emm1/Sequence Type 28 Strains of Group A Streptococci That Express covR at Early Stationary Phase Are Associated with Increased Growth and Earlier SpeB Secretion

Bacterial strains.The GAS clinical isolates analyzed in this study were randomly selected from the collection consecutively archived between 1994 and 2003 at National Cheng Kung University Hospital, Tainan, Taiwan (Table 1). GAS strain NZ131 (M49) was described previously (43). SF370 (M1T1) is strain ATCC 700294. All GAS strains were grown in tryptic soy broth (Becton, Dickinson and Company, Sparks, MD) supplemented with 0.5% yeast extract (TSBY).

RNA extraction and Northern hybridization.RNA extraction from GAS was performed by the method described previously (12). Briefly, the bacterial pellet was washed with ice-cold 0.2 M sodium acetate twice and resuspended in buffer (100 mM of Tris-HCl [pH 7.0], 1 mM EDTA, 25% [wt/vol] glucose) containing 40 μl of lysozyme (20 mg/ml) and 10 μl of mutanolysin (1 U/μl; Sigma Chemicals, St. Louis, MO) and then incubated at 37°C for 1 h. After incubation, bacterial pellets were collected and resuspended in 500 μl of acetic buffer (20 mM sodium acetate [pH 5.5], 1 mM EDTA, 0.5% [wt/vol] sodium dodecyl sulfate) with 500 μl of hot phenol. After vortexing, the mixtures were centrifuged at 14,000 × g for 20 min. The upper aqueous layer was collected, and RNAs were precipitated with isopropanol. RNAs were dissolved in diethyl pyrocarbonate-H₂O and treated with DNase (Promega, Madison, WI) at 37°C for 2 h. After DNase treatment, RNAs were obtained by using phenol-chloroform extraction and isopropanol precipitation. Northern hybridization analysis was performed as described previously (12). The probes used for detecting covR, speB, sagA, emm, and scpA expression are described in Table 2. After hybridization, the membranes were washed and the hybridized RNA transcripts were detected by a LAS3000 Intelligent Dark Box (Fujifilm, Japan). All Northern hybridization analyses were performed at least twice independently.

Western hybridization.Three hundred microliters of bacterial culture supernatant was mixed with 1.2 ml of acetone and incubated at −20°C for 1 h to precipitate the total protein. The precipitated protein was collected by centrifugation at 14,000 × g for 20 min at 4°C. The protein pellet was dissolved in 50 mM NaOH, and the protein concentration was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Two micrograms of total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to polyvinylidene difluoride membranes. The membrane was blocked with phosphate-buffered saline (PBS) containing 5% (wt/vol) skim milk at 37°C for 2 h and probed with anti-C192S mouse serum prepared as previously described (28). After three washes with PBS containing 0.05% Tween 20 (PBST), the membrane was incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody (Chemicon International, Inc., Temecula, CA). After incubation, the membrane was washed with PBST solution three times and then was treated with chemiluminescent substrate (Pierce ECL Western blotting substrate; Thermo Fisher Scientific, Inc., Rockford, IL) to visualize the signal. The signal was detected with the LAS 3000 Intelligent Box (Fujifilm, Japan). All Western hybridization analyses were performed at least twice independently.

Bacterial growth curve with kinetic parameters.To obtain bacterial growth curves, GAS strains were cultured in TSBY broth at 37°C for 16 h. One milliliter of overnight culture was transferred to 100 ml of fresh TSBY and incubated at 37°C without agitation. The optical density of the bacterial culture at 600 nm (OD₆₀₀) was measured by UV/visible spectrophotometer (GE Healthcare, United Kingdom) after 3 to 17 h of incubation.

