tRNA Modification by GidA/MnmE Is Necessary for Streptococcus pyogenes Virulence: a New Strategy To Make Live Attenuated Strains

Bacterial strains.The bacterial strains and plasmids used in this study are listed in Table 1. Molecular cloning experiments were performed with Escherichia coli DH5α which was cultured in Luria-Bertani broth (38) at 37°C. For routine culture of S. pyogenes, Todd-Hewitt medium (BBL) supplemented with 0.2% yeast extract (Difco) (THY medium) was employed. Unless otherwise indicated, for growth of S. pyogenes for SpeB activity assays, Western blot analyses, and preparation of RNA for real-time reverse transcription (RT)-PCR C medium was used (37). For growth in liquid media, S. pyogenes was cultured at 37°C in sealed tubes without agitation. To produce solid media, Bacto agar (Difco) was added to a final concentration of 1.4% (wt/vol). When appropriate, antibiotics were added to the media at the following concentrations: spectinomycin, 100 μg/ml for E. coli and S. pyogenes; chloramphenicol, 15 μg/ml for E. coli and 3 μg/ml for S. pyogenes; kanamycin, 50 μg/ml for E. coli and 500 μg/ml for S. pyogenes; and erythromycin, 750 μg/ml for E. coli and 1 μg/ml for S. pyogenes.

Manipulation and computational analyses of DNA.Plasmid DNA was isolated via standard techniques and used to transform S. pyogenes as described previously (5). Restriction endonucleases, ligases, and polymerases were used according to the manufacturer's recommendations. The fidelity of all constructs derived by PCR was confirmed by DNA sequencing analyses. All references to genomic loci and predictions of protein function are based on the genome of S. pyogenes strain SF370 (14) and were supported by interrogation of the nonrepetitive sequence database at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/ ) using a gapped BLAST algorithm (2).

Construction of EZ-Tn5<ΩErm>.For application of Tn5-based “transposome” mutagenesis (22) to S. pyogenes, the BamHI site of pMOD-2<MCS> (Epicenter Biotechnologies) was used to insert a BamHI fragment that contains the ΩErm element of pJDM-STM (30). The resulting plasmid was designated pMOD-2::Tn5<ΩErm>, and the resulting transposable element was designated EZ-Tn5<ΩErm> (Fig. 1A). The erythromycin resistance gene of the ΩErm element can be used for selection in both E. coli and S. pyogenes (30).

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FIG. 1.

(A) Transposable element EZ-Tn5<ΩErm>. The structure of the EZ-Tn5<ΩErm> transposon developed for transposome mutagenesis (22) is shown. The ΩErm element contains an erythromycin resistance determinant (erm) that is flanked by strong transcription and translation terminators (indicated by “Ω”) (30). The ΩErm element is flanked by the left and right mosaic ends of Tn5 (arrowheads ME_L and ME_R). For clarity, only a limited region of plasmid pMOD-2::Tn5<ΩErm> (dashed line), a derivative of pMOD-2<MCS> (Epicenter Biotechnologies), is shown. Restriction sites: Pv, PvuI; B, BamHI. (B) Reduced SpeB expression as a result of transposon insertion in gidA. The organization of the chromosomal region containing gidA is shown at the top. The arrows indicate individual open reading frames and their orientations and are labeled based on the annotation of the S. pyogenes SF370 genome (14). The predicted proteins encoded by these open reading frames and their putative functions are shown at the bottom. The markers above gidA indicate the sites of EZ-Tn5<ΩErm> insertion in three mutants (TM-GidA1 to TM-GidA3) that resulted in reduced expression of the SpeB cysteine protease. The specific sites of transposon insertion are as follows: TM-GidA1, within a lysine codon (L368); TM-GidA2, following an alanine codon (A16); and TM-GidA3, within a serine codon (S179).

Transposome mutagenesis.A gel-purified PvuI fragment of pMOD-2::Tn5<ΩErm> that included Tn5<ΩErm> (Fig. 1A) was mixed in vitro with the EZ-Tn5 transposase according to the directions of the manufacturer (Epicenter Biotechnologies). Electroporation was then used to introduce the transposome complex into competent cells of S. pyogenes that had been prepared by a standard method (5) and then washed once at room temperature in a 15% (vol/vol) glycerol solution containing 1 mM EDTA to remove Mg²⁺. This additional step was required for stabilization of the transposon-transposase complex prior to its introduction into the streptococcal cell (22). Transformants with insertions of Tn5<ΩErm> were then recovered following overnight incubation at 37°C on THY medium plates supplemented with erythromycin. For mutants of interest, the transposon insertion site was identified by direct sequencing of chromosomal DNA using a transposon-specific primer (SqTM-r) (see Table S1 in the supplemental material) as described previously (18).

