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FEMS Microbiol Lett. Author manuscript; available in PMC 2009 July 1. Published in final edited form as: doi: 10.1111/j.1574-6968.2008.01171.x. | PMCID: PMC2561273 NIHMSID: NIHMS55793 |
Inter- and intraserotypic variation in the Streptococcus pyogenes Rgg regulon Alexander V. Dmitriev,1,2 Emily J. McDowell,1 and Michael S. Chaussee1 1 Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, Vermillion, South Dakota, USA 2 Department of Molecular Microbiology, Institute of Experimental Medicine, Saint-Petersburg, Russia Human isolates of Streptococcus pyogenes, a Gram-positive bacterium, are characterized by significant genetic and phenotypic variation. The rgg locus, also known as ropB, is a global transcriptional regulator of genes associated with metabolism, stress responses, and virulence in S. pyogenes strain NZ131 (serotype M49). To assess the breadth of the Rgg regulon, the rgg gene was inactivated in three additional strains representing serotypes M1 (strains SF370 and MGAS5005) and M49 (strain CS101). Changes in gene expression were identified in the post-exponential phase of growth using Affymetrix NimbleExpress Arrays. The results identified an Rgg core-regulon consisting of speB and adjacent hypothetical protein gene, spy2040, and a variable and strain-specific sub-regulon, ranging in size from a single gene (spy1793) in strain MGAS5005 to 43 genes in strain SF370. Thus, both interserotypic and intraserotypic variation is characteristic of the Rgg regulon in S. pyogenes. Keywords: Streptococcus pyogenes, Rgg, transcriptional regulation Streptococcus pyogenes (group A streptococcus) is a Gram-positive pathogen that exhibits significant phenotypic diversity, which is likely to contribute to the wide variety of clinical outcomes associated with human infection. The clinical severity of infection ranges from asymptomatic colonization to severe invasive diseases, such as streptococcal toxic shock syndrome and necrotizing fasciitis. Moreover, post-infection sequelae including rheumatic fever, glomerulonephritis, and neurological disorders contribute substantially to the morbidity and mortality associated with the pathogen (Cunningham, 2002). Clinical isolates of S. pyogenes are differentiated into more than 100 emm-types based on variability of the 5′-region of the emm gene ( http://www.cdc.gov/ncidod/biotech/strep/M-ProteinGene_typing.htm), which encodes the LPXTG-anchored adhesin and antiphagocytic M protein ( Fischetti, 1989). More generally, clinical isolates can be differentiated into two classes (Class I and Class II) based on reactivity with antibodies directed against the C repeat region of the M protein and the organization and composition of genes proximal to emm in the chromosome. For example, class I, but not class II, strains possess the gene encoding the streptococcal inhibitor of complement (SIC), which contributes to virulence in murine models ( Lukomski et al., 2000). Class II, but not class I, strains possess the gene encoding serum opacity factor (SOF), which also contributes to virulence and promotes adherence ( Timmer et al., 2006). Class I strains are associated with invasive diseases and acute rheumatic fever, while class II strains are associated with pyoderma and acute glomerulonephritis ( Cunningham, 2000). Although differences in the host response are clearly important, specific strains of S. pyogenes are more likely to cause certain diseases than others, which indicates that strain-variable genetic elements contribute to the disease process. The complete genome sequences of more than 12 strains of S. pyogenes have been determined. These include isolates from invasive disease episodes ( Ferretti et al., 2001; Beres et al., 2006), rheumatic fever ( Smoot et al., 2002; Holden et al., 2007), and puerperal sepsis ( Green et al., 2005). The genome content of the strains is heterogeneous, mostly due to various bacteriophages ( Banks et al., 2002) and integrated conjugative elements ( Beres & Musser, 2007). These strain-variable elements contribute to the so-called pan-genome ( Tettelin et al., 2005), or metagenome ( Beres & Musser, 2007), which is estimated to consist of approximately 2,500 genes ( Lefébure & Stanhope, 2007). Rgg, also known as RopB, ( Chaussee et al., 1999; Lyon et al., 1998) is one member of a family of transcriptional regulators (TIGR01716) encoded in the genomes of some species of low G+C Gram-positive bacteria. Inactivation of rgg in the class II strain NZ131 (serotype M49) is associated with changes in the transcript levels of 706 genes compared to the parental wild-type strain ( Dmitriev et al., 2006). Many of these genes encode known or putative virulence