Heterogeneity in the Polarity of Nra Regulatory Effects on Streptococcal Pilus Gene Transcription and Virulence

Bacterial culture.Unless otherwise specified, the M53 strain Alab49 and its isogenic mutants were grown at 30 or 37°C with 5% CO₂ in Todd-Hewitt broth supplemented with 1% yeast extract (THY). Bacterial growth was monitored by the optical density at 600 nm (OD₆₀₀).

Mutant construction.Inactivation of nra and msmR in Alab49 (Fig. 1) was achieved by allelic exchange mutagenesis following transformation of bacteria with purified linear DNA containing the kanamycin resistance gene (aphA3) flanked by sequences upstream and downstream of the target gene. Linear DNA cassettes were constructed by PCR-based fusion assembly and used to transform Alab49 by electroporation (23). Primers used for construction of mutants are listed in Table S1 in the supplemental material. Transformants were selected on THY-blood agar plates containing 500 μg/ml of kanamycin and evaluated for replacement of the target gene by PCR-based mapping and nucleotide sequence determination. All mutants of Alab49 were confirmed to have growth curves in THY broth identical to that for wt Alab49.

Chromosomal replacement in the Δnra mutant, generating the Alab49 nra::aad9 construct, was achieved following exchange of the aphA3 gene of the Δnra mutant with the intact nra gene plus a spectinomycin resistance gene (aad9) (20) positioned downstream (Fig. 1). The linear DNA cassettes used for transformation contained the juxtaposed nra and aad9 genes flanked by DNA corresponding to regions lying upstream and downstream of nra in wt Alab49, allowing for double crossover by homologous recombination. Transformants were selected on THY-blood agar plates containing 200 μg/ml of spectinomycin, and the spectinomycin-resistant clones were subsequently screened for loss of aphA3 by a failure to grow in the presence of kanamycin. The resulting Alab49 nra::aad9 construct was confirmed by PCR-based mapping and nucleotide sequence determination of the entire replaced region, extending beyond the sites of crossover.

Humanized mouse model for impetigo.The hu-skin SCID mouse model for streptococcal impetigo was implemented as previously described in extensive detail (23, 33, 35, 36). Briefly, human neonatal foreskin was engrafted onto the hind flanks of C.B.-17 scid mice, which fail to reject the xenografts. Healed skin grafts were gently scratched with a scalpel blade and inoculated with 50 μl of bacteria in THY broth. The inoculated bacteria had been grown freshly to mid-logarithmic or stationary phase and diluted as appropriate. The actual inoculum doses were ascertained by serial dilutions performed in duplicate, with the numbers of CFU being averaged. Mid-logarithmic phase was defined as the point of approximately half-maximal OD₆₀₀. Stationary-phase cultures were incubated for 24 h. Inoculated skin grafts were occluded with a bandage. At 7 days postinoculation, the human skin grafts were surgically removed from mice and split, and each portion was weighed. One weighed portion of the graft was evaluated for the number of CFU released following vigorous vortex mixing, with serial dilutions performed in duplicate. The other portion of the skin was used for histopathology; it was formalin fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Using a severity scale ranging from 0 to 3, each tissue was scored for both the extent of inflammation and destruction of the epidermal layer; scores from two investigators, who were blinded to experimental treatment and outcome, were added (for a scale of 0 to 6) and then averaged. Also at the time of biopsy, spleens were removed and cultured to measure systemic infection by GAS; in this study, all spleen cultures were negative for bacterial growth.

Purification of RNA.Bacteria were grown overnight in THY broth, diluted 1:100 in fresh THY broth, and grown to mid-logarithmic (OD₆₀₀ = 0.350 ± 0.05) or stationary (OD₆₀₀ = 0.850 ± 0.05) phase. Total RNA was extracted using RNAprotect RNA stabilization reagent (Qiagen). Bacteria were harvested by mixing 1 volume (2.5 ml) of culture with 2 volumes of RNAprotect and then were centrifuged, and the pellet was resuspended in RNase-free Tris-EDTA buffer containing lysozyme (2 mg/ml), mutanolysin (2.5 U/μl), and proteinase K (60 × 10⁻³ active unit/ml), and incubated at room temperature for 15 min; cells were lysed by adding Qiazol lysis reagent preheated at 65°C, followed by chloroform extraction. The aqueous phase containing RNA was purified using an RNeasy kit (Qiagen) according to the manufacturer's instructions. Samples were treated with DNase I, using an RNase-free DNase set (Qiagen) to remove potential traces of DNA in the sample; the absence of contaminating DNA was verified by failure to amplify the purified RNA samples prior to cDNA synthesis, using Taq polymerase and oligonucleotide primers targeting recA. The A₂₆₀/A₂₈₀ ratio of each RNA sample was measured to determine concentration and assess purity (i.e., a ratio of >1.8).

