus as a positive control for growth inhibition, a laboratory strain of Staphylococcus epidermidis as a negative control for inhibition (growth across the original streak), wild-type HSC5, and the SalY mutant strain. Both HSC5 and the SalY mutant were inhibited for growth in the region of the S. salivarius streak.

Zebrafish model. Zebrafish were maintained according to established protocols in the laboratory (33). Prior to injection, zebrafish were anesthetized in Tris-buffered tricaine (3-amino benzoic acidethylester; Sigma), pH 7.0, 168 µg/ml. Using a U-100 ultrafine insulin syringe with a 29-gauge needle (BD Scientific), an inoculum of 1 x 105 CFU log-phase bacteria was injected into the dorsal muscle for an intramuscular (i.m.) injection or just in front of the ventral fins for an intraperitoneal (i.p.) injection as described previously (33).

Determining LD50. The 50% lethal dose (LD50) for infection of zebrafish by i.m. routes of infection was determined for each mutant. The fish were challenged with bacterial suspensions ranging from 1 x 102 to 1 x 106 CFU. At least three separate experiments were performed with six fish per group to determine the LD50 (n = 18). The LD50 of wild-type HSC5 is 3 x 103 CFU in an i.m. infection. The LD50 is calculated based on survival data using the method of Reed and Muench (40).

CI assay in vivo. S. pyogenes strains were grown separately to mid-log phase (optical density at 600 nm of 0.300). Cultures of 1 x 108 CFU bacteria were washed and resuspended in 1 ml of fresh TP. Wild-type bacteria and a single mutant strain were then mixed at a 1:1 ratio. The culture was brought to 1 x 107 CFU/ml in TP, and 1 x 105 CFU was injected i.m. into the zebrafish. The input inoculum was serially diluted and plated on solid media both with and without the appropriate antibiotics to determine the input ratio. At 24 h postinjection, spleens were recovered from the euthanized fish. The organs were homogenized and plated on solid media both with and without the appropriate antibiotics to determine the output ratio. The competitive index (CI) was determined by dividing the output ratio of the mutant strain to the wild-type strain by the input ratio of the mutant to the wild type. A minimum of eight fish were used in all the CI experiments.

Dissection of fish. Dissected spleens were placed in a 1.5-ml microcentrifuge tube in 300 µl of phosphate-buffered saline (PBS; pH 7.2) and homogenized with a microcentrifuge tube tissue grinder (Kontes, Fisher Scientific). Serial dilutions of homogenates were performed in PBS, and the numbers of CFU were determined by plating the dilutions on solid medium with the appropriate selective antibiotics.

Histology of S. pyogenes-infected zebrafish. Zebrafish were injected as described above. At specified time points, the zebrafish were euthanized and whole fish were fixed in 10% formalin for 24 h. Next, the zebrafish were washed with increasing concentrations of ethanol. Following a final 2-h wash in 100% ethanol, the fish were incubated in toluene for another 24 h. The fish were then placed in tissue cassettes and 60°C paraffin including tissue infiltration medium. The fish were incubated in a 60°C water bath for at least 1 hour, the paraffin was changed, and the fish were incubated for 48 h in the same water bath. Finally, paraffin was added without the tissue infiltration medium, and the cassettes were cooled. Five-micrometer sections were prepared on microscope slides. The slides were stained using a standard hematoxylin and eosin staining procedure.

RAW 264.7 cell culture. RAW 264.7 cells (39), a cultured murine macrophage cell line, were cultured in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), penicillin-streptomycin (Invitrogen) at 100 U/ml and 100 µg/ml, respectively, and ampicillin (Sigma) at 50 µg/ml.

Gentamicin protection assay. RAW 264.7 cells were seeded at 1 x 106 cells per well into a 24-well plate. The following day, cells were washed three times with PBS. Washed log-phase bacteria suspended in DMEM plus 10% FBS were added at 1 x 106 CFU/ml per well, a multiplicity of infection (MOI) of 1. Following a brief centrifugation, 5 min at 1,000 rpm with a slow start and stop at room temperature, the plate was incubated at 37°C in a 5% CO2 incubator for 1 h. Each well was then washed five times with PBS to remove any nonadherent bacterial cells. Fresh DMEM plus 10% FBS was added, supplemented with 100 µg/ml gentamicin (Invitrogen), to each well, and the plate was incubated for an additional hour. Aliquots were taken from supernatants and plated to determine that all extracellular bacteria were killed by the 1-hour gentamicin treatment. Following the gentamicin treatment, the plate was washed three times with PBS. At this point, one set of wells were lysed by the addition of 1 ml of sterile double-distilled water to establish the 0-h time point. The resulting lysate was serially diluted and plated to enumerate the bacteria surviving intracellularly. Fresh DMEM plus 10% FBS containing no antibiotics was added to each remaining well. At 2-, 4-, 8-, 12-, 16-, 20-, and 24-h time points, aliquots were taken from the supernatants and cells were lysed and plated to enumerate intracellular and emerging bacteria at each time point.

TEM. RAW 264.7 cells infected with S. pyogenes strains were examined using transmission electron microscopy (TEM). RAW 264.7 cells were grown in 75-cm2 tissue culture flasks and infected at an MOI of 1 with S. pyogenes strains. The infection procedure was the same as described above. At 2-, 4-, and 24-hour time points, cells were scraped and processed for TEM. The cells were fixed using a fixative at a 1:1:1 ratio that consisted of 2% OsO4, 2.5% glutaraldehyde in 0.1 M phosphate buffer, and 0.2 M Sorenson's phosphate buffer. Cells were incubated in the fixative for 1.5 h in an ice bath. Cells were pelleted and resuspended in fresh fixative and incubated for an additional 1.5 h. Following a series of three washes in cold 0.1 M phosphate buffer, the cells were treated with increasing concentrations of ethanol to wash and dehydrate the samples. The dehydration process was continued with two additional treatments of 100% propylene oxide for 30 min each. The samples were next infiltrated with increasing concentrations of Epon-Araldite plastic, and finally the samples were embedded in curing molds using fresh Epon-Araldite. The molds were allowed to polymerize at room temperature for 24 h, at 40°C for 24 h, at 60°C for 8 h, and at 80°C for 24 h. Ultrathin slices of the samples were prepared. For viewing the samples, a JEOL JEM 1010 transmission electron microscope with a Gatan slow-scan charge-coupled device camera and digital imaging software was used.

