Partial loss of CovS function in Streptococcus pyogenes causes severe invasive disease
© Tatsuno et al.; licensee BioMed Central Ltd. 2013
Received: 7 January 2013
Accepted: 20 March 2013
Published: 28 March 2013
CovRS (or CsrRS) is a two-component regulatory system that regulates the production of multiple virulence factors in Streptococcus pyogenes. covS mutations are often found in isolates recovered from mice that have been experimentally infected with S. pyogenes and covS mutations enhance bacterial virulence in an invasive infection mouse model. In addition, covS mutations were detected more frequently in a panel of clinical isolates from severe invasive streptococcal infections than those from non-severe infections. Thus, covS mutations may be associated with the onset of severe invasive infections.
Known covS mutations were divided into two groups: (i) frameshift mutations that caused a deletion of functional regions and (ii) point mutations that caused single (or double) amino acid(s) substitutions. Frameshift mutations are frequent in mouse-passaged isolates, whereas point mutations are frequent in clinical isolates. The functions of CovS proteins with a single amino acid substitution in clinical isolates were estimated based on the streptococcal pyrogenic exotoxin B (SpeB) production and NAD+-glycohydrolase (NADase) activity, which are known to be regulated by the CovRS system. Point mutations partially, but not completely, impaired the function of the covS alleles. We also investigated some of the benefits that a partial loss of function in covS alleles with point mutations might confer on clinical isolates. We found that covS knockout mutants (ΔcovS strains) had an impaired growth ability in a normal atmosphere in Todd Hewitt broth compared with parental isolates having wild-type or point-mutated covS.
The loss of CovS proteins in S. pyogenes may confer greater virulence, but bacteria may also lose the ability to respond to certain external signals recognized by CovS. Therefore, point mutations that retain the function of CovS and confer hypervirulence may have natural selective advantages.
Streptococcus pyogenes is a Gram-positive bacterium that infects the upper respiratory tract, including the tonsils and pharynx, which is responsible for post-infection diseases such as rheumatic fever and glomerulonephritis. S. pyogenes also causes severe invasive diseases including necrotizing fasciitis [1–5].
S. pyogenes is exclusively a human pathogen and it possesses many virulence factors that help it to resist host defense systems. The production of these factors is thought to be precisely regulated in response to host environmental conditions such as different infection sites or host immune system induction levels [6–8]. In prokaryotes, the regulation of protein production in response to fluctuating environmental conditions depends primarily on two-component regulatory systems, which consist of a sensor histidine kinase and its cognate response regulator . Thirteen two-component regulatory systems have been described in S. pyogenes, of which the CovRS system (also known as the CsrRS system) mediates the control of several virulence factors [10–15]. Specific isolates from mice infected with S. pyogenes exhibited enhanced virulence in mice owing to spontaneous covR or covS mutations [10, 15, 16]. In addition, covS mutations were detected more frequently in a panel of clinical isolates from severe invasive streptococcal infections than in a panel of clinical isolates from non-streptococcal toxic shock syndrome [10, 16–18]. Thus, Ikebe et al.  suggested that covS mutations are closely associated with the onset of streptococcal toxic shock syndrome.
The strains used for experimental murine infections [10, 15, 16] and clinical isolates  frequently have the M1 serotype, which is the most widely disseminated global serotype [19–21]. Engleberg et al. showed that most covS mutations were frameshift or nonsense mutations in isolates from mice infected with the M1 strain. In contrast, all of the spontaneous changes in CovS detected in clinical M1 isolates  resulted from single amino acid substitutions. Thus, we were interested in why this difference occurred and we hypothesized that it was related to the use of animal-passaged isolates in the first study whereas the latter used clinical isolates. Several covS mutations have been reported in other studies [10, 16, 18] in addition to the two mentioned previously [15, 22]. In the current study, we first reviewed the different types of covS mutations. This suggested that most of the spontaneous changes in CovS detected in clinical M1 isolates resulted from single amino acid substitutions, whereas most of the covS mutations detected in animal-passaged isolates were frameshift mutations. We also showed that covS mutations comprising single amino acid substitutions in the clinical isolates partially, but not completely, impaired the functions of CovS. Finally, we present some new findings and discuss why covS mutations in clinical isolates are preferentially single amino acid substitutions, whereas animal-passaged isolates tend to have frameshift mutations.
Results and discussion
Classification of covS mutations
Assessment of the function of CovS with an amino acid substitution using two-dimensional gel electrophoresis (2-DE)
Evaluation of the function of CovS with an amino acid substitution based on its NADase activity
NADase activity of S. pyogenes strains
3.4 ± 0.7
93.5 ± 3.5
62.9 ± 4.6
57.0 ± 3.6
114.3 ± 8.7
59.8 ± 2.6
60.5 ± 5.4
59.4 ± 4.8
106.7 ± 3.7
105.0 ± 3.2
103.5 ± 6.7
201.9 ± 2.8
130.4 ± 3.4
176.7 ± 8.9
1529ΔcovS (pLZ-covS1529 H437R)
114.5 ± 6.8
162.7 ± 11.0
184.8 ± 6.9
186.7 ± 4.2
2.4 ± 0.16
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
On the basis of our results and those from previous studies we concluded that the loss of covS increases the virulence of S. pyogenes (which is advantageous in vivo). However, the loss of covS also impaired the growth ability of this organism in THY broth (which is disadvantageous in vitro). Therefore, the CovRS system may confer benefits in stages when virulent gene expression is not required. The expression of many genes is precisely regulated so they are expressed only when required; e.g., catabolite repression. Therefore, partial attenuation of the CovRS system to promote resistance to the host defense system appears to be a wise choice for survival in nature.
We did not determine the components recognized by the CovS sensor proteins in our experimental conditions; i.e., THY broth, natural atmosphere, or 5% CO2. However, it was probably not the Mg2+ ion, which was suggested previously , because we did not add Mg2+ ion to THY broth. Therefore, we propose that CovS can sense other signals in addition to the Mg2+ ion.
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
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 . 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 . 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 . 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 , 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
pLZ-covS1529, pLZ-covS1529I30L, and pLZ-covS1529E428G were constructed as described previously . 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  (pLZ-covRSGT01). 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-covRSGT01 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 . 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.
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.
Availability of supporting data
There are two supplementary tables.
Todd Hewitt yeast.
We thank Hideyuki Matsui for technical assistance, and Drs. M. Ohnishi, M. Ato, T. Ikebe for their helpful advices. This study was supported by JSPS KAKENHI Grant number 21790425 and 24590531, a grant from Ohyama Health Foundation, and a grant from the 24th General Assembly of the Japanese Association of Medical Sciences (Medical Science Promotion Fund). The authors would like to thank Enago (http://www.enago.jp) for the English language review.
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