qRT-PCR.cDNA was synthesized from 1 μg RNA by using SuperScript III first-strand synthesis supermix for quantitative real-time PCR (qRT-PCR) with random oligonucleotide primers (Invitrogen) according to the manufacturer's instructions. RT-PCR was performed with SYBR green ER qPCR supermix in 96-well MicroAmp optical reaction plates (Applied Biosystems), using an ABI Prism 7900 HT sequence detection system (Applied Biosystems). Primers for RT-PCR (see Table S1 in the supplemental material) were designed to amplify internal regions (72 to 139 bp) within the open reading frames of selected genes. The recA gene was used as an internal reference transcription control to normalize expression data for each target gene. Unless indicated otherwise, each gene target was tested in duplicate from three or more RNA templates prepared from independent bacterial cultures. Relative expression of each gene was determined by the $$mathtex$$\(2^{{-}({\Delta}{\Delta}C_{T})}\)$$mathtex$$ method (22). Briefly, the average cycle threshold number (C_T) of each target gene was normalized to the C_T value of the recA gene for each experiment, and the difference was expressed as ΔC_T (ΔC_T = target gene C_T − recA C_T). The relative expression ratio for each gene in each construct was calculated as the product of $$mathtex$$\(2^{{-}({\Delta}{\Delta}C_{T})}\)$$mathtex$$, in which ΔΔC_T = mean ΔC_T(mutant) − mean ΔC_T(wt). A difference in transcript abundance of >2-fold or <0.5-fold was chosen as the threshold value for alterations that are likely to be biologically significant.

Mutanolysin extraction.Cell wall extracts of GAS were prepared using mutanolysin as described previously (23); unless otherwise specified, cells were harvested following growth at 30°C to mid-logarithmic phase (OD₆₀₀ = 0.350 ± 0.05) or stationary phase (16 h). Extracts from 50 ml of THY broth culture were reduced to a final volume of 400 μl in protoplast buffer.

Immunoblots.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on gradient gels (4 to 15% acrylamide) under reducing conditions. Rabbit sera raised against recombinant fusion polypeptides (23) prepared in Escherichia coli and originally derived from strain Alab49 were used at a dilution of 1:1,000.

Cysteine proteinase activity.Measurement of secreted cysteine proteinase activity in broth culture supernatants was determined by an azocasein assay, as described previously (23), following bacterial growth in C broth medium for 20 h at 37°C. To ensure that differences in enzymatic activity were not attributable to differences in bacterial cell density, the OD₆₀₀ of each culture was assessed. Activity was calculated based on an A₃₆₆ value of 0.155 being equivalent to 10 activity units (U) per ml of culture supernatant (35).

Bacterium-bound plasmin activity.The activation of human plasminogen on the surfaces of bacterial cells was determined by a method adapted from a previously described method (32). Overnight cultures grown in THY broth were washed in phosphate-buffered saline and resuspended in 1/10 the original volume; 100 μl of concentrated cells, equivalent to ∼10⁸ CFU, was added to a mixture of 2.8 ml THY broth and 1.2 ml fresh human plasma and incubated at 37°C for 1 h. Reactions were terminated by centrifugation at 4°C. The bacterial pellets were washed in ice-cold phosphate-buffered saline containing 0.1% Tween 20 and placed on ice. Plasmin activity associated with the bacteria was measured using a method adapted from a previously described method (17). Each pellet was incubated in 100 μl of plasmin substrate solution, prepared by mixing 2 volumes of chromogenic substrate H-D-Val-Leu-Lys-ρ-nitroanilide (S2251; Sigma) stock solution (5 mg/ml in water) with 3 volumes of 32 mM Tris (pH 7.5)-1.77 M NaCl solution for 15 min at 37°C.

HA content.The hyaluronic acid (HA) content of GAS, attributable to the polysaccharide capsule, was measured according to previously described methods (36).

Statistical analysis.Statistical significance was calculated using GraphPad Prism (San Diego, CA), by the unpaired t test (two-tailed) and the Mann-Whitney U test. Both the t test and the Mann-Whitney U test are conservative and may slightly underestimate the significance of differences between groups with a small sample size.