Directed mutagenesis and complementation.Insertional inactivation of specific genes and complementation analyses were conducted using a standard recombinational strategy (8). For mutagenesis, an approximately 0.7-kbp fragment of the gene of interest that lacked both its 5′ and 3′ ends was amplified by PCR, inserted into pSPC18 (37), and then introduced into E. coli. The plasmid was purified and then used to transform S. pyogenes. Since pSPC18 cannot replicate in S. pyogenes, most transformants resistant to spectinomycin have undergone a single homologous recombination event that results in disruption of the targeted gene (8). For complementation of a GidA⁻ mutant, an intact copy of gidA was introduced into the chromosome of the mutant as follows. A chromosomal segment containing the entire predicted gidA open reading frame and putative promoter region was amplified by PCR and inserted into the integrational plasmid pCIV2 (34). Transformation followed by recombination of the resulting plasmid into the mutant's chromosome resulted in introduction of an intact copy of gidA. Details for each integrating plasmid are shown in Table 1, and the sequences of primers used for amplification of gene segments are shown in Table S1 in the supplemental material. The chromosomal structure of each mutant was confirmed by PCR using primers for the appropriate sequences. To ensure that mutant phenotypes were specifically due to gene disruption and not due to a nonspecific effect of integration of pSCP18 into the chromosome, a strain with pSPC18 inserted into the intergenic region downstream of recF (SPy_2204) (9), which is referred to here as HSC5spc (Table 1), was included in selected analyses.

Analysis of protein expression.Analyses of cell growth and use of protease indicator medium, Western blot analysis of SpeB secretion, and quantitative measurement of SpeB activation and SpeB cysteine protease activity were all performed as described previously (37). Sample preparation and experiments with antisera for Western blot analyses to quantitate expression of streptolysin O (SLO), NAD glycohydrolase (SPN), mitogenic factor (MF), and RopA-hemagglutinin (RopA-HA) were conducted as described elsewhere (22, 35). Cells were fractionated as described previously (36), and M protein in cell wall fractions was analyzed by Western blotting using an antiserum developed against a peptide (QELAKKEEQNKISDASRKG) that was shown to be involved in streptococcal cell aggregation (16). The level of expression of a specific protein in various mutants was expressed relative to its expression in the wild type by comparison of band intensities in digital images of Western blots analyzed using IMAGEJ software (1).

Analysis of transcription.RNA from the various streptococcal strains was isolated and analyzed using real-time RT-PCR as described previously (27), employing the primers listed in Table S1 in the supplemental material. Transcript abundance was normalized to the results for recA, and the data presented below are the means and standard deviations derived from at least two independent experiments that were performed on different days, with each individual sample analyzed in triplicate. Comparison of global transcriptional profiles using DNA microarrays was conducted as described in detail elsewhere (6, 13, 36).

Infection of macrophages.Murine bone marrow-derived macrophages were obtained by a standard procedure (7) and were cultured in RPMI 1640 containing l-glutamine (Cellgro Mediatech, Inc.) with 20% (vol/vol) heat-inactivated fetal bovine serum (BioWhittaker, Inc.), 30% (vol/vol) L-cell conditioned medium (a source of macrophage colony-stimulating factor), penicillin G (50 IU ml⁻¹), and streptomycin (50 μg ml⁻¹). Macrophages were cultured for 8 days, harvested with ice-cold phosphate-buffered saline, resuspended in macrophage medium (Dulbecco's minimal essential medium [BioWhittaker, Inc.] containing 50 mM HEPES, 10% fetal bovine serum, and 8 mM l-glutamine), and then plated at a density of 2.5 × 10⁶ cells per well in a six-well plate (catalog no. 3506; Corning Inc.). For infection, overnight cultures of S. pyogenes in THY medium were harvested, washed twice with phosphate-buffered saline, and then resuspended in macrophage medium to an optical density at 600 nm of 0.76. Portions of this suspension ranging from 50 to 100 μl were then added directly to wells containing macrophages. Final multiplicities of infection were determined by plating the streptococcal suspensions on THY agar following brief sonication to disrupt the streptococcal chains. In selected experiments, streptococci were inactivated by exposure to UV light (254 nm, 500 mJ/cm²) prior to incubation with macrophages. This dose exceeds the lethal dose for S. pyogenes (44), which was routinely confirmed by plating on THY agar.

