Classification of covS mutations

We investigated all previously reported covS mutations. The covS mutations were found most frequently in M1 isolates rather than any other serotypes [10, 15, 18, 22], and our unpublished data]. The covS mutations detected in M1 strains were divided into two groups: (i) frameshift or nonsense mutations that caused a deletion in functional regions and (ii) point mutations that caused single (or double) amino acid(s) substitutions. Of the 34 covS mutations 25 were detected in isolates from mice infected with the M1 strain were frameshift mutations (Figure 1A and Additional file 1). In contrast, 16 of 29 covS mutations detected in a panel of clinical isolates comprised single (or double) amino acid(s) substitutions (Figure 1B and Additional file 2). Thus, significantly more frameshift mutations were detected in mouse-passaged isolates, whereas point mutations were most frequent in clinical isolates (P < 0.05, Fisher’s exact test).

Assessment of the function of CovS with an amino acid substitution using two-dimensional gel electrophoresis (2-DE)

Theoretically, it is possible that a single amino acid substitution has no effect on CovS function, whereas a large deletion may affect domains that are critical for its function. However, we previously observed that all four clinical isolates (strains GT01, K2, AP04, and AP06) with covS alleles comprising single amino acid substitutions had lower SpeB production than a clinical isolate having the wild-type covS when the culture supernatant proteins were analyzed by 2-DE [22]. It is known that covS positively regulates speB expression [11, 13, 14], which suggests that mutated covS alleles degrade the function of CovS [22]. Thus, we were interested in how the functions of CovS proteins with single amino acid substitutions were degraded in clinical isolates. Thus, we deleted the covS _GT01 allele encoding CovS_GT01A206S (a substitution of Ala206 with Ser) from the GT01 isolate. As shown in Figure 2, the resulting GT01ΔcovS had lower SpeB production than the parental GT01 isolate, suggesting that CovS_GT01A206S did not completely lose its function.

Evaluation of the function of CovS with an amino acid substitution based on its NADase activity

Next, we attempted to complement 1529ΔcovS with wild-type covS ₁₅₂₉ or derivatives, which were cloned into plasmid vector pLZ12-Km2. Wild-type covS ₁₅₂₉ from isolate 1529 was cloned into pLZ-covS₁₅₂₉ and it reduced the NADase activity by 71.5 U from 201.9 U of 1529ΔcovS (pLZ12-Km2: control vector) to 130.4 U of 1529ΔcovS (pLZ-covS₁₅₂₉). In contrast, the NADase activity levels in pLZ-covS₁₅₂₉I30L, pLZ-covS₁₅₂₉E428G, and pLZ-covS₁₅₂₉A206S encoding mutated covS alleles from isolates K2, AP04 (or AP06), and GT01 were reduced by 39.2 U, 17.1 U, and 15.2 U, respectively (Table 1 and Figure 2). Thus, the pLZ-covS₁₅₂₉I30L, the pLZ-covS₁₅₂₉E428G, and the pLZ-covS₁₅₂₉A206S certainly retained their abilities to reduce NADase activity, but the abilities were lower than that of the pLZ-covS₁₅₂₉ with wild-type covS.

These results suggest that amino acid substitutions, such as I30L, E428G, and A206, partially impaired the function of CovS.

Benefits of partially impaired CovS

According to previous studies [10, 16–18], CovS negatively regulates the expression of certain virulent genes; therefore, a mutation in covS may increase the virulence in mouse models of infection where it plays a crucial role in the onset of severe invasive infections. However, the loss of CovS function means that S. pyogenes can no longer adjust to environmental fluctuations. For example, environmental Mg²⁺ is thought to be recognized by the CovS sensor protein [24]. Therefore, the partial loss of CovS function may be favorable in nature, but not under laboratory conditions. This hypothesis led us to investigate the benefits of covS in S. pyogenes. Previously, Trevino et al.[25] showed that a covS mutated strain had a lower growth ability than the parental wild-type strain in human saliva, but not in Todd Hewitt broth, which is the standard broth used to culture S. pyogenes. We were interested in the factor present in human saliva that is recognized by CovS; therefore, we repeated this experiment using the isolates 1529, SF370, and GT01. Bacteria were cultured under essentially the same conditions as those described previously [25]. However, the CFU (colony forming units)/ml for overnight THY broth cultures of strains 1529ΔcovS, SF370ΔcovS, and GT01ΔcovS were lower than or similar to those of their parental strains; i.e., 1529, SF370, and GT01, respectively (Figure 3A), whereas the CFU/ml for overnight THY broth cultures of the isogenic mutant 2221covS::7 bp was four times that of the parental strain MGAS2221 reported in the previous study [25]. Thus, we observed two discrepancies: (i) between our results using isolates 1529, SF370, and GT01 (Figure 3A), and the previous results based on isolate MGAS2221 [25] and (ii) between our results with isolate 1529 (or GT01) and SF370. These discrepancies may be because of strain specificities. We did not have strain MGAS2221, so we further investigated the discrepancy between strains SF370 and GT01 or 1529. First, we analyzed the growth curves of the covS mutated strains. SF370ΔcovS, 1529ΔcovS, and GT01ΔcovS all showed delayed growth compared with that of their parental strains; i.e., SF370, 1529, and GT01, respectively (Figures 4A–C). Thus, there was no discrepancy between strains SF370, GT01, and 1529 in terms of their growth kinetics. In addition, GT01 exhibited delayed and advanced growth compared with strain 1529 (or SF370) and strain GT01ΔcovS (Figures 4D and C), which was consistent with our hypothesis that the A206S substitutions partially impaired the function of CovS.