LIVE/DEAD staining of RAW 264.7 cells. RAW 264.7 cell viability following infection with S. pyogenes was determined by using a LIVE/DEAD BacLight bacterial viability kit (Invitrogen/Molecular Probes). The Syto9 green fluorescent nucleic acid stain works independently of the level of membrane damage and stains all cells green. The propidium iodide stains the nuclei of cells with compromised cell membranes. RAW 264.7 cells were loaded onto a 24-well plate with 1 x 106 cells/well into wells containing sterile glass coverslips. RAW 264.7 cells were infected as described above and were mounted with BacLight mounting medium (Invitrogen/Molecular Probes) onto microscope slides. The slides were examined under epifluorescence with a Zeiss Axioskop 40 microscope.

Construction of the salY complement. The full-length salY gene and ribosomal binding site were PCR amplified using HSC5 chromosomal DNA as a template. The primers used in this reaction included 5' salY EcoRI-CCG GAA TTC GAT AGA GGA GAG TAA TAT C and 3' salY PstI-AAA ACT GCA GCA TAT TAT TTA CTT AAT CG. The resulting fragment was cloned downstream of the streptococcal rofA promoter on the pLZ12 vector (13, 34). This expression vector was propagated in E. coli TOP10 cells (Invitrogen), and the plasmid was purified using a plasmid midi prep kit (QIAGEN). The correct construct was confirmed by PCR and sequencing. The purified plasmid DNA was transformed into the SalY mutant using an established protocol (5). The wild-type strain and the SalY mutant were transformed with the empty vector lacking the salY gene as controls. Restoration of expression of the full-length salY gene was confirmed by reverse transcription-PCR (data not shown).

Cytospins of zebrafish spleens. Zebrafish were injected i.m. with 1 x 105 CFU as described above. At 16 h postinjection, the fish were euthanized and dissected spleens were gently homogenized in PBS by being passed through a 27-gauge needle (BD Scientific). The homogenate was loaded onto the funnel of a cytospin column with a glass microscope slide and centrifuged for 5 min at 700 rpm in a cytospin centrifuge (Shandon, Thermo). The centrifugation step applies a single layer of fish splenic cells to the microscope slide. The slides were then stained using DifQuik (Fisher) stain for visualization.

Crude cell lysate assay. RAW 264.7 cells were seeded onto a 24-well plate at 2 x 106 cells/well. Separate groups of cells were stimulated overnight with gentamicin-killed wild-type bacteria for the purpose of activating the cells. A control group of unstimulated cells was also included. The stimulated and unstimulated RAW 264.7 cells were washed one time with PBS, and six wells of 2 x 106 cells/well were lysed in 1 ml sterile double-distilled water. The bacteria were grown to mid-log phase, and 1 x 108 CFU were washed and resuspended in 1 ml PBS. Bacteria were added to 1 ml DMEM supplemented with 10% FBS at a concentration of 3.33 x 106 CFU. The crude cell lysate was added to the bacterial cultures in DMEM plus 10% FBS at a 1:9 ratio. The cultures were incubated at 37°C in 1.5-ml tubes for 7 h. At the specified time point, the cultures were serially diluted and plated to enumerate the bacteria.

CAR-treated zebrafish. The zebrafish carrageenan (CAR) procedure was adapted from CAR treatment for mice (11, 46, 55). A 10-mg/ml stock solution of CAR (Sigma) in warm PBS was prepared. Zebrafish were incubated in 100 µg/ml streptomycin for a minimum of 24 h to reduce the commensal bacterial load. Zebrafish were then injected i.p. with 100 µg CAR. Control fish were injected i.p. with 10 µl of PBS. All experimental fish were immediately injected i.m. with the appropriate bacterial strain. At time points of 2, 4, 8, 12, 16, 20, and 24 h, zebrafish treated with either CAR or PBS were euthanized and the dorsal muscles (at the site of injection) and spleens were dissected and homogenized. The homogenate was serially diluted and plated to enumerate the bacteria recovered from each tissue sample.

To confirm that macrophages were depleted by CAR in the zebrafish, cytospin preparations of spleens removed from treated and nontreated fish were evaluated at 16 h postinjection. To account for slide-to-slide variations, the number of macrophages present was determined in relation to the number of red blood cells (RBCs) present in the same field of view, as CAR has no effect on RBCs. Three slides per condition (CAR treated or PBS treated) were evaluated, and at least 15 fields of view were enumerated per slide. For PBS-treated zebrafish, the number of macrophages averaged 3.61% (±0.35% standard error of the mean [SEM]) of the RBCs present, while the CAR-treated zebrafish averaged only 0.885% (±0.276% SEM) of the RBCs present per field of view (P < 0.0001).

Statistical analysis. Statistical significance of data was determined using the StatView statistical analysis software and the two-tailed paired t test function (SAS Institute Inc., Cary, NC).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transposon mutagenesis screen. In an effort to identify membrane and secreted streptococcal proteins that may be specifically involved in the necrotizing fasciitis and myonecrosis disease state, we employed the zebrafish infectious disease model to screen a library of transposon mutants. As mentioned above, the clinical syndromes reported for human patients with these diseases are highly similar to what we observe in the zebrafish. The library was generated using the previously described genetic tool TnFuZ, which has an alkaline phosphatase gene (phoZ) inserted in the Tn4001 transposable element (10). The phoZ gene lacks a signal sequence and acts as a reporter gene in this assay. Transformants that were positive for alkaline phosphatase activity identified mutants with an insertion of the TnFuZ element into a gene encoding a secreted protein. Multiple transformations resulted in 94 alkaline phosphatase-positive transformants, which were subsequently screened through the zebrafish infectious disease model. Zebrafish infections were performed by i.m. injection of 1 x 105 CFU (~100 times the LD50) as described previously (33). That fish survived infection with an alkaline phosphatase-positive mutant suggested the possibility that an important virulence factor had been disrupted by the transposon insertion. Seven mutants were identified as being significantly attenuated for virulence compared to the wild-type strain and were sequenced to identify the location of the transposon insertion by comparison to S. pyogenes genomes (NCBI; http://www.ncbi.nlm.nih.gov/BLAST/). Virulence attenuation was measured by two methods: the LD50 was measured for each of the mutants and a CI assay was performed to determine how well the mutant competed directly with the wild-type strain in vivo (Table 2).