The bacterial growth curve was fitted by using a modified logistic model to estimate the growth parameters asymptotes (A), maximum specific growth rate (μ_m), and lag time (λ), as described by Zwietering et al. (47). A is defined as the maximal value of bacterial growth, μ_m is defined as the tangent in the inflection point of the bacterial growth curve, and λ is defined as the x-axis intercept of this tangent. Experiments in duplicate were performed, and the averages were used for estimating growth parameters.

emm typing and PFGE typing. emm typing and PFGE typing were performed as previous described (46). The PFGE fragment patterns were interpreted in accordance with the criteria of Tenover et al. (41). PFGE fragment patterns were compared with the use of GelCompar II software (Unimed Healthcare, Inc., Houston, TX), by using the unweighted pair group method using average linkages (UPGMA) based on the Dice coefficient with a position tolerance of 1.5%. PFGE-based clusters were defined as isolates with a genetic relatedness of >80% on a dendrogram.

MLST.GAS genomic DNA was prepared as previously described (12). Seven housekeeping genes of GAS (gki, gtr, murI, mutS, recP, xpt, and yiqL) were amplified and sequenced using the primers and conditions described in the MLST database (www.mlst.net ). New housekeeping allele sequences and STs generated from this study were submitted to the MLST database. To generate dendrograms, sequences of the seven housekeeping genes used in the MLST scheme were joined into a 3,134-bp concatenated sequence and a dendrogram was constructed from concatenated sequences by the UPGMA method and the Kimura two-parameter nucleotide substitution model with 1,000 bootstrap trials (25). A clonal cluster was defined by bootstrap value of >95 and further verified by the eBURST program (eburst.mlst.net ).

Statistics.Statistical analysis was performed by using Student's t test. A P value of <0.05 was taken as significant.

covR expression pattern in SF370 and NZ131.To investigate the covR expression pattern in SF370 (emm1) and NZ131 (emm49) during bacterial growth, the RNAs were extracted from SF370 and NZ131 at different phases of growth and covR expression was analyzed by Northern hybridization with an alkaline phosphatase-labeled covR probe. covR and covS consist of an operon, the covR- and covS-cotranscribed RNA (covRS), and covR monocistronic transcripts (covR) were detected by a covR probe (Fig. 1A) (12). The results showed that SF370 had maximal covR RNA expression at the early stationary phase (after 5 h of incubation), whereas NZ131 had its maximal covR expression in the exponential phase of growth (Fig. 1A and B). No difference was found in covRS-cotranscribed RNA between SF370 and NZ131 (Fig. 1A).

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FIG. 1.

covR expression patterns and growth rates in SF370 and NZ131. (A) covR expression pattern during bacterial growth in SF370 and NZ131. The RNAs were extracted after 3, 5, and 7 h of incubation, and covR expression in SF370 and NZ131 was analyzed by Northern hybridization with an alkaline phosphatase-labeled covR probe. covR/S represents the covRS-cotranscribed RNA; covR represents the covR monocistronic transcripts. The lower panels show the 23S rRNA and 16S rRNA as the internal controls. (B) Growth curves of SF370 and NZ131. The bacterial density was measured by spectrophotometer at OD₆₀₀ after 3 to 17 h of incubation. The data presented are from three independent experiments.

Two different covR expression patterns are distributed in GAS.Nineteen clinical strains collected from 1994 to 2003 were analyzed for their covR expression pattern by Northern hybridization with an alkaline phosphatase-labeled covR probe. Bacteria were cultured in TSBY broth, and covR expression was analyzed after 3, 5, and 7 h of incubation. We found nine strains expressed covR RNA maximally at the early stationary phase (covR-ST), and 10 strains expressed covR RNA at the exponential phase (covR-E). Among the covR-E strains, six different emm types were found (two of emm4, four of emm12, one of emm22, one of emm73, one of emm89, and one of emm95). All of the emm1 strains were the covR-ST phenotype. In addition, one emm75 strain and one emm89 strain were the covR-ST type (Table 1).

The growth curves of the covR-E and covR-ST strains.SF370 and NZ131 expressed covR RNA at different phases of growth (Fig. 1A). In addition, the growth curves of SF370 and NZ131 had different characteristics (Fig. 1B). Apparently, SF370 had the better growth activity and spent less time reaching the stationary phase than did NZ131 (Fig. 1B). To further demonstrate that the covR expression pattern was associated with bacterial growth activity, we analyzed the growth curves of 21 strains (SF370, NZ131, and 19 clinical isolates) (Table 1). Bacteria were cultured in TSBY broth at 37°C, and the bacterial density was determined by spectrophotometer at 600 nm after 3, 5, 7, and 9 h of incubation. The average of the bacterial density of covR-E or covR-ST strains at each time point was calculated and plotted on a chart to represent the growth curve of covR-E and covR-ST strains (Fig. 2A). This chart showed that the covR-ST strains had faster growth and reached the stationary phase faster than did the covR-E strains (Fig. 2A).