The growth abilities of SF370 and its isogenic covS mutant SF370ΔcovS in THY broth differed from each other when evaluated on the basis of, but not the CFU/ml in overnight cultures (Figure 3A), the growth curves (Figure 4A). This new discrepancy may have occurred because the overnight culture, but not the growth curve, was conducted in 5% CO₂, which was the condition described in a previous study [25]. Therefore, we prepared overnight cultures of wild-type SF370 (SF370wt) and SF370ΔcovS in natural atmosphere (NA) conditions. As shown in Figure 3B, the CFU/ml for SF370ΔcovS was lower than that of its parental strain SF370. Finally, we performed supplementary and supporting experiments to test the reliability of this study. covS and covRS cloned into a plasmid vector complemented the delayed growth of 1529ΔcovS (Figure 5). pLZ-covS₁₅₂₉ and pLZ-covRS₁₅₂₉ increased the CFU/ml for overnight THY broth cultures of strain 1529ΔcovS (Figure 6). As shown in Figure 7, 1529ΔcovS was hypervirulent in a mouse infection model compared with the parental strain 1529 (P<0.01), as shown with other strains and their isogenic ΔcovS mutants in previous studies [10, 16].

Bacterial strains

Streptococcal strains were isolated as the causative organisms in patients from Japan [22, 23]. S. pyogenes (GAS) strain SF370, which was the most prevalent database reference isolate (accession number NC_002737), was provided by J. J. Ferretti [26, 27]. Streptococcal strains were cultured in brain–heart infusion (E-MC62, EIKEN Chemical Co., Tokyo, Japan) supplemented with 0.3% yeast extract (BD, Sparks, MD, USA), (BHI-Y) broth or Todd Hewitt broth (BD, Sparks, MD, USA) supplemented with 0.2% yeast extract broth (THY) unless otherwise stated.

Production of covS knockout strains

We constructed S. pyogenes strain 1529ΔcovS as described previously [28]. Strains GT01ΔcovS and SF370ΔcovS were constructed using the same strategy [28].

Two-dimensional gel electrophoresis (2-DE)

Each bacterial isolate was cultured in BHI-Y at 37°C overnight without agitation. Exoproteins from the culture supernatant were prepared as described previously [22]. In brief, all sample pellets derived from bacterial culture supernatant were dissolved in dehydration solution, which consisted of 7.8 M urea, 2 M thiourea, 2% CHAPS, 0.6% dithiothreitol, and 0.5% IPG buffer. The samples were loaded onto 13 cm Immobiline DryStrip gels (pH 3–10, GE Healthcare Biosciences Co. Piscataway, NJ, USA). The first-dimensional electrophoresis conditions were carried out according to the manufacture’s instruction. Second-dimensional SDS-PAGE separation was performed as described previously [22]. The experiments were repeated at least 3 times to confirm their reproducibility.