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  		TABLE 2. Transposon mutants attenuated for survival in the zebrafish model

 
One of the most attenuated mutants isolated had an insertion in the emm gene, encoding the M protein (Table 2). Numerous studies have indicated the importance of the membrane-bound M protein in S. pyogenes virulence (reviewed in reference 9). One of the primary functions determined for the M protein is the inhibition of phagocytosis. The isolation of a mutant strain with an insertion in a well-known, membrane-bound S. pyogenes virulence factor in the zebrafish animal model validated the ability of the screen to identify proteins important for pathogenesis.

Two of the attenuated mutants were found to have insertions in the salivaricin locus, a putative lantibiotic-producing operon. The Ac1 mutant had an insertion in the salY gene, encoding a putative ABC transporter with an LD50 of 7 x 105 CFU, which was more than 2 logs higher than the wild-type LD50 of 3 x 103 CFU (Table 2). The CI for the Ac1 mutant was 0.008, indicating a virulence that was 2 logs lower than that of the wild-type strain. The Cc1 mutant, with an insertion in the salK gene, encoding a putative histidine kinase, was also highly attenuated, with an LD50 of 1 x 105 CFU and a CI of 0.020.

Comparative analysis of the salivaricin locus. The salivaricin locus was sequenced in order to compare the HSC5 serotype M14 sequence to other known S. pyogenes and S. salivarius sequences and to determine the location of the Ac1 and Cc1 insertions (Fig. 1). Sequencing results revealed that the salA gene, encoding the SalA prepropeptide, was intact, with the same 51-amino-acid sequence as other S. pyogenes strains, which have 5 amino acid residue changes from the S. salivarius SalA sequence (44). However, downstream of salA, two single-nucleotide mutations, which subsequently introduce two stop codons, were found in the salM coding sequence (Fig. 1). SalM is responsible for the dehydration and cyclization or the thioether linkage formation of the SalA prepropeptide in S. salivarius (37, 54). The introduction of two stop codons, at codons 176 and 513 of the 944-amino-acid protein, most likely results in a truncated, nonfunctional protein, which would also result in the loss of the ability to process the SalA peptide into the active lantibiotic form.


Figure 1
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  		FIG. 1. sal locus structure of S. pyogenes, strain HSC5, serotype M14. Horizontal arrows depict genes within the locus and the direction of transcription. Vertical arrows identify sites of transposon insertions. Open arrows illustrate changes in open reading frames in HSC5 compared to S. salivarius (see text).

 
Immediately downstream of salM are the salT1 and salT2 genes. The salT gene has a single-nucleotide deletion that results in a frame shift compared to the S. salivarius salT, producing two separate open reading frames. The same frame shift mutation is also found in the serotype M3 S. pyogenes strain MGAS315 (54). In S. salivarius, the full-length salT gene codes for a bifunctional protein with two separate domains. The N-terminal domain is responsible for cleavage of the leader sequence of SalA (HSC5 salT1), while the C-terminal domain (HSC5 salT2) functions in the transport of the processed propeptide (54).

The salX, salY, salK, and salR genes are conserved in the HSC5 M14 serotype as well as among the other published sequences of S. pyogenes serotypes. The SalX and SalY proteins have homology to ABC transporters, ATPase and permease, respectively, and have been proposed to act in immunity to salivaricin in S. salivarius (51), although this function has not been experimentally confirmed. SalK and SalR have homology to a two-component regulatory system. Conservation of these genes in the absence of a functional lantibiotic suggests that there is a selective pressure to retain these genes. Alignment of the SalY amino acid sequences from all available S. pyogenes genomes with the HSC5 SalY revealed high conservation among strains (between 98 and 100% identical) (data not shown). The HSC5 SalY sequence was 90% identical to that of S. salivarius, although 49 of 635 amino acid residues of S. salivarius were different from those of all the S. pyogenes strains examined (data not shown). Of the 49 amino acid changes from the S. salivarius sequence, 20 resulted in a change in the charge of the residue. Fourteen of the 20 amino acids that have a change in charge are located in a predicted extracellular loop in the putative 10-transmembrane domain protein (http://bioweb.pasteur.fr/seqanal/interfaces/toppred). The high number of amino acid changes in S. pyogenes compared to the changes in S. salivarius may represent a shift in specificity for binding and/or export of the substrate by SalY due to either a change in charged residues or conformational changes induced.

Using an S. salivarius strain (Carolina Biologicals) that was positive for the sal locus by PCR, a deferred-antagonism assay was performed as previously described (49) (data not shown). Results confirmed that the HSC5 strain is susceptible to inhibition from secreted products produced by S. salivarius as reported for nearly all S. pyogenes strains tested (42, 44).

Bacterial survival assay in the zebrafish animal model. In this report, our analysis focuses on the most highly attenuated strain, the Ac1 mutant with an insertion in the salY gene, which we will refer to as the SalY mutant strain. Initial analysis of the infected dorsal muscle tissue sections from the SalY mutant 24 h postinfection revealed major differences from a wild-type infection (Fig. 2A and B). Dorsal muscle tissue from an infection with the wild-type strain shows the characteristic necrosis of the muscle tissue and the absence of infiltration of inflammatory cells with large aggregates of bacteria as has been reported previously (33) (Fig. 2A). In sharp contrast is the histology from an infection with the SalY mutant, which shows massive infiltration of inflammatory cells to the site of infection and no aggregates of bacteria (Fig. 2B).


Figure 2
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  		FIG. 2. Zebrafish infections. The histologies of zebrafish dorsal muscle tissue sections infected i.m. are shown 20 h postinjection with 1 x 105 CFU wild-type bacteria (A) and with 1 x 105 CFU SalY mutant bacteria (B). Magnification, x1,000. Samples were stained using a standard hemotoxylin and eosin staining protocol. Arrows in panel A point to aggregates of bacteria. Arrowheads in panel B point to inflammatory cells at the site of infection. (C) Counts of bacterial CFU recovered from zebrafish spleens. Each indicated strain was injected i.m. into zebrafish dorsal muscles at 1 x 105 CFU. The fish were euthanized, and spleens were removed for bacterial isolation at the indicated time points. Zebrafish were injected i.p. with sterile PBS to act as a negative control for later experiments (see Fig. 5C and text). All assays were done twice with six fish per time point (n = 12). Error bars represent ±SEMs. WT, wild type; *, P < 0.05.