Cytokine assays.Following 4 h of infection of macrophage cultures, the supernatant fluids were harvested and then sterilized by filtration (Millex-GP; 0.22 μm; Millipore Corp). Cytokine levels in these fluids were determined using a commercial antibody-based multiplex bead assay (Bio-Plex, Mouse Cytokine 23-Plex; catalog no. 171-F11241; Bio-Rad Laboratories) to measure the 23 different murine cytokines that are listed in Fig. 8 and Fig. S2 in the supplemental material. For this analysis the directions of the manufacturer (Bio-Rad Laboratories) were followed, and the analysis was conducted by the High Speed Cell Sorter Core Lab at the Siteman Cancer Center of Washington University. The data presented are the means and standard errors derived from three different infections, which were conducted on different days, and each experiment was analyzed in duplicate. Any differences in the mean values for specific cytokines induced in response to mutants compared with the wild type were tested for significance by the unpaired t test (19), and the null hypothesis was rejected when the P value was <0.05.

Murine subcutaneous infection model.The abilities of selected mutants to cause disease in soft tissue were evaluated by subcutaneous infection of 6- to 8-week-old SKH1 hairless mice (Charles River Labs) as described previously (4). Various strains were analyzed using groups containing five mice, and each experiment was repeated at least twice on different days. The area of the draining ulcers that formed was documented every 24 h by digital photography, and the precise area for each ulcer was calculated from the digital record using MetaMorph image analysis software (version 4.6; Universal Imaging Corp.). Any differences in the areas of ulcers between experimental groups was tested for significance by the Mann-Whitney U test (19), and the null hypothesis was rejected when the P value was <0.05.

Microarray data accession number.The microarray data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo ) under GEO Series accession number GSE9678.

Transposome mutagenesis identified the gidA gene.We adapted a Tn5-based transposome mutagenesis strategy to screen for a mutant with reduced virulence factor expression (see Materials and Methods). This method has the advantages that it is simple, target site selection is random, and, because the inserted element itself does not encode transposase, insertions are stable in the absence of continued antibiotic selection (22). Introduction of a transposome complex generated with EZ-Tn5<ΩErm> (Fig. 1A) into S. pyogenes HSC5 produced transformants at an efficiency between 10³ and10⁴ CFU/μg DNA. An insertion library was then examined for mutants that displayed reduced activity of the secreted SpeB cysteine protease following overnight culture on a protease indicator medium (see Materials and Methods). Approximately 4,000 colonies were examined, and nine mutants with reduced activity were identified that were subsequently found to express cysteine protease activity at levels between 5 and 20% of the wild-type level when they were examined using a quantitative proteolysis assay with a casein substrate (see Materials and Methods) following growth in two different media (THY and C media). Identification of the transposon insertion loci (see Materials and Methods) revealed that six mutants had insertions in the gene encoding RopB, which is a positive transcriptional regulator of SpeB, and three mutants had insertions at different sites within the gidA open reading frame (SPy_2185) (Fig. 1B), whose orthologous gene products have been implicated in the flavin adenine dinucleotide (FAD)-dependent modification of tRNAs decoding two-family box triplet codons (47). Mutation of gidA in E. coli causes cell elongation and slow growth in rich media supplemented with glucose (45). However, the S. pyogenes mutants did not exhibit any measurable defect in either the growth rate or cell morphology upon culture in several rich media (THY and C media) in the absence or presence of additional glucose (0.5%, wt/vol) (data not shown). Also, like the genomes of many bacterial species (46), the S. pyogenes genome encodes a protein that is highly homologous to, although somewhat smaller than, GidA (SPy_1173) (20.1% identical and 55.1% similar). Insertional inactivation of SPy_1173 did not lead to any observable defect in SpeB biogenesis (data not shown).