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FIG. 2.

Growth curve characteristics of strains exhibiting two different covR expression patterns. Strains having covR expressed at the exponential phase are defined as the covR-E strains, and those with covR expressed at the early stationary phase are defined as the covR-ST strains. (A) Growth curves of the covR-E and covR-ST strains. The bacterial density (measured by spectrophotometer at OD₆₀₀) of the covR-E or covR-ST strains after 3, 5, 7, and 9 h of incubation was recorded, and the average value was calculated. The bacterial growth curve was plotted according to the calculated average values. Data for the growth kinetics for each strain were from two independent experiments. (B) Maximal specific growth rate (μ_m) of the covR-ST and covR-E strains. The mathematic model of bacterial growth curve was described in Materials and Methods. Statistical analysis was performed with Student's t test. *, P < 0.05.

To demonstrate the growth curve characteristics mathematically, we used the mathematic model to analyze the bacterial growth kinetics of our clinical isolates. The results showed the lag time (λ of 4.096 for covR-E strains and 4.234 for covR-ST strains) and the maximal value of bacterial growth (A of 1.977 for covR-E strains and 1.947 for covR-ST strains) were no different between covR-E and covR-ST strains (P > 0.5). However, the maximum specific growth rate (μ_m) of the covR-ST strains was significantly higher than that of the covR-E strains (Fig. 2B) (P < 0.05).

speB and sagA expression in covR-ST and covR-E strains.Gene expression in GAS is controlled by growth-phase regulation (9, 43). The transcription of speB and sagA is restricted to the stationary phase, and expression of emm and scpA is restricted to the exponential phase. We hypothesized that the covR-ST strains, which had better growth activity, would express speB and sagA genes earlier than did the covR-E strains. To verify this hypothesis, SF370 and A-20 (covR-ST strains) and NZ131 and GAS516 (covR-E strains) were cultured for 6 to 10 h in TSBY broth and speB and sagA transcripts were analyzed by Northern hybridization with the alkaline phosphatase-labeled speB and sagA probes, respectively. The results showed that speB expression in SF370 was detected after 6 h of incubation, whereas speB expression in NZ131 was only detected after 10 h of incubation (Fig. 3A). Similarly, A-20 speB expression was detected after 6 h of incubation, and GAS516 speB expression was detected after 8 h of incubation (Fig. 3B). However, the sagA expression pattern in these strains was similar. Expression was detected after 6 h of incubation and increased after 8 and 10 h of incubation in SF370, NZ131, and GAS516 (Fig. 3C).

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FIG. 3.

speB and sagA expression patterns in covR-ST and covR-E strains. (A and B) speB expression pattern during bacterial growth. (C) sagA expression pattern during bacterial growth. RNAs were extracted after 6, 8, and 10 h of incubation, and speB and sagA expression was analyzed by Northern hybridization with the alkaline phosphatase-labeled speB and sagA probes, respectively. SF370 and A-20 are the covR-ST strains, and NZ131 and GAS516 are the covR-E strains. The lower panels show 23S rRNA and 16S rRNA as the internal controls. (D) SpeB protein in culture supernatant of SF370 (upper panel) and NZ131 (lower panel). The total proteins were collected from bacterial culture supernatant at different culture time points. The SpeB protein in culture supernatant was analyzed by Western hybridization with anti-C192S (SpeB protease mutant recombinant protein) mouse serum. The zymogen form of SpeB is 42 kDa, and the mature form of SpeB is 28 kDa.