 
Since the SalY mutant was significantly attenuated in the zebrafish model (Table 2) and the histology from a SalY mutant infection was strikingly different from the histology from a wild-type infection, the levels of bacterial loads were examined over a 24-h time period. Zebrafish were infected with the wild-type strain, the SalY mutant strain, or the M protein (Emm) mutant strain. The Emm mutant is highly attenuated in the zebrafish animal model, with an LD50 of 5 x 105 CFU, which is comparable to the attenuation of the SalY mutant, with an LD50 of 7 x 105 CFU (Table 2). At 2, 4, 8, 12, 16, 20, and 24 h after i.m. injection, the zebrafish were euthanized and the spleens were aseptically removed and gently homogenized. The homogenate was serially diluted and plated to enumerate bacteria. The numbers of CFU of all three strains stayed between 102 and 104 until the 12-hour time point (data not shown). After 12 h, the wild-type strain shows more than a 1-log increase in number of CFU, and the count remains relatively constant throughout 24 h (Fig. 2C). The Emm mutant starts to show a dramatic drop in number of CFU after the 12-hour time point and is almost cleared by 24 h. Similarly, the SalY mutant starts decreasing in number of CFU after 12 h, with a final CFU count greater than 2 logs, or 131-fold, lower than the wild-type count by 24 h (Fig. 2C).

SalY attenuation in zebrafish and murine macrophages. Initial characterization of the SalY mutant demonstrated that the strain was deficient for survival in the RAW 264.7 murine macrophage tissue culture assay (Fig. 3A). This was not due to a metabolic defect in growth, as the SalY mutant strain grows as well as the wild-type strain in normal laboratory media (THYB and TP) and a minimal medium (data not shown). RAW 264.7 cells were infected at an MOI of 1 with the wild type or the mutant strain as described in Materials and Methods. At the indicated time points, aliquots were taken from supernatants and the cells were lysed. Lysates and supernatants were serially diluted for enumeration of intracellular and emerging bacteria. Similar numbers of CFU were recovered from infections with the two strains at early time points; however, by 8 h after treatment with gentamicin, the number of wild-type CFU started increasing while the number of SalY mutant CFU started decreasing. By 24 h, there were approximately 1,000-fold fewer SalY mutant CFU recovered than wild-type CFU (Fig. 3A). Complementation of the SalY mutant was accomplished by expressing the salY gene from a plasmid, resulting in the recovery of wild-type levels of CFU by 24 h (data not shown).


Figure 3
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  		FIG. 3. (A) Bacteria recovered from in vitro macrophage infection. Each indicated strain was added to RAW 264.7 macrophages at an MOI of 1. At the indicated time points after a 1-hour gentamicin treatment to kill extracellular bacteria (0 time point), macrophages were lysed and viable bacteria were enumerated from supernatants and lysates. Assays were done twice in quadruplicate (n = 8). Error bars represent ±SEMs. (B) TEMs of RAW 264.7 cells infected with the S. pyogenes wild type (top) or the SalY mutant (bottom). Wild-type-infected cells at 24 h postinfection show multiple cocci in cytoplasm or released from dead cells. SalY mutant-infected RAW 264.7 cells at 24 h postinfection show a large empty vacuole and a single cocci in a vacuole (black arrows). WT, wild type.

 
Examination of infected RAW 264.7 cells by TEM was consistent with the above-described tissue culture results. At early time points after infection, there was very little difference observed between cultures infected with the wild type or the SalY mutant (data not shown). However, by 24 h, most of the wild-type-infected macrophages were lysed or contained many intracellular cocci in the cytoplasm (Fig. 3B, top panels). In contrast, the SalY-infected macrophages exhibited an activated morphology, and the few cocci found were in large vacuoles (Fig. 3B, bottom panels). Similarly, using fluorescent stains to measure cell membrane permeability (LIVE/DEAD BacLight; Molecular Probes) demonstrated that infection with the wild-type strain resulted in cell death by 24 h postinfection, while the macrophages infected with the SalY mutant showed an activated morphology and had cell membranes that were impermeable to the propidium iodide DNA stain (data not shown).

Crude cell lysates inhibit bacterial growth. To determine whether an inhibitory factor produced by the RAW 264.7 cells was having an effect on the S. pyogenes strains, crude cell lysates of unstimulated and stimulated RAW 264.7 cells were prepared and incubated with the bacterial cultures. The unstimulated RAW 264.7 cells were seeded on a 24-well plate and incubated overnight under normal tissue culture conditions. Stimulation of the RAW 264.7 cells was accomplished by incubation overnight with gentamicin-killed wild-type bacteria. The following day, the RAW 264.7 cells were lysed and the whole-cell crude lysates were added to bacterial cultures in DMEM plus 10% FBS. Bacterial survival was assessed after a 7-hour incubation at 37°C (Fig. 4). When sterile double-distilled water was added to the cultures alone (in the absence of cell lysates), there was no difference between the levels of wild-type and mutant growth. The addition of either stimulated or unstimulated cell lysates to the wild-type culture resulted in about 60% growth inhibition compared to the growth in nontreated cultures, which correlates with the growth observed in cultures where CFU numbers stay relatively constant until the 8-h time point (Fig. 3A). However, the degree of inhibition was significantly greater for the SalY mutant (Fig. 4). Not only was the growth of the SalY mutant inhibited, but the number of recovered bacterial CFU after the 7-hour incubation was reduced from the starting inoculum from the addition of both the stimulated and unstimulated cell lysates, indicating that some cell deaths had occurred. The crude cell lysate data further support the hypothesis that the macrophages are able to inhibit, and even kill, the SalY mutant strain at 7 h postinfection, while the wild-type strain is less susceptible to this inhibition.


Figure 4
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  		FIG. 4. Crude whole-cell lysate was added to bacterial cultures grown in DMEM with 10% FBS. RAW 264.7 cells were lysed in sterile double-distilled water. The graph shows bacterial survival of the wild type (WT) and the SalY mutant strain at 7 h after the addition of cell lysates or water alone. The cell lysate groups include RAW 264.7 cells that had not been activated or RAW 264.7 cells activated for 24 h with gentamicin-killed wild-type bacteria. The graph shows the results of assays done in triplicate on three different days. Error bars represent ±SEMs.