Characterization of the gidA chromosomal region.Examination of the genomic data available for multiple S. pyogenes strains revealed that the structure of the gidA chromosomal region is strongly conserved and that this region includes genes whose products are implicated in the function of ribosomes or DNA polymerase or have an unknown function (Fig. 1B), all of which may contribute to the biogenesis of SpeB and other virulence factors. Since the EZ::Tn5<ΩErm> element includes strong transcription and translation terminators to ensure generation of strongly polar insertions (Fig. 1A), it was necessary to examine the contribution of the adjacent genes to the GidA⁻ phenotype. Insertion duplication mutations constructed to inactivate gidA and the open reading frame immediately downstream (SPy_2184) (Fig. 2) produced two mutants that both had reduced expression of SpeB (HSC5::pKHC100 and HSC5::pKHC102) (Fig. 2). However, when the amounts of SpeB protein that were secreted into the supernatant were compared using a Western blot analysis, the two mutants had distinct phenotypes. When tested after 10 h of culture, the GidA⁻ mutant secreted a reduced amount of SpeB protein, like the original transposon insertion mutants. In contrast, the SPy_2184 mutant secreted wild-type levels of the protease zymogen at this time point but failed to process the inactive precursor to the active protease (data not shown). A comparison of the transcript levels of the genes downstream of gidA (SPy_2184 and rplI) (Fig. 1B) between the wild type and a transposon insertion mutant (TM-GidA1) (Fig. 1B) revealed no significant differences when a real-time RT-PCR was used (GidA⁻ mutant/wild-type strain expression ratio for SPy_2184, 0.92 ± 0.17; GidA⁻ mutant/wild-type strain expression ratio for rplI, 0.91 ± 0.30), showing that insertions into gidA are not polar for expression of downstream genes. Taken together, these data suggested that both GidA and SPy_2184 are required for SpeB biogenesis but contribute to different steps. A requirement for both genes was confirmed by construction of an additional insertion duplication mutation that placed the integrated plasmid between the intact gidA and SPy_2184 open reading frames. The resulting strain (HSC5::pKHC101) (Fig. 2) expressed nearly wild-type levels of SpeB proteolytic activity. Finally, reintroduction of an intact copy of gidA into the chromosome of a GidA⁻ mutant (see Materials and Methods) resulted in a complemented strain whose protease activity more closely resembled that of the wild type than that of the GidA⁻ mutant on protease indicator plates (Fig. 3A) and which produced SpeB activity in culture supernatants at nearly wild-type levels (Fig. 3B). Based on these data, we concluded that the reduced-expression phenotype observed in the transposon insertion mutants was due to the loss of gidA.

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FIG. 2.

Confirmation of the contribution of gidA to SpeB expression. The integrational plasmids shown at the top were used to disrupt various regions of the gidA locus in S. pyogenes HSC5. The broken ends of the plasmid-encoded boxes indicate that a gene segment lacks the 5′ or 3′ end of the corresponding chromosomal open reading frame, and these regions are indicated by the open boxes above the open reading frames. Chromosomal structures resulting from the single recombination events (labeled a, b, and c) are indicated below the solid arrow, and the designations and genotypes of the resulting strains are shown on lines a, b, and c. The open reading frame labeled aad9 encodes a spectinomycin resistance determinant. The SpeB cysteine protease activities produced by these strains were determined relative to the activity produced by the wild-type strain (HSC5) (WT) and are indicated in the box on the right. The data are the means and standard errors of the means derived from at least three independent experiments.

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FIG. 3.

Reconstitution of disrupted gidA restores SpeB expression. An intact copy of gidA was inserted into the chromosome of a GidA⁻ mutant (see Materials and Methods for details). (A) Protease production on indicator medium. The indicated strains were patched on protease indicator medium and examined following overnight incubation. A zone of clearing resulted from protease activity. (B) Quantitative assessment of protease activity. The SpeB cysteine protease activities in supernatants of 12-h cultures are shown relative to the SpeB activity of the wild-type strain. The data are the means and standard errors of the means derived from at least three independent experiments. The following strains were used: wild type (WT), HSC5; GidA⁻ mutant (GidA⁻), HSC5::pKHC100; and GidA⁻ complemented (Comp.), HSC5::pKHC100::pKHC103.