factors, such as the hyaluronic acid capsule, C5a peptidase, streptokinase, streptolysins S and O, and mitogenic factor ( Chaussee et al., 2001; Dmitriev et al., 2006). Corresponding phenotypic differences identified in the mutant strain include: (i) tolerance to penicillin-mediated killing and thermal and oxidative stressors ( Chaussee et al., 2004; Chaussee et al., 2006); (ii) catabolism of arginine during the exponential phase of growth in the presence of glucose ( Chaussee et al., 2003); (iii) increased production of DNase, NADase, and SLO; (iv) an inability to grow in chemically defined media (CDM) containing sucrose, fructose or mannose as the primary carbon source; and (v) decreased frequency of prophage NZ131.1 induction ( Dmitriev et al., 2006). Thus, Rgg is an important global regulator of genes associated with metabolism, stress response, and virulence in strain NZ131. The purpose of this study was to assess the breadth of the Rgg regulon in additional isolates of S. pyogenes including two M1 serotypes representing class I strains and an additional M49 serotype representing class II strains. DNA microarrays identified an Rgg core-regulon and a variable and strain-specific sub-regulon, which suggests that diversity among regulatory circuits contributes to the phenotypic diversity of S. pyogenes. Bacterial strains, plasmids, and growth conditions The wild-type S. pyogenes strains MGAS5005, SF370, and CS101 have been described ( Ferretti et al., 2001; Sumby et al., 2005; Haanes et al., 1992). S. pyogenes was grown at 37 °C in a 5% CO 2 atmosphere without agitation in either Todd-Hewitt broth (Becton Dickinson, Sparks, MD) containing 0.2% (wt/vol) yeast extract or CDM ( Dmitriev et al., 2006). Escherichia coli strain DH5α was purchased from Gibco-BRL (Gaithersburg, MD), and the suicide cloning vector pVA891-2 was kindly provided by H. Malke ( Malke et al., 1994). DNA techniques Plasmid DNA was isolated from E. coli using Plasmid Midi Kit (QIAGEN, Valencia, CA). From agarose gels, DNA was isolated using the QIAquick PCR Purification Kit (QIAGEN). PCR products were purified using DNA Clean & Concentrator (Orange, Calif.). Most of other routine molecular techniques were done as previously described ( Sambrook et al., 1989). Insertional inactivation of rgg The rgg gene was insertional inactivated in each strain as previously described ( Chaussee et al., 1999). Briefly, the entire rgg gene was amplified using the primers RGG+1 (5′-CTG GAG CTG TTG AGA TAA ACT AC-3′) and RGG-4 (5′-GGC TAT TGA CCT TAT GCA CC-3′), and digested with EcoRI and HindIII, which had corresponding restriction sites within rgg. The resulting 592 bp fragment was cloned into the vector pVA891-2. Following E. coli transformation, the recombinant plasmid was isolated and used to transform S. pyogenes. Transformants were selected on agar plates containing 2.5 μg mL −1 of erythromycin. Insertional inactivation was confirmed in each strain by nucleotide sequencing and Southern blotting, as previously described ( Chaussee et al., 1999). The heterologous DNA inserted into the rgg locus was identical in all three rgg mutant strains and identical to the previously described NZ131 rgg mutant ( Chaussee et al., 1999). Pulsed field gel electrophoresis (PFGE) analysis Chromosomal DNA for PFGE analysis was isolated and digested with SmaI as described ( Elliott et al., 1998). Fragments were separated in 1% agarose gel in 0.5 × TBE buffer (GeneLine apparatus, Beckman Instruments, Calif.) using the following conditions: 5 min, 170 V, 5 s pulse time; 16 h, 200 V, 40 s pulse time; and 8 h, 200 V, 8 s pulse time, and visualized with ethidium bromide, 0.5 μg mL −1. DNA microarray analysis RNA was isolated from 40 ml cultures of S. pyogenes in the post-exponential phase of growth () with an RNeasy Mini Kit (QIAGEN). Affymetrix NimbleExpress Arrays designed based on the S. pyogenes strain SF370 genome sequence ( Ferretti et al., 2001) were purchased from Affymetrix (Santa Clara, CA). In addition to the SF370 genome sequence, 660 known streptococcal bacteriophage genes were included in the array platform. Together, the arrays consisted of 3,203 qualifiers representing 2,354 predicted S. pyogenes ORFs, 804 intergenic region probes, and 45 control oligos used for spike-ins. Microarray hybridization and analysis of the data was done as recently published ( Dmitriev et al., 2006). The average signal intensity value of each gene was transformed to a log 2 (log base 2) value. The change between two experimental conditions ( n-fold) was calculated by taking the ratio of the signal intensity (difference of the log 2 value) between experimental conditions. Present and absent calls were assessed and statistically significant