 
Examination of zebrafish macrophages. To determine if this phenomenon played a role in virulence in vivo, zebrafish macrophages were examined during an infection with both the wild-type and SalY mutant strains. At 16 h after i.m. injection, the fish were euthanized, the spleens were aseptically removed and gently homogenized, and cytospin preparations were made of the lysates. Wild-type-infected zebrafish showed evidence of infected splenic cells, and while some chains of cocci were found to be extracellular, the majority of the bacteria were localized intracellularly, within macrophages (Fig. 5A). The SalY-infected zebrafish showed macrophages that were highly activated with large empty vacuoles (Fig. 5B), and almost no SalY cocci could be visualized in the spleen homogenate samples (the total number of each strain isolated from spleens is reported in Fig. 2C). These observations for the infected zebrafish splenic macrophages correlated with those in the murine macrophage tissue culture assay, in that a mutation in the salY gene results in an inability to survive in macrophages.


Figure 5
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  		FIG. 5. Zebrafish infections. Panels A and B show zebrafish spleen cytospin preparations 16 h after i.m. injection. (A) Images of splenic cells from wild-type-infected zebrafish. Multiple intracellular cocci can be visualized. (B) Images of splenic macrophages from SalY-infected zebrafish illustrating the actively changing morphology and the absence of cocci. Magnification, x1,000. (C) Bacterial CFU recovered from spleens of CAR-treated zebrafish. Prior to injection of bacterial strain, zebrafish were injected i.p. with 100 µg of CAR. Each indicated strain was injected i.m. into zebrafish dorsal muscles at 1 x 105 CFU. The fish were euthanized, and spleens were removed for bacterial isolation at the indicated time points. All assays were done twice with six fish per time point (n = 12). Error bars represent ±SEMs. WT, wild type; *, P < 0.05. For controls, zebrafish were also treated with PBS prior to infection, the results for which are shown in Fig. 2C.

 
Infections in macrophage-depleted zebrafish. To substantiate that the SalY mutant attenuation was specific to a susceptibility to macrophage-killing in vivo, zebrafish were treated with CAR to specifically deplete macrophages. CAR is a high-molecular-weight sulfated polysaccharide generated from marine plants that has been demonstrated to specifically deplete macrophages without affecting neutrophil activity in mice and guinea pigs (21, 31). Spleen homogenates from CAR-treated zebrafish without infection were examined first to confirm that CAR functions in zebrafish as has been shown for mice (11, 18) (see Materials and Methods). Zebrafish were injected i.p. with 100 µg CAR or with sterile PBS as a negative control prior to i.m. injection of a specific S. pyogenes strain. To confirm that the increased virulence of the SalY mutant in the zebrafish was due to the depletion of macrophages and not simply to the toxicity of the CAR treatment, the Emm mutant was included in the CAR assays. If the increase in virulence observed in SalY-infected, CAR-treated zebrafish is the result of toxicity from the compound, then the Emm mutant would also exhibit the same level of virulence. The levels of bacterial loads over time were examined by infecting CAR-treated zebrafish with the three indicated strains (Fig. 5C). Comparison of the results from the two different infections reveals that the bacterial loads of all strains are very similar up to 8 h (data not shown). Between 8 and 12 h, the wild-type strain shows a 10-fold increase in bacterial load in the CAR-treated fish compared to the bacterial load in PBS-treated zebrafish, showing that the loss of macrophages also benefits survival of the wild-type strain (compare Fig. 2C and 5C). However, the SalY mutant shows a 55-fold increase in CFU count compared to the CFU count at the 12-hour time point in the fish without CAR. Similarly, the wild type exhibits an increase of less than 10-fold for the next three time points, while the SalY mutant shows 47-fold, 85-fold, and 165-fold increases in CFU counts for the 16-, 20-, and 24-h time points, respectively, compared to results at the same time points for the fish without CAR (Fig. 2C and 5C). While the Emm mutant CFU count was higher overall in the CAR-treated fish than in the PBS-treated fish, the CFU count was still approximately 50-fold lower than that of the SalY mutant and more than 2 logs lower than the CFU count of the wild-type strain. This attenuation most likely was due to the susceptibility of the Emm mutant to neutrophil killing because of the loss of the antiphagocytic M protein, while the SalY mutant still expresses the M protein.

Wild-type-infected zebrafish exhibit a large white lesion at the site of injection in the dorsal muscle, while infection with either the SalY mutant or the Emm mutant does not result in a lesion (Fig. 6A). However, CAR treatment of zebrafish restores the ability of the SalY mutant to cause excessive necrosis to a level similar to that seen in the wild-type-infected zebrafish (Fig. 6B). Conversely, in spite of CAR treatment, injection of the Emm mutant results in very little necrosis and no lesion development, suggesting that the restoration of virulence observed with the SalY mutant is due specifically to the depletion of the macrophages.


Figure 6
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  		FIG. 6. Images of zebrafish injected i.m. with the wild type (WT), the SalY mutant, or the Emm mutant at 20 h postinjection. (A) Zebrafish treated i.p. with PBS; (B) zebrafish treated i.p. with CAR.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In recent years, S. pyogenes, a prototypical extracellular pathogen, has demonstrated the ability to survive in the intracellular environment (23, 24, 27-29, 36, 41, 51, 53). This ability would offer several advantages, such as escaping many of the toxic immune factors that are produced by the host during an infection or antibiotic treatment. On the other hand, this new environment also presents many challenges to an organism not adapted to that setting, creating a selective pressure for acquisition of new mechanisms for survival. Intracellular survival mechanisms would be particularly important within phagocytic cells, where survival strategies must include escaping the multiple cytotoxic mechanisms used to process and present foreign antigens. Previously described factors involved in the intracellular survival of S. pyogenes in host cells include streptolysin O, which contributes to escape from the endocytic vacuole and evasion of lysosomal killing (14, 32), and SpeB, specifically in macrophages (51). In addition, the M protein functions in antiphagocytosis and is necessary for survival in neutrophils (48) and for inhibiting the fusion of azurophilic granules with the phagosome (2, 47). The SalY mutant has retained both streptolysin O and SpeB activity (data not shown) and still expresses the M protein, further suggesting that the attenuated phenotype in macrophages associated with this mutant was due to the loss of function of SalY and not a loss of these previously described factors.