Transcription of speB is delayed in GidA⁻ mutants.Expression of speB is known to be highly regulated at the level of transcription, and this gene is expressed in a growth phase-dependent pattern (26). Since GidA⁻ mutants produced a reduced amount of SpeB protein at the time points described above, it was of interest to determine if the loss of gidA had any effect on the levels or timing of speB transcription. The growth patterns of the strains (the wild type, the GidA⁻ mutant, and the SPy_2184 mutant) were almost identical (Fig. 4; see Fig. S1 in the supplemental material), so the levels of speB transcript abundance of the strains at different time points were examined by real-time RT-PCR. The levels of the speB message in the wild type exhibited the expected growth phase pattern, with initiation at the onset of stationary phase and an increase over the next several hours, followed by a gradual decline (Fig. 4). The SPy_2184 mutant, which had reduced SpeB activity but not a reduced level of secreted SpeB protein, exhibited a pattern identical to that of the wild type (Fig. 4). In contrast, the GidA⁻ mutant displayed an aberrant pattern of expression. At the onset of stationary phase, the abundance of the speB transcript was over 60-fold less than that of the wild type (Fig. 4). The transcript became more abundant over time, until it reached a level that was not significantly different from the wild-type level when it was measured in 10-h cultures (Fig. 4).

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FIG. 4.

Disruption of gidA results in altered pattern of speB transcription. The relative abundance of the speB message was determined over the course of growth in several strains using real-time RT-PCR. For each strain, transcript abundance is shown relative to the abundance of the speB message in the wild-type strain at 6 h. For clarity, only the growth pattern of the wild-type strain is shown, as the growth characteristics of all strains were indistinguishable under these conditions (see Fig. S1 in the supplemental material). The transcription data are the means and standard deviations of three independent experiments. The following strains were used: wild type (WT), HSC5; GidA⁻ mutant (GidA⁻), HSC5::pKHC100; and SPy_2184 mutant (2184⁻), HSC5::pKHC102. OD₆₀₀, optical density at 600 nm.

Disruption of mnmE showed the same phenotype as mutation of gidA.In addition to GidA, modification of U34 in tRNAs decoding two-family box triplet codons requires additional enzymes, including MnmE (4, 8, 14, 47). This relationship is supported by the observation that GidA and MnmE form a heterotetrameric complex in E. coli (47). To examine whether the SpeB expression defect of GidA⁻ mutants was specific to gidA or was a consequence of the loss of the ability to modify tRNA, a strain lacking mnmE was constructed (see Materials and Methods). A Western blot analysis revealed that the MnmE⁻ mutant had a SpeB expression defect identical to that of the GidA⁻ mutant (Fig. 5A), indicating that the entire pathway for U34 modification may be important for SpeB biogenesis.

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FIG. 5.

Overexpression of RopB restores SpeB expression in GidA pathway mutants. (A) Western blot analysis to detect SpeB in strains containing a plasmid that overexpresses RopB (pRopB-HA) or in the absence of a plasmid (no plasmid). Note that multiple proteolytic cleavages are required for activation of the 46-kDa SpeB zymogen to the 28-kDa active protease. (B) Western blot analysis to determine the amount of RopB-HA present in the indicated strains (panel a), with protein levels quantitated by densitometry and expressed relative to the wild-type level (line b). Panel c shows the relevant region of the membrane stained with Coomassie blue for comparison of the amounts of total protein present in each lane. The following strains were used: wild type (WT), HSC5; GidA⁻ mutant (GidA⁻), HSC5::pKHC100; and MnmE⁻ mutant (MnmE⁻), HSC5::pKHC104.

GidA influences SpeB expression via the transcription regulator RopB.In Shigella flexneri, mutation of several genes involved in tRNA modification results in a reduction in expression of selected virulence genes as a consequence of poor translation efficiency of the transcription regulator VirF (13). Expression of such virulence factors can be rescued in Shigella mutants by overexpression of VirF (13). In S. pyogenes, transcription of speB is under control of the transcription activator RopB (26). In the GidA⁻ mutant, the abundance of the ropB transcript was unchanged from the abundance in the wild type (Fig. 6), suggesting that the tRNA processing mutants may not efficiently translate this transcription regulator. In this case, overexpression of RopB should rescue speB expression, similar to overexpression of VirF in Shigella tRNA modification mutants. To test this, the GidA⁻ and MnmE⁻ mutants were transformed with a plasmid that expresses high levels of a RopB protein that has been modified to include an influenza HA tag at its carboxy terminus. This construct produces a functional RopB, as demonstrated by its ability to efficiently complement a ropB deletion (26). When these transformed strains were analyzed, it was found that overexpression of RopB restored the amount of SpeB protein secreted by the mutants to wild-type levels (Fig. 5A, compare “no plasmid” to “pRopB-HA”). Even though the amount of RopB that the mutant strains produced was sufficient to rescue SpeB expression, these strains produced less than 50% of the amount of RopB-HA made by the wild-type strain transformed by the overexpression plasmid (Fig. 5B). These data are consistent with the idea that the loss of GidA- and MnmE-dependent tRNA modification alters the pattern of speB transcription via an effect on the translation efficiency of the ropB message.