genes (T-test; P value ≤ 0.05) were considered to be differently expressed. All of the microarray data are available through the Gene Expression Omnibus data repository via accession numbers GSE 7335 and 7341. Determination of SpeB, NADase, DNase and SLO cytolytic activities Quantitative reverse transcription (RT)-PCR Oligonucleotide primers and TaqMan probes used in the study were previously published ( Dmitriev et al., 2006). In addition, primers and fluorescent probes for the phosphoribosylglycinamide formyltransferase purN gene (5′-CTT GGC CTA TGA GAG GCG TAT T-3′, 5′-CCG TGG GCA CCT GGA A-3′, and 5′-TCA ATA TTC ACC CAG CCT ACC TGC CTG AA-3′) and the streptolysin S sagA gene (5′-TTG CTC CTG GAG GCT GCT-3′, 5′-CTT CCG CTA CCA CCT TGA GAA T-3′, and 5′-ACC ACT TCC AGT AGC AAT TGA GAA GCA ACA AG-3′) were designed with Primer Express 2.0 software (ABI Prism, PE Biosystems, Framingham, Mass.) and purchased from Sigma-Genosys (The Woodlands, TX). Amplification and detection were done with the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems) using TaqMan One-Step RT-PCR Master Mix reagents (Roche, Indianapolis, In.), as previously described ( Chaussee et al., 2003). Sequence analysis The rgg genes from S. pyogenes strains MGAS5005, SF370, and CS101 were sequenced using ABI Prism 377 Perkin-Elmer Sequencer and Big Dye Terminator Kit (Applied Biosystems) with the primers RGG+1 and RGG-4. The data were deposited into the GenBank database under accession numbers DQ009036 and DQ176644. The deduced Rgg proteins of all three strains were identical to that of the NZ131 strain. The 498 bp regions upstream the rgg genes sequenced with the primers 5′-CGG CAA ATA CTG GGT TAG CAA GA-3′ and 5′-GGA TGC CTA ATG AAT TCA ACG GTT T-3′ were also identical in all the strains. Expression of virulence factors in the rgg mutant strains To assess the Rgg regulon in S. pyogenes, three widely studied strains, representing both class I (MGAS5005 and SF370, serotypes M1) and class II (CS101, serotype M49) organisms, were selected for study. Inactivation of rgg in strains CS101, MGAS5005, and SF370 as described in the Materials and Methods, abrogated SpeB expression (), similar to previously described mutants created in strains NZ131 and HSC5 ( Chaussee et al., 1999; Lyon et al., 1998). Southern blotting showed that the heterologous DNA inserted into only the rgg locus (data not shown). These results indicate that Rgg-dependent activation of speB expression is conserved among both class I and II clinical isolates of S. pyogenes. The SLO, NADase, and DNase activities, which were elevated in the NZ131 rgg mutant strain ( Dmitriev et al., 2006), were similarly measured in strains MGAS5005, SF370, and CS101 and the corresponding rgg mutant derivatives. Despite variation in SLO, NADase, and DNase activities among the wild-type strains, no difference between wild-type and corresponding rgg mutant strains was observed (data not shown). Growth of wild-type and the rgg mutant strains in Todd-Hewitt broth and CDM Inactivation of rgg in strain NZ131 is associated with altered growth in Todd-Hewitt broth and an inability to use non-glucose carbohydrates as the primary carbon and energy sources when grown with CDM ( Chaussee et al., 2003; Dmitriev et al., 2006). To determine if similar changes occurred in strains CS101, MGAS5005, and SF370, the growth of wild-type strains and mutant derivatives was examined. Growth of wild-type CS101 and MGAS5005 strains was identical to the corresponding mutant strains in both Todd-Hewitt broth () and CDM containing 2% glucose, sucrose or fructose (data not shown). In contrast, the SF370 rgg mutant strain had a slightly shorter lag period and different growth yield compared to the parental strain when grown with Todd-Hewitt broth () or CDM containing various carbohydrates as the primary carbon and energy source (). Thus, while there was a minor effect on growth in strain SF370 following rgg inactivation, mutant strains retained the ability to use sucrose, glucose and fructose as the primary carbon and energy source during growth with CDM, in contrast to results previously obtained with strain NZ131 ( Dmitriev et al., 2006). DNA microarray analysis of the wild-type and rgg mutant strains To identify Rgg-regulated genes, the transcriptomes of CS101, MGAS5005, and SF370 wild-type and rgg mutant strains were analyzed. Our initial experiments revealed few differences in the transcriptomes of MGAS5005 rgg mutant and CS101 rgg mutant compared to corresponding wild-type strains during exponential phase (data not shown). These data are in agreement with earlier observations in strain NZ131, in which rgg inactivation alters a larger number of genes during the post-exponential