Similar observations from human infections correlate with the data presented here and strongly support the hypothesis that S. pyogenes can endure macrophage-killing mechanisms. Thulin et al. (51) demonstrated that viable S. pyogenes organisms were localized intracellularly in phagocytic cells, primarily macrophages, in patients with soft tissue infections (51). Biopsy specimens from these patients were obtained after antibiotic treatment, and high levels of viable bacteria could still be recovered from these samples, suggesting that because of the intracellular survival of S. pyogenes, phagocytic cells may serve as a reservoir for this organism. The data presented here are consistent with the results from the human biopsy specimens and provide some insight into how S. pyogenes survives in this cell type. Therefore, further characterization of the SalY mutant will add to our knowledge of the pathways involved in the intracellular survival of group A streptococcus in macrophages.

Many gram-positive organisms produce lantibiotics, and the salivaricin A lantibiotic has been well characterized in S. salivarius (37, 42, 44, 52, 54). Synthesis of a lantibiotic provides an offensive strategy when bacteria are inhabiting an environment where competition for colonization is intense, such as the highly colonized oral cavity. In this environment, the production of salivaricin A would be highly advantageous for S. pyogenes, as colonization of the oral pharynx is necessary for the onset of pharyngitis, one of the many diseases caused by this pathogen. However, if a strain has evolved to become more invasive, thereby colonizing tissues or environments that are normally sterile, then the need for synthesis of a lantibiotic against other organisms would be less important. This hypothesis is supported by the discovery that almost all S. pyogenes strains tested to date do not produce a functional SalA lantibiotic (42, 44). However, the sal locus has been highly conserved in almost all S. pyogenes strains examined, suggesting the possibility that a selective pressure for retaining the locus exists.

The SalA peptide has been shown to have a dual function, as a lantibiotic in the processed form and as a signaling peptide for transcriptional regulation of the locus (52). Retention of the structural gene for SalA suggests that even though the peptide is not processed into the active lantibiotic (due to mutations in the processing protein, SalM) (Fig. 1), it may still be produced and secreted for either the signaling process for transcriptional regulation or another unknown function. Supporting the role of the sal locus in virulence are data from a recent study that demonstrated that HSC5 salA expression was increased 15-fold, as shown by microarray analyses, in the muscle tissue of a murine model for S. pyogenes infection (26). According to the HSC5 sal operon sequence analysis, the only putative promoter driving transcription of the salA-salY genes is found at the beginning of the locus, upstream of salA, consistent with the concomitant increased expression of downstream genes during the murine muscle infection. Therefore, since most S. pyogenes strains are not producing an active lantibiotic, the function of the locus may have evolved to serve a different purpose. In support of this hypothesis, antibiotic peptides, which are cationic, low-molecular-weight, heat-stable bacteriocins (19), share a great deal of similarity with antimicrobial peptides produced by eukaryotic cells, in that the peptides are similar in size and cationic and amphiphilic in nature (43, 56). Furthermore, in phagocytic cells, some antimicrobial peptides are constitutively present (56). Because of the significant amino acid changes in the SalY protein sequence of S. pyogenes compared to that of S. salivarius, as well as 14 of the changes resulting in a change in charge located on the extracellular domain of the protein, the substrate specificity of this putative transporter may have changed from salivaricin A to a macrophage factor, perhaps an antimicrobial peptide, that is detrimental to S. pyogenes intracellular survival. The data presented here support the hypothesis that the function of SalY in S. pyogenes may have evolved to provide a novel mechanism for intracellular survival.

The ability of pathogens to sense and adapt to cationic antimicrobial peptides is well established in the literature. The PhoP/PhoQ quorum-sensing system was found to sense sublethal concentrations of cationic antimicrobial peptides in both Salmonella enterica serovar Typhimurium and Yersinia pestis intracellularly in macrophages, and genes under PhoP/PhoQ regulation were identified as being necessary for the survival of both organisms in macrophages (1, 12). Changes following PhoP activation include synthesis of cation transporters (45). The pathogen Burkholderia pseudomallei has been shown to be resistant to cationic bactericidal peptides, and this resistance contributes to the ability to survive intracellularly in phagocytic cells (4, 20). An efflux pump responsible for the resistance of B. pseudomallei to aminoglycosides and macrolides was found to be necessary for intracellular survival. Although a direct correlation has not been made, the efflux pump may be able to expel host antimicrobial peptides found in the intracellular environment (7). Using this scenario, the SalY protein may have evolved through changes in specific substrate binding to function as an immunity protein for antimicrobial peptides produced by macrophages during an infection. The locus, therefore, would be retained to support this immunity function, particularly in strains that have adapted to the intracellular environment.

Collectively, the results presented here introduce a role for the sal locus in virulence. With the introduction of two random mutations in one locus (salY and salK), it is clear that this lantibiotic locus contributes significantly to S. pyogenes pathogenesis. We propose a model whereby a lantibiotic operon has evolved to function in protection from the host during infection. The evidence for such a model is (i) the conservation of the immunity and regulatory portions of the locus in the majority of S. pyogenes strains in the absence of an active lantibiotic, (ii) the loss of function of the putative immunity protein for protection against salivaricin A for virtually all S. pyogenes strains, and (iii) the changes in charged amino acid residues in the SalY immunity transporter protein, suggesting a change in substrate specificity. Further analysis of the sal locus in virulence will add to our knowledge of how an extracellular pathogen has adapted to survive in the intracellular environment and thus escape clearance by the host immune system.


   ACKNOWLEDGMENTS
 
The technical assistance provided by Donna Runft and Anne Kizy in this project is greatly appreciated.

This work was supported by Public Health Service grant AI52141 from the NIAID of the National Institutes of Health.


