To investigate the function of the CsrRS TCS in GBS, we previously constructed mutants in the background of GBS type Ia strain 515 and type V strain 2603 (12
). A nonpolar inactivating mutation was introduced into csrR
) or csrS
). Analysis of expression patterns of a limited number of genes revealed increased expression of cylE
(β-hemolysin/cytolysin) and scpB
(C5a peptidase) and reduced expression of cfb
(CAMP factor) in the csrR
mutants in both strain backgrounds and similar, though less extreme, changes in the csrS
). In the present study, we used GBS genomic microarrays as a more comprehensive means to investigate genome-wide changes in gene expression that result from inactivation of csrR
in the same two GBS strain backgrounds.
Inactivation of csrR or of csrS was associated with altered expression of a large number of genes in both strain backgrounds (Table ). Using as a threshold a twofold change in gene expression between the mutant and wild type, we found evidence of CsrRS regulation of 134 genes in strain 2603 and 80 genes in strain 515. One significant difference between the 2603 and 515 strains was the presence of 12 IS1381 transposase subunits found to be up-regulated in 2603ΔcsrR. A glycosyltransferase (SAG_1548, SAG_1551) in the up-regulated and cotranscribed SAG_1548 to -1555 is disrupted by an IS1381 transposase (SAG_1549, SAG_1550). Although the IS1381 transposase genes are on the opposite strand relative to the cotranscribed