phase of growth compared to the exponential phase ( Dmitriev et al., 2006). Therefore, to identify the largest number of Rgg-regulated genes in MGAS5005, CS101, and SF370 strains, RNA was isolated from post-exponential phase cultures and gene transcripts were measured with Affymetrix NimbleExpress Arrays. Inactivation of rgg in strains MGAS5005, CS101, and SF370 was associated with a 2-fold or more ( P ≤ 0.05) change in the expression of 3, 13, and 45 loci, respectively ( Table 1). In strain MGAS5005, 1 and 2 gene transcripts were more abundant and less abundant, respectively, in the mutant strain compared to the wild-type strain. In strain CS101, 10 and 3 gene transcripts were more and less abundant, respectively, in the mutant strain compared to the wild-type strain. Finally, in strain SF370, 28 and 17 gene transcripts were more abundant and less abundant, respectively, in the mutant strain compared to the wild-type strain ( Table 1). The known or putative functions of the majority of Rgg-regulated genes in all of the strains were associated with replication, transcription and translation, and metabolism. Quantitative RT-PCR was used to validate the microarray data ( Table 2). | Table 1Transcriptome changes (2-fold or greater; P values ≤ 0.05) associated with rgg nactivation during post-exponential growth |
| Table 2Correlation between results obtained with DNA microarrays and quantitative TaqMan RT-PCR |
Surprisingly, the only genes similarly affected by rgg inactivation in all three strains were speB and spy2040, which are co-transcribed ( Neely et al., 2003; Ma et al., 2006). In addition, only the expression of purine metabolism genes ( spy25- spy28) in strain SF370 and the peptidyl prolyl isomerase gene ( prsA) in strain CS101 were altered by more than 5-fold following rgg inactivation ( Table 1). The results indicate that Rgg influences transcription in strains MGAS5005, SF370, and CS101 during the post-exponential phase of growth; however, the magnitude and extent of transcriptional changes is not nearly as great as those previously identified in strain NZ131 ( Dmitriev et al., 2006). Inactivation of rgg alters transcription of regulatory genes in strains SF370 and CS101, but not in strain MGAS5005 Inactivation of rgg in strain NZ131 altered the transcript levels of 20 genes encoding transcriptional regulatory proteins ( Dmitriev et al., 2006). Such perturbation of other regulatory networks presumably contributes to the genomewide changes associated with rgg inactivation in this strain ( Chaussee et al., 2002; Dmitriev et al., 2006). In contrast, rgg inactivation altered the abundance of three regulatory gene transcripts ( sagA, regR and gntR) in strain SF370, and only one ( regR) in CS101. No change in regulatory gene expression was detected in the rgg mutant of strain MGAS5005 ( Table 1). Thus, the number of differences in the expression of regulatory genes in rgg mutant strains correlated with the number of differences in structural gene expression ( Table 1). The results indicate that many genes in the Rgg regulon are controlled by strain-specific secondary mechanisms that remain to be elucidated. Rgg core- and sub-regulons Strain CS101 (serotype M49; class II) was selected to facilitate the identification of intraserotypic variation in the Rgg regulon by comparing the results to those previously obtained with the strain NZ131 (serotype M49; class II) ( Dmitriev et al., 2006). In addition, strains MGAS5005 and SF370 (serotypes M1; class I) were selected to identify potential differences within serotype M1 and to identify potential interserotypic and class differences in the regulon by comparing the results to those obtained with M49 strains. The genome sequences of MGAS5005, SF370, and NZ131, but not CS101, have been determined ( Ferretti et al., 2001; McShan et al., 2006; Sumby et al., 2005). The M1 strains have similar PFGE patterns of chromosomal DNA, which were different from those of M49 strains (data not shown). Furthermore, MGAS5005 and SF370 belong to the same Multi Locus Sequence Type ST28 ( http://www.mlst.net), and their Rgg regulons were expected to be similar. Surprisingly, the Rgg regulon varied significantly not only among the serotypes, but even among strains of the same serotype and Multi Locus Sequence Type. Only speB and adjacent spy2040 (encoding a 56 amino acid hypothetical protein) gene transcripts were less abundant in all of the rgg mutant strains ( Table 1), indicating that these co-transcribed genes ( Neely et al., 2003; Ma et al., 2006; Dmitriev et al.; 2006) comprise the Rgg core-regulon. Rgg binds to DNA in the promoter region of speB ( Neely et al., 2003), indicating that the core-regulon is directly regulated by