   FOOTNOTES
 
* Corresponding author. Mailing address: Immunology & Microbiology Dept., Wayne State University School of Medicine, 540 East Canfield St., Detroit, MI 48201. Phone: (313) 577-1314. Fax: (313) 577-1155. E-mail: mneely{at}med.wayne.edu Back

{triangledown} Published ahead of print on 18 June 2007. Back

Editor: A. Camilli


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Bader, M. W., W. W. Navarre, W. Shiau, H. Nikaido, J. G. Frye, M. McClelland, F. C. Fang, and S. I. Miller. 2003. Regulation of Salmonella typhimurium virulence gene expression by cationic antimicrobial peptides. Mol. Microbiol. 50:219-230.[CrossRef][Medline]
1a. Bakleh, M., L. E. Wold, J. N. Mandrekar, W. S. Harmsen, H. H. Dimashkieh, and L. M. Baddour. 2005. Correlation of histopathologic findings with clinical outcome in necrotizing fasciitis. Clin. Infect. Dis. 40:410-414.[CrossRef][Medline]
2. Bauer, S., and H. Tapper. 2004. Membrane retrieval in neutrophils during phagocytosis: inhibition by M protein-expressing S. pyogenes bacteria. J. Leukoc. Biol. 76:1142-1150.[Abstract/Free Full Text]
3. Bisno, A. L., and D. L. Stevens. 1996. Streptococcal infections of skin and soft tissues. N. Engl. J. Med. 334:240-245.[Free Full Text]
4. Burtnick, M. N., and D. E. Woods. 1999. Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin B resistance. Antimicrob. Agents Chemother. 43:2648-2656.[Abstract/Free Full Text]
5. Caparon, M. G., and J. R. Scott. 1991. Genetic manipulation of the pathogenic streptococci. Methods Enzymol. 204:556-586.[Medline]
6. Carapetis, J. R., A. C. Steer, E. K. Mulholland, and M. Weber. 2005. The global burden of group A streptococcal diseases. Lancet Infect. Dis. 5:685-694.[CrossRef][Medline]
7. Chan, Y. Y., and K. L. Chua. 2005. The Burkholderia pseudomallei BpeAB-OprB efflux pump: expression and impact on quorum sensing and virulence. J. Bacteriol. 187:4707-4719.[Abstract/Free Full Text]
8. Cockerill, F. R., III, R. L. Thompson, J. M. Musser, P. M. Schlievert, J. Talbot, K. E. Holley, W. S. Harmsen, D. M. Ilstrup, P. C. Kohner, M. H. Kim, B. Frankfort, J. M. Manahan, J. M. Steckelberg, F. Roberson, W. R. Wilson, et al. 1998. Molecular, serological, and clinical features of 16 consecutive cases of invasive streptococcal disease. Clin. Infect. Dis. 26:1448-1458.[Medline]
9. Fischetti, V. A. 1989. Streptococcal M protein: molecular design and biological behavior. Clin. Microbiol. Rev. 2:285-314.[Abstract/Free Full Text]
10. Gibson, C. M., and M. G. Caparon. 2002. Alkaline phosphatase reporter transposon for identification of genes encoding secreted proteins in gram-positive microorganisms. Appl. Environ. Microbiol. 68:928-932.[Abstract/Free Full Text]
11. Goldmann, O., M. Rohde, G. S. Chhatwal, and E. Medina. 2004. Role of macrophages in host resistance to group A streptococci. Infect. Immun. 72:2956-2963.[Abstract/Free Full Text]
12. Grabenstein, J. P., H. S. Fukuto, L. E. Palmer, and J. B. Bliska. 2006. Characterization of phagosome trafficking and identification of PhoP-regulated genes important for survival of Yersinia pestis in macrophages. Infect. Immun. 74:3727-3741.[Abstract/Free Full Text]
13. Granok, A. B., D. Parsonage, R. P. Ross, and M. G. Caparon. 2000. The RofA binding site in Streptococcus pyogenes is utilized in multiple transcriptional pathways. J. Bacteriol. 182:1529-1540.[Abstract/Free Full Text]
14. Hakansson, A., C. C. Bentley, E. A. Shakhnovic, and M. R. Wessels. 2005. Cytolysin-dependent evasion of lysosomal killing. Proc. Natl. Acad. Sci. USA 102:5192-5197.[Abstract/Free Full Text]
15. Hanski, E., P. A. Horwitz, and M. G. Caparon. 1992. Expression of protein F, the fibronectin-binding protein of Streptococcus pyogenes JRS4, in heterologous streptococcal and enterococcal strains promotes their adherence to respiratory epithelial cells. Infect. Immun. 60:5119-5125.[Abstract/Free Full Text]
16. Herbomel, P., B. Thisse, and C. Thisse. 1999. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 126:3735-3745.[Abstract]
17. Hidalgo-Grass, C., M. Dan-Goor, A. Maly, Y. Eran, L. A. Kwinn, V. Nizet, M. Ravins, J. Jaffe, A. Peyser, A. E. Moses, and E. Hanski. 2004. Effect of a bacterial pheromone peptide on host chemokine degradation in group A streptococcal necrotising soft-tissue infections. Lancet 363:696-703.[CrossRef][Medline]
18. Ishizaka, S., S. Kuriyama, and T. Tsujii. 1989. In vivo depletion of macrophages by desulfated iota-carrageenan in mice. J. Immunol. Methods 124:17-24.[CrossRef][Medline]
19. Jack, R. W., J. R. Tagg, and B. Ray. 1995. Bacteriocins of gram-positive bacteria. Microbiol. Rev. 59:171-200.[Abstract/Free Full Text]
20. Jones, A. L., T. J. Beveridge, and D. E. Woods. 1996. Intracellular survival of Burkholderia pseudomallei. Infect. Immun. 64:782-790.[Abstract]
21. Kaneda, N., H. Kawauchi, and G. Mogi. 1991. Role of phagocytes in antimicrobial defence of the dle ear. Auris Nasus Larynx 18:331-342.[Medline]
22. Kaul, R., A. McGeer, D. E. Low, K. Green, B. Schwartz, et al. 1997. Population-based surveillance for group A streptococcal necrotizing fasciitis: clinical features, prognostic indicators, and microbiologic analysis of seventy-seven cases. Am. J. Med. 103:18-24.[CrossRef][Medline]
23. Kobayashi, S. D., K. R. Braughton, A. R. Whitney, J. M. Voyich, T. G. Schwan, J. M. Musser, and F. R. DeLeo. 2003. Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils. Proc. Natl. Acad. Sci. USA 100:10948-10953.[Abstract/Free Full Text]
24. LaPenta, D., C. Rubens, E. Chi, and P. P. Cleary. 1994. Group A streptococci efficiently invade human respiratory epithelial cells. Proc. Natl. Acad. Sci. USA 91:12115-12119.[Abstract/Free Full Text]