To further clarify the speB expression pattern in SF370 and NZ131, SpeB protein secretion was detected by Western hybridization. SF370 and NZ131 were cultured in TSBY broth for 3 to 20 h, and SpeB protein in culture supernatant was analyzed by Western hybridization with anti-C192S mouse serum (C192S is a protease-negative recombinant SpeB protein). The results showed that the zymogen form of SpeB protein (42 kDa) was detected in SF370 culture supernatant after 8 h of incubation, whereas it was detected after 12 h of incubation in NZ131 (Fig. 3D). In addition, after 12 h of incubation, SF370 accumulated more SpeB protein in culture supernatant than did NZ131 (Fig. 3D).

emm and scpA expression in covR-E and covR-ST strains. emm and scpA expression was restricted to the exponential phase of growth. We also hypothesized that covR-ST strains expressed these genes in a more restricted pattern during bacterial growth than did covR-E strains. The SF370 and A-20 strains (covR-ST) and NZ131 (covR-E) were cultured for 3 to 9 h, and emm and scpA transcripts were analyzed by Northern hybridization with alkaline phosphatase-labeled emm1 and scpA probes, respectively. The results showed that SF370, A-20, and NZ131 maximally expressed emm and scpA genes at 5 h of incubation and decreased expression after 7 h of incubation (data not shown).

MLST analysis.We found that all emm1 strains expressed covR at the early stationary phase of growth. Therefore, we hypothesized that the strains expressing covR at the early stationary phase were clonally related. To verify this hypothesis, MLST analysis was used to analyze all 21 strains. The allelic profile, emm type, and epidemiologic information of strains in this study were posted on the MLST database (www.mlst.net ). Four new MLST STs (465, 466, 467, and 468) were found in the present study. Thirteen STs were found in 21 GAS strains. Among them, all emm1 strains were ST28 (Table 1). Strains 663 and 276, which expressed covR at the early stationary phase, were ST468 and ST384, respectively.

The MLST-based dendrogram analysis showed four major clonal clusters (Fig. 4). Eight emm1 strains with identical STs (ST28) were defined as cluster 1 (Fig. 4). Cluster 2 and cluster 3 were composed of four emm12 strains with three different STs (36, 465, and 467) and two emm4 strains with two different STs (391 and 39), respectively (Fig. 4). L257 (emm95/ST400) was cluster 4. Furthermore, strains that shared more than five identical housekeeping loci were classified into one clonal cluster by eBURST analysis. eBURST analysis showed similar classification results except for cluster 4 (L257; data not shown). Six strains (276, 625, 516, 663, L259, and NZ131) were singletons (Fig. 4). Other than strains 276 and 663, clusters 2, 3, and 4 and other singleton strains all expressed covR at the exponential phase of growth (Fig. 4).

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FIG. 4.

MLST-based dendrogram of 21 GAS strains and their STs and emm types. The dendrogram was generated by comparing the concatenated housekeeping genes sequence by UPGMA with the Kimura two-parameter method. A clonal cluster (no. 1 to 4) was verified by a bootstrap value of >95 and eBURST analysis. The value shown on nodes of the branches is the bootstrap value. The scale bar represents the sequence similarity. Asterisks indicate the strains with covR expression at the early stationary phase of growth.

PFGE type analysis.A PFGE-based dendrogram showed four major clusters (Fig. 5). The largest cluster (cluster P2) comprised 6 emm1/ST28 strains. Another emm1/ST28 strain, L395, has >75% genetic relatedness to cluster P2 strains (Fig. 5). Strains SF370 (emm1/ST28), 276 (emm89/ST384), and 663 (emm75/ST468), expressed covR at the early stationary phase and were not related to any specific clusters. Clusters P1 and P4 comprised two emm4 and three emm12 strains, respectively (Fig. 5). Cluster P3 comprised emm73 and emm95 strains with different STs. Except for cluster P2 strains, which expressed covR at the early stationary phase, cluster P1, P3, and P4 strains all expressed covR at the exponential phase of growth (Table 1).

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FIG. 5.

Dendrogram and PFGE band patterns of SmaI-digested chromosomal DNA of 21 GAS isolates. The dendrogram was constructed by using UPGMA based on the Dice coefficient with a position tolerance of 1.5%. Clusters P1 to P4 were defined by genetic relatedness of >80% on a dendrogram. Asterisks indicate the strains with covR expression at the early stationary phase.