Nra is a positive regulator of pilus gene transcription in an M53 strain.Studies of an M49 strain (isolates designated CS101 and 591; emm pattern E) demonstrated that Nra functions as a repressor of genes encoding FCT region surface proteins that give rise to pili (16, 30). In this report, the role of Nra in pathogenesis of a classical emm pattern D skin strain, Alab49 (M53), is evaluated. An Alab49 mutant lacking the nra gene (Δnra) was constructed by allele exchange mutagenesis with the aphA3 gene, encoding kanamycin resistance. The nra gene is located within the chromosomal FCT region (Fig. 1). Like the M49 strain, Alab49 has the FCT-3 form of the FCT region; the FCT regions of both strains are ∼11 kb in length and include genes involved in pilus structure and biosynthesis (cpa through fctB), the transcriptional regulatory gene msmR, and prtF2, encoding a fibronectin-binding protein (13).

The Δnra mutant was compared to the Alab49 wt strain in the level of RNA transcripts corresponding to FCT region genes following growth to mid-logarithmic or stationary phase in THY broth in the presence of 5% CO₂. Relative transcript abundance was measured by qRT-PCR. The Δnra mutant displayed an ∼3- to 10-fold decrease in transcripts encoding proteins involved in pilus structure and assembly (cpa through fctB) at both stages of growth (Table 1). Thus, Nra is a positive regulator of pilus gene expression in Alab49, a finding that contrasts with its function in the M49 strain, where Nra acts as a negative regulator of pilus gene transcription (14, 26, 30). The opposite regulatory effect was observed despite the finding that the Nra products of the M53 and M49 strains share 99.6% amino acid sequence identity (4).

As a control, the aphA3 gene in the Δnra mutant was chromosomally replaced with the wt nra gene, generating the Alab49 nra::aad9 construct; the aad9 gene conferring spectinomycin resistance was inserted between the 3′ end of nra and a putative transcriptional termination site (30) lying downstream of nra and upstream of hsp33 (Fig. 1). The data show that transcription of the cpa through fctB genes was restored to wt levels in Alab49 nra::aad9 (Table 1). The possibility that genetic alterations within and around the nra locus lead to polar effects on the hsp33 gene lying downstream of nra seems unlikely because differences in the abundance of hsp33 transcripts were not observed for the Δnra mutant or Alab49 nra::aad9 relative to the wt (Table 1).

qRT-PCR data also show that the relative abundances of msmR and prtF2 transcripts were largely unaltered in the Δnra mutant of Alab49 compared to the wt (Table 1). The lack of an Nra-mediated effect on prtF2 in the M53 strain contrasts with the finding for the M49 strain, whereby Nra is a repressor of prtF2 transcription (16, 30).

Consistent with the qRT-PCR findings on transcript levels (Table 1), immunoblot analysis of mutanolysin extracts from the Δnra mutant showed a marked loss in the quantity of the ladder-like polymer structure characteristic of pili compared to that for wt Alab49 (Fig. 2A). In the control nra::aad9 construct, the wt phenotype was restored. Similar findings were obtained when antiserum directed to either recombinant FctA (rFctA) or rFctB was used on immunoblots (see Fig. S1 in the supplemental material). In contrast to the bands for pilus-associated surface proteins, the anti-rPrtF2-reactive bands (Fig. 2B) appeared unaltered in the Δnra mutant, consistent with the finding that Nra has no measurable effect on RNA transcript levels for prtF2 (Table 1).

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FIG. 2.

Deletion of nra results in loss of pilus-like structures. Immunoblots were treated with antiserum raised to rCpa (A) or rPrtF2 (B). Mutanolysin extracts subject to SDS-PAGE were prepared from wt Alab49 (lanes 1 and 2), the Δnra mutant (lanes 3 and 4), and Alab49 nra::aad9 (lanes 5 and 6). Extracts from cells grown to mid-logarithmic phase (4 h at 30°C) are shown in lanes 1, 3, and 5; extracts from cells grown to stationary phase (16 h at 30°C) are shown in lanes 2, 4, and 6. To account for the increase in bacterial cell mass at stationary phase, the loading volume of the samples obtained from mid-logarithmic-phase cultures was increased 2.5-fold, a value which corresponds to the observed increase in OD₆₀₀ for the stationary-phase cultures relative to that for mid-logarithmic-phase cultures. Antisera to rFctA and rFctB showed staining patterns highly similar to those observed with anti-rCpa serum, with a loss of ladder-like structures in the Δnra mutant (see Fig. S1 in the supplemental material). Because PrtF2 is completely degraded by SpeB in stationary-phase cultures (23), only extracts from mid-log-phase cultures are shown; also, the protease inhibitor cocktail was not included during the extraction procedure. Molecular size markers are indicated in kilodaltons.