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FIG. 6.

Nearly normal transcription of multiple virulence factors in a GidA⁻ mutant. The relative levels of abundance of the messages for the indicated virulence factors in a GidA⁻ mutant were determined by real-time RT-PCR following 6 h of culture in C medium and are expressed as changes relative to the wild-type strain. Lines indicate expression that was not different from the wild-type expression (dashed line) and expression that was twofold different from the wild-type expression (solid lines). The chromosomal locus and the primers used for analysis of each gene are listed in Table S1 in the supplemental material. The transcription data are the means and standard errors of the means derived from at least two independent experiments. The following strains were used: wild type, HSC5Spc; and GidA⁻ mutant, HSC5::pKHC100.

Reduced production of multiple secreted virulence proteins.A sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based comparison of cytosolic, cell membrane, and cell wall fractions of GidA⁻ and MnmE⁻ mutants and the wild type did not reveal any obvious differences in protein profiles (data not shown), consistent with the lack of any overall growth defect in the mutants. Transcription of speB occurs during the early stationary phase of growth; however, many virulence factors are expressed in growing cells. Examination of transcription profiles in growing cells using a microarray analysis indicated that the loss of GidA had a minimal impact on the global transcriptome, with only about 2% of the transcripts examined altered at a level of twofold or greater. Of these, only 0.5% (seven transcripts) were altered at levels greater than threefold and none were altered at levels greater than fivefold (see Tables S2, S3, and S4 in the supplemental material). A lack of an effect on transcription of virulence-associated genes was confirmed by a real-time RT-PCR analysis of a subset of messages, including several messages that encode transcription regulators implicated in virulence (mga, ropB, and covR) and a panel of additional factors representing both intracellular and secreted proteins. This analysis revealed that transcription of this panel of genes varied only about twofold or less from the wild-type transcription at 6 h postinoculation (Fig. 6). In contrast, examination of the levels of several secreted virulence proteins, including SPN, SLO, the surface M protein, and MF, demonstrated that both the GidA⁻ and MnmE⁻ mutants expressed each of these proteins at levels that were less than 50% of the wild-type levels in the exponential phase (Fig. 7). Taken together, these data suggest that a reduction in expression of multiple virulence proteins, such as SPN, SLO, M protein, and MF, in the exponential phase is influenced at the level of translation by GidA and MnmE and that there may be a greater impact on virulence factor production than on the production of housekeeping proteins that are involved in cell growth.

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FIG. 7.

GidA pathway mutants have reduced expression of several virulence proteins. The results of Western blot analyses to detect the levels of expression of the indicated virulence proteins in several GidA pathway mutants are shown. Strains were cultured in THY medium and harvested at the point of maximal expression of each factor in the wild-type strain (6 h for SPN, SLO, and M protein; 16 h for MF). A cysteine protease inhibitor (10 μM E64) was added to culture media to inhibit possible degradation of virulence proteins by SpeB. Expression levels were determined by densitometry and are expressed relative to expression in the wild-type strain (indicated below each panel). The data are the means and standard deviations of two independent experiments. The following strains were used: wild type (WT), HSC5Spc; GidA⁻ mutant (GidA⁻), HSC5::pKHC100; and MnmE⁻ mutant (MnmE⁻), HSC5::pKHC104.