Production of covR knockout strains

To construct the plasmid for the covR knockout mutant, the 5^′ end of covR (fragment 1) was amplified using the oligonucleotide primer cov R-n6 (5^′-GGCTAGCCTTTAGAGAATATGGTTACT-3^′) with an Nhe I restriction site and primer cov R-c2 (5^′-TCCCCCGGGCTTTGTCATTTATACCAACC-3^′) with an Sma I restriction site, while the 3^′ end of covR (fragment 2) was amplified using the primer cov R-n7 (5^′-TCCCCCGGGGAGAAATAAGTCATATGGAA-3^′) with an Sma I restriction site and primer cov S-c10 (5^′-GGACTAGTATGTAAAATTAGAGTCCACC-3^′) with an Spe I restriction site. Fragment 2 was digested with Sma I and Spe I before its insertion into multicloning site 2 in the plasmid pFW12 [29]. The resulting plasmid was digested using Nhe I and Sma I, and the spc1 DNA fragment containing aad9 (promoterless spectinomycin resistance gene), which was obtained from a Sma I-digested fragment of pSL60-1 [29], and the Nhe I-Sma I-digested fragment 1 were inserted. This plasmid, covR::aad9/pFW12, was a suicide vector for S. pyogenes. To prepare competent cells, strains 1529 and GT01 were harvested in the early to mid-log phase (OD₆₆₀, 0.4) and washed twice with 0.5 M sucrose buffer. The suicide vector construct, covR::aad9/pFW12, was transformed into strains 1529 and GT01 via electroporation. The conditions for electroporation were 1.25 kV/mm, 25-μF capacitance, and 200-Ω resistance, and it was performed using a GenePulser II instrument (Bio-Rad, Hercules, CA). After incubation at 37°C for 3 h, competent cells were spread onto BHI agar plates containing 0.3% yeast extract and spectinomycin (final concentration, 100 μg/ml). Selected colonies were cultured from the plates. The cultured bacteria were washed once with saline, resuspended in 10 mM Tris-1 mM EDTA, and boiled for 10 min. Genomic DNA was obtained from the supernatant of the boiled bacteria. The double-crossover replacement was analyzed by PCR using genomic DNA. Successful double-crossover replacement was further confirmed by DNA sequencing.

Quantification of the NADase activity in the bacterial supernatant

NADase activity was determined using the method of Stevens et al. [30] as described previously [31].

Plasmids

pLZ-covS₁₅₂₉, pLZ-covS₁₅₂₉I30L, and pLZ-covS₁₅₂₉E428G were constructed as described previously [22]. To construct pLZ-covS₁₅₂₉A206S, the DNA fragment was amplified using the oligonucleotide primers covR-n2 (5^′-CTTTAGAGAATATGGTTACT-3^′), covS-c2 (5^′-GTAATTACATTTTGGACAAC-3^′), and GT01 genomic DNA as templates with TaKaRa Ex Taq DNA polymerase (Takara, Ohtsu, Japan). The fragment consisted of covR _GT01 , covS _GT01 , and their 5^′-noncoding region, which possibly contained the promoter region. This fragment was cloned into the pGEM-T vector (Promega, Madison, WI, USA). The resultant plasmid was digested with Eco RI and ligated into the same site in the pLZ12-Km2 plasmid [32] (pLZ-covRS_GT01). To construct a plasmid containing only the covS _GT01 region, inverse PCR was conducted using two primers, covR-c2Sma (5^′-TCCCCCGGGCTTTGTCATTTATACCAACC-3^′) and covR-n7Sma (5^′-TCCCCCGGGGAGAAATAAGTCATATGGAA-3^′), with pLZ-covRS_GT01 plasmid DNA as template and Prime-STAR HS DNA polymerase (Takara) to eliminate the covR region. This blunt-ended PCR product was treated with T4 polynucleotide kinase (Takara) and self-ligated. The resultant plasmid was pLZ-covS₁₅₂₉A206S. pLZ-covRS₁₅₂₉ encoding the covRS ₁₅₂₉ operon of isolate 1529 was constructed as described previously [22]. All of the covRS DNA sequences were confirmed by sequencing.

Mouse model of invasive skin tissue infection

All animal studies conducted comply with federal and institutional (the Committee on the Ethics of Animal Experiments of the Nagoya City University) guidelines. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Nagoya City University (Permit Number: H23M-07). All efforts were made to minimize suffering.

The ability of S. pyogenes to cause local skin lesions and necrosis in mice after skin inoculation was assessed using a similar procedure to that described previously [23, 33]. Three-week-old female ICR mice (10–12 g) were anesthetized with sevoflurane and the skin of the left flank was laid bare by separating the hair with an alcohol swab, unless indicated otherwise. Bacteria (0.2 ml; 2 × 10⁷ CFU/mouse) grown in BHI-Y were injected immediately beneath the surface of the skin using a 27-gauge needle so a superficial bleb appeared below the skin surface. The number of CFU injected was verified in each experiment by plating bacteria on BHI-Y or sheep blood agar plates and counting the CFU.

Statistical analysis

The survival times were assessed using a log-rank comparison. The R program was used for the statistical analysis http://bioinf.wehi.edu.au/software/russell/logrank/webcite. P ≤ 0.05 was considered significant.