The Alab49 wt strain and mutants also underwent T-type agglutination analysis following growth at 30°C; both the wt and Alab49 nra::aad9 were T type T3/13/B, whereas the Δnra mutant was T nontypeable (TNT) (data not shown). Prior studies showed that cpa, fctA, and prtF2 each contributes to the T type in Alab49 (23), whereas the M49 strain is TNT (13).

The positive regulatory effect of Nra in Alab49 was shown following mutanolysin extraction of bacteria grown to 37°C, whereby pilus-like structures were absent in the Δnra mutant but restored to wt levels in Alab49 nra::aad9 (see Fig. S2 in the supplemental material). Therefore, the effect of Nra on pilus gene expression is not a consequence of growth temperature.

Nra is required for superficial skin infection.Past studies showed that both cpa and prtF2 are required for virulence of strain Alab49 in a humanized SCID mouse model for skin infection following bacterial growth in THY broth to the mid-logarithmic stage but not to the stationary phase (23). In this model, engrafted human skin is gently scratched, bacteria are topically applied, and the infection site is occluded with a bandage. Important features of the humanized mouse model for superficial skin infection by GAS include high specificity (i.e., skin strains, such as Alab49, are more virulent than throat strains) and high sensitivity (i.e., low doses of a virulent strain yield infection) (23, 33, 35, 36). A drawback is that the humanized mouse model does not readily allow for high-throughput analysis, and thus, sample sizes are small and measures of statistical significance are conservative.

In this study, the Δnra mutant was compared to wt Alab49 for virulence at the skin. All 18 skin grafts inoculated with wt Alab49, tested over a wide range of doses and cultured in THY broth to either mid-log or stationary phase, displayed a net increase in CFU at 7 days postinoculation (Fig. 3A). The net reproductive growth of bacteria at the skin is the primary outcome measure of virulence and is highly concordant with histological alterations that resemble human cases of impetigo (23, 33, 35, 36).

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FIG. 3.

Skin infection in the humanized mouse at 7 days postinoculation. Bacteria grown to either mid-logarithmic (filled symbols) or stationary (open symbols) phase in THY broth culture were used to inoculate scratched human skin engrafted on SCID mice. The inoculum dose (log₁₀ CFU) is depicted on the x axis. The net change (increase or decrease) in log₁₀ CFU recovered from a graft at biopsy relative to the inoculum dose is shown on the y axis. Each data point represents an inoculated skin graft. The strains used were wt Alab49 (A), the Δnra mutant (B), and Alab49 nra::aad9 (C). The differences in inoculum doses tested for the wt and each mutant were not significant (Mann-Whitney U test).

In sharp contrast to those inoculated with wt Alab49, nearly all skin grafts (16 of 17 grafts [93%]) inoculated with the Δnra mutant and cultured to either mid-log or stationary phase showed a net loss in CFU at 7 days postinoculation (Fig. 3B). The differences in the magnitude of the net change in CFU between the wt and the Δnra mutant inoculated at either growth stage were highly significant (P < 0.01, by both unpaired t test and Mann-Whitney U test [two-tailed]). Furthermore, there was no significant difference in the net change in CFU for the Δnra mutant following inoculation of a mid-log-phase versus stationary-phase culture. The data show that Nra is required for streptococcal virulence at the skin. In addition, Nra-dependent virulence is not influenced by the growth phase of the bacterial inoculum, unlike the previous finding for Cpa (23).

As a control, the nra::aad9 construct was tested for virulence at the skin, and its virulence was restored to wt levels (Fig. 3C) (differences were not significant).

Histopathology scores on the severity of tissue damage and inflammation were compared for tissue grafts displaying a net increase (n = 26) versus a decrease (n = 13) in CFU (four grafts were excluded from histological analysis due to poor tissue processing). The data show highly significant differences in histopathology scores between the two groups (P = 0.0042; two-tailed unpaired t test), with mean average scores of 2.02 and 4.78 for tissues having a net loss and gain in CFU, respectively.

Nra does not alter Mga expression in the M53 strain.In the M49 strain, Nra functions as a negative regulator of mga transcription (14, 30). Mga is a key transcriptional regulator of numerous virulence genes, including emm and speB (15, 31). Therefore, effects of Nra on the expression of mga may indirectly influence the transcription of other virulence genes.