Comparison of cytokine profiles from infected macrophages.It has been shown that S. pyogenes induces a unique cytokine activation program in macrophages (21) and that macrophages have an important role in controlling S. pyogenes infection in a murine model of disease (20). Since macrophages are also a key cell element of the adaptive immune response, the influence of GidA on streptococcus-macrophage interaction was assessed. Murine bone marrow-derived macrophages were infected with either the wild type or the GidA⁻ mutant or with these strains following UV treatment to block growth. After 4 h of infection, supernatant fluids were monitored for the presence of a panel of 23 different cytokines and chemokines (see Materials and Methods). Overall, the wild type and the GidA⁻ mutant, whether they were UV treated or not treated, induced identical patterns of expression of 21 cytokines at levels of twofold or greater (Fig. 8; see Fig. S2 in the supplemental material). The cytokines not stimulated by both the mutant and the wild type included interleukin 1α (IL-1α) and eotaxin (see Fig. S2 in the supplemental material). For the induced cytokines, the magnitudes of induction were also similar for the wild type and the GidA⁻ mutant (see Fig. S2 in the supplemental material), with two exceptions. For both untreated and UV-inactivated cultures, the GidA⁻ mutant induced tumor necrosis factor alpha (TNF-α) and IL-12(p40) at levels that were less than 50% of the levels induced by the wild type (Fig. 8). Since IL-12(p40) is a subunit of both IL-12 and IL-23 and since the IL-12(p70) subunit of IL-12 was not differentially expressed (Fig. 8), it was likely that IL-23, rather IL-12, was differentially induced. This was confirmed by real-time RT-PCR analysis of the IL-23(p19) subunit, which showed that this transcript was present in macrophages infected by the GidA⁻ mutant at a level that was only 22% ± 15% of the level induced in macrophages infected with the wild type.

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FIG. 8.

Cultured macrophages produce reduced levels of TNF-α and IL-12(p40) in response to GidA⁻ mutants. The levels of production of the indicated cytokines following 4 h of infection of cultured murine bone marrow-derived macrophages are shown. Cytokine levels were measured using an antibody-based multiplex bead assay (Bio-Plex; Bio-Rad Laboratories). Macrophage cultures were infected at a multiplicity of 10 streptococci per macrophage or were not infected (Uninfect.). Streptococcal strains were used immediately following culture (Untreated) or were inactivated by treatment with UV radiation (UV-treated) immediately prior to infection. An asterisk indicates that expression was significantly decreased compared to the wild type (P < 0.05), and ND indicates that the level of expression was below the level of detection of the assay. In addition to the expression of IL-12(p70) shown, there was no difference in expression between the wild type and the mutant for the 21 other cytokines analyzed in this experiment (see Fig. S2 in the supplemental material). The data are the means and standard errors of the means from three independent experiments. The following strains were used: wild type (WT), HSC5Spc; and GidA⁻ mutant (GidA⁻), HSC5::pKHC100.

GidA⁻ mutants are highly attenuated in a murine model of soft tissue infection.The ability of the GidA⁻ mutants to cause disease in soft tissue was evaluated using the murine subcutaneous model. For the strain used in this study (HSC5), injection into the subcutaneous tissue of an SKH1 hairless mouse results in a local draining ulcer, whose area reaches the maximum value 3 days postinfection and which then goes on to heal over the next 10 to 14 days (4). In a comparison of the abilities of a GidA⁻ mutant (TM-GidA1) and the wild type to cause disease in this model, it was found that the latter produced lesions of the expected size by day 3 postinfection (Fig. 9). In contrast, the GidA⁻ mutant was significantly attenuated in the ability to cause lesions. While a few infected mice exhibited small lesions, the majority of mice infected with the mutant did not exhibit any visible lesions at day 3 postinfection or at any subsequent time point. Identical results were obtained with a second GidA⁻ mutant (HSC5::pKHC100) (data not shown). These data indicate that the reduction in virulence factor expression observed in vitro in the GidA⁻ mutants translates into a severely attenuated ability to cause disease in soft tissue.

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FIG. 9.

GidA⁻ mutants are highly attenuated. The ability of a GidA⁻ mutant to cause disease in the murine subcutaneous ulcer model is shown. Virulence was evaluated on the basis of the area of the ulcer produced at the time when ulcer formation was maximal in the wild-type strain (3 days postinfection). The circles and squares represent ulcer areas in individual mice for the wild type and GidA⁻ mutant, respectively. The solid bars indicate the mean values for the strains, and P value as determined by a Mann-Whitney U test statistic is indicated above the bracket. The data shown are pooled data from two independent experiments. The following strains were used: wild type (WT), HSC5Spc; and GidA⁻ mutant (GidA⁻), HSC5::pKHC100.