25. Lieschke, G. J., A. C. Oates, M. O. Crowhurst, A. C. Ward, and J. E. Layton. 2001. Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish. Blood 98:3087-3096.[Abstract/Free Full Text]
26. Loughman, J. A., and M. Caparon. 2006. Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a model for in vivo gene expression. J. Bacteriol. 188:399-408.[Abstract/Free Full Text]
27. Marouni, M. J., A. Barzilai, N. Keller, E. Rubinstein, and S. Sela. 2004. Intracellular survival of persistent group A streptococci in cultured epithelial cells. Int. J. Med. Microbiol. 294:27-33.[CrossRef][Medline]
28. Medina, E., O. Goldmann, A. W. Toppel, and G. S. Chhatwals. 2003. Survival of Streptococcus pyogenes within host phagocytic cells: a pathogenic mechanism for persistence and systemic invasion. J. Infect. Dis. 187:597-603.[CrossRef][Medline]
29. Medina, E., M. Rohde, and G. S. Chhatwal. 2003. Intracellular survival of Streptococcus pyogenes in polymorphonuclear cells results in increased bacterial virulence. Infect. Immun. 71:5376-5380.[Abstract/Free Full Text]
30. Meehl, M. A., J. S. Pinkner, P. J. Anderson, S. J. Hultgren, and M. G. Caparon. 2005. A novel endogenous inhibitor of the secreted streptococcal NAD-glycohydrolase. PLoS Pathogens 1:e35.[CrossRef]
31. Mitsuyama, M., K. Nomoto, and K. Takeya. 1982. Direct correlation between delayed footpad reaction and resistance to local bacterial infection. Infect. Immun. 36:72-79.[Abstract/Free Full Text]
32. Nakagawa, I., A. Amano, N. Mizushima, A. Yamamoto, H. Yamaguchi, T. Kamimoto, A. Nara, J. Funao, M. Nakata, K. Tsuda, S. Hamada, and T. Yoshimori. 2004. Autophagy defends cells against invading group A Streptococcus. Science 306:1037-1040.[Abstract/Free Full Text]
33. Neely, M. N., J. D. Pfeifer, and M. Caparon. 2002. Streptococcus-zebrafish model of bacterial pathogenesis. Infect. Immun. 70:3904-3914.[Abstract/Free Full Text]
34. Neely, M. N., W. R. Lyon, D. L. Runft, and M. Caparon. 2003. Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes. J. Bacteriol. 185:5166-5174.[Abstract/Free Full Text]
35. O'Brien, K. L., B. Beall, N. L. Barrett, P. R. Cieslak, A. Reingold, M. M. Farley, R. Danila, E. R. Zell, R. Facklam, B. Schwartz, and A. Schuchat. 2002. Epidemiology of invasive group A streptococcus disease in the United States, 1995-1999. Clin. Infect. Dis. 35:268-276.[CrossRef][Medline]
36. Osterlund, A., R. Popa, T. Nikkila, A. Scheynius, and L. Engstrand. 1997. Intracellular reservoir of Streptococcus pyogenes in vivo: a possible explanation for recurrent pharyngotonsillitis. Laryngoscope 107:640-647.[CrossRef][Medline]
37. Patton, G. C., and W. A. van der Donk. 2005. New developments in lantibiotic biosynthesis and mode of action. Curr. Opin. Microbiol. 8:543-551.[CrossRef][Medline]
38. Phelps, H. A., and M. N. Neely. 2005. Evolution of the zebrafish model: from development to immunity and infectious disease. Zebrafish 2:87-103.[Medline]
39. Ralph, P., and I. Nakoinz. 1977. Antibody-dependent killing of erythrocyte and tumor targets by macrophage-related cell lines: enhancement by PPD and LPS. J. Immunol. 119:950-954.[Abstract/Free Full Text]
40. Reed, L. J., and H. Muench. 1938. A simple method of estimation of fifty percent endpoints. Am. J. Hyg. 27:493-497.
41. Rohde, M., E. Muller, G. S. Chhatwal, and S. R. Talay. 2003. Host cell caveolae act as an entry-port for group A streptococci. Cell. Microbiol. 5:323-342.[CrossRef][Medline]
42. Ross, K. F., C. W. Ronson, and J. R. Tagg. 1993. Isolation and characterization of the lantibiotic salivaricin A and its structural gene salA from Streptococcus salivarius 20P3. Appl. Environ. Microbiol. 59:2014-2021.[Abstract/Free Full Text]
43. Sahl, H. G., and G. Bierbaum. 1998. Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria. Annu. Rev. Microbiol. 52:41-79.[CrossRef][Medline]
44. Simpson, W. J., N. L. Ragland, C. W. Ronson, and J. R. Tagg. 1995. A lantibiotic gene family widely distributed in Streptococcus salivarius and Streptococcus pyogenes. Dev. Biol. Stand. 85:639-643.[Medline]
45. Soncini, F. C., E. García Véscovi, F. Solomon, and E. A. Groisman. 1996. Molecular basis of the magnesium deprivation response in Salmonella typhimurium: identification of PhoP-regulated genes. J. Bacteriol. 178:5092-5099.[Abstract/Free Full Text]
46. Sosroseno, W., and E. Herminajeng. 2002. The role of macrophages in the induction of murine immune response to Actinobacillus actinomycetemcomitans. J. Med. Microbiol. 51:581-588.[Abstract/Free Full Text]
47. Staali, L., S. Bauer, M. Morgelin, L. Bjorck, and H. Tapper. 2006. Streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes. Cell. Microbiol. 8:690-703.[CrossRef][Medline]
48. Staali, L., M. Morgelin, L. Bjorck, and H. Tapper. 2003. Streptococcus pyogenes expressing M and M-like surface proteins are phagocytosed but survive inside human neutrophils. Cell. Microbiol. 5:253-265.[CrossRef][Medline]
49. Tagg, J. R., and L. V. Bannister. 1979. "Fingerprinting" beta-haemolytic streptococci by their production of and sensitivity to bacteriocine-like inhibitors. J. Med. Microbiol. 12:397-411.[Abstract]
50. Taylor, F. B. J., A. E. Bryant, K. E. Blick, E. Hack, P. M. Jansen, S. D. Kosanke, and D. L. Stevens. 1999. Staging of the baboon response to group A streptococci administered intramuscularly: a descriptive study of the clinical symptoms and clinical chemical response patterns. Clin. Infect. Dis. 29:167-177.[Medline]
51. Thulin, P., L. Johansson, D. E. Low, B. S. Gan, M. Kotb, A. McGeer, and A. Norrby-Teglund. 2006. Viable group A streptococci in macrophages during acute soft tissue infection. PLoS Med. 3:e53.[CrossRef][Medline]