The effect of Nra on mga transcription in strain Alab49 was assessed by qRT-PCR. The data indicate that the relative abundances of mga transcripts were nearly equivalent for the Δnra mutant and wt Alab49 (Table 2). The lack of Nra-mediated regulation of mga in the M53 strain contrasts with reported findings on the M49 strain (14, 30).

SpeB is a secreted cysteine protease playing a critical role in skin infection by Alab49 (23, 35). Nra is a positive regulator of speB transcription in an M49 strain (14, 26) but had no measurable effect on speB in strain Alab49 (Table 2). The observed similarity in transcript levels for speB between wt Alab49 and the Δnra mutant was confirmed by a phenotypic test that measures secreted cysteine protease activity (see Fig. S3 in the supplemental material).

Streptokinase (Ska) and the plasminogen-binding M protein (PAM; also known as M53) are critical determinants of impetigo caused by Alab49 (36). However, the relative transcript abundances of the ska and pam (i.e., emm53) genes were highly similar in wt Alab49 and the Δnra mutant (Table 2). The qRT-PCR findings for ska and pam in wt Alab49 versus the Δnra mutant were confirmed by assays that measure secreted Ska via immunoblotting (Fig. 4A) and that determine bacterium-bound plasmin activity (Fig. 4B).

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FIG. 4.

Streptokinase production and bacterium-bound plasmin activity. (A) The immunoblot was treated with antiserum to rSka. Bacterial culture supernatants subject to SDS-PAGE were concentrated following growth to mid-log (4 h) (lanes 1, 3, and 5) or stationary (16 h) (lanes 2, 4, and 6) phase. wt Alab49 (lanes 1 and 2), the Δnra mutant (lanes 3 and 4), and Alab49 nra::aad9 (lanes 5 and 6) are shown. (B) Bacterial surface-bound plasmin assay. Each culture was tested in triplicate. The Δska and Δpam mutants were previously described (36).

The nra::aad9 construct was highly similar to both wt Alab49 and the Δnra mutant in the measures of non-FCT-region gene expression and activity of their products (Table 2; see Fig. S3 and S4 in the supplemental material). The HA capsule content was also nearly equivalent for wt Alab49, the Δnra mutant, and Alab49 nra::aad9; mean average yields were 17.95, 19.45, and 16.34 fg of HA per CFU, respectively.

MsmR is a repressor of nra and pilus gene transcription in the M53 strain.For the M49 strain, in which Nra functions as a negative regulator of pilus gene expression, the FCT region gene product MsmR is an activator of nra, prtF2, and pilus gene transcription (28).

To better understand the function of MsmR in the M53 strain, the msmR gene was inactivated in Alab49 (ΔmsmR), and the mutant was compared to the wt for transcription regulatory effects. Table 3 shows qRT-PCR results demonstrating that MsmR has a repressive effect on nra and pilus gene transcription in Alab49, in contrast to its reported activity in the M49 strain. The repressive effect of MsmR in Alab49 is slight; an immunoblot of the polymeric ladder corresponding to pilus proteins was only slightly increased in the ΔmsmR mutant (see Fig. S4 in the supplemental material).

Like the case for the M49 strain, MsmR is a transcriptional activator of prtF2 transcription in the M53 strain (Table 3). The activating effect of MsmR on prtF2 transcript levels was >10-fold. Phenotypic differences in extractable PrtF2 were readily visible by immunoblotting for wt Alab49 versus the ΔmsmR mutant (Fig. 5). The magnitude of the effect of MsmR on prtF2 transcription was much greater than its effect on either nra or pilus gene transcription.

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FIG. 5.

Deletion of msmR results in loss of PrtF2. The immunoblot was treated with antiserum raised to rPrtF2. Mutanolysin extracts subjected to SDS-PAGE were prepared from wt Alab49 (lane 1) or the ΔmsmR mutant (lane 2). Extracts were from cells grown to mid-logarithmic phase (4 h at 30°C) in the absence of the protease inhibitor cocktail.

The repressive effect of msmR on cpa and fctA transcription seemed to be somewhat less apparent at an early stage in exponential growth (Table 3). Similarly, the activating effect of nra on cpa and fctA transcription also appeared to be less pronounced at early logarithmic phase (see Table S3 in the supplemental material). Thus, both MsmR and Nra appear to exert their effects on pilus expression as the bacterium begins to approach the midpoint of logarithmic growth.