Rgg. Other genes, which are influenced by Rgg, varied from a single gene ( spy1793) in strain MGAS5005 to 43 genes in the strain SF370. These genes can be considered as part of the Rgg sub-regulon, which is strain-variable. Rgg interacts with other regulons The strains used in this study were selected, in part, because they encode an identical Rgg polypeptide, have identical rgg promoter regions, and have similar levels of rgg expression, as determined with TaqMan quantitative RT-PCR (data not shown). Thus, the strain-associated differences in the regulon are not the result of Rgg sequence variation or rgg transcript levels, as previously observed for other streptococcal regulatory proteins ( Vickerman et al., 2003; Vahling & McIver, 2005; Loughman & Caparon, 2007). An association between the number of Rgg-regulated transcription factors and the total number of Rgg-regulated structural genes suggests that many changes in the sub-regulon are due to indirect effects associated with the perturbation of other regulatory circuits. For example, inactivation of rgg in strain NZ131 alters the expression of several regulatory genes ( Dmitriev et al., 2006), each of which is present in the genomes of strains SF370 and MGAS5005; however, only sagA, regR, and gntR expression was altered following inactivation of rgg in strain SF370. All chromosomally encoded regulatory proteins present in strain SF370 are also encoded by strain 5005, suggesting that the differences in the sub-regulon between the strains is not due to compositional variation in the set of chromosomally encoded regulatory genes. Of course allelic variation or differential expression of regulatory proteins, including novel regulators such as LacD.1 ( Loughman & Caparon, 2006), might contribute to strain-associated differences in the Rgg sub-regulon. In addition, S. pyogenes strains are polylysogenic and may differ in the number and types of bacteriophages present in the chromosome. For example, three bacteriophages (370.1, 370.2, and 370.4) are present in the SF370 strain but absent in the MGAS5005 strain. Similarly, two bacteriophages (5005.1 and 5005.3) are present in strain MGAS5005 but absent in the SF370 strain. Each prophage encodes several regulatory proteins involved in structural gene expression and lysogeny. Given that Rgg shares similarity with bacteriophage-encoded regulators, it is possible that the strain-associated differences in bacteriophage-encoded regulatory proteins influences the Rgg sub-regulon; although, further experiments are clearly necessary to test this hypothesis. Regulon variation in human bacterial pathogens The diverse clinical manifestations associated with S. pyogenes are due, in part, to variation in the gene content of strains. In addition, strain- or isolate-specific variation in gene expression described in human pathogens significantly contributes to phenotypic diversity and significantly impacts host-pathogen interactions ( Kwinn et al., 2007). In Streptococcus pneumoniae, inactivation of a response regulator gene ( rr09) altered the transcript levels of 102 and 80 genes in strains D39 and TIGR4, respectively; however, the expression of only 7 of these genes were similarly affected in the two strains ( Hendriksen et al., 2007). Similar diversity has been described in the BvgAS regulon of Bordetella bronchiseptica and Bordetella pertussis ( Cummings et al., 2006). In S. pyogenes, the global transcriptional regulator Mga promotes the expression of a number virulence factors, including M protein ( emm) and the C5a peptidase ( scpA) ( Hondorp & McIver, 2007). Mga influences the expression of 204, 201, and 37 genes in strains of serotypes serotypes M4, M1, M6, respectively. Notably, only emm, scpA, and spy2036 were similarly affected by mga inactivation in all three strains ( Ribardo & McIver, 2006). The variation in Rgg regulon, in conjunction with variation in the Mga regulon, indicates that significant diversity exists among virulence-associated regulatory circuits of S. pyogenes. Additional information related to the molecular basis for such variation is thus necessary to understand the regulation of virulence factor expression in S. pyogenes. In summary, we identified Rgg-regulated genes in strains representing class I and II organisms (M1 and M49 serotypes). The results show both inter- and intraserotypic variation in the Rgg regulon, which was consistent with results of biochemical, microbiological and quantitative real-time PCR assays. Such plasticity in regulatory circuits may provide pathogens with a means to adapt rapidly to changes in host-pathogen interactions. We thank A. Erkine, K. Weaver, A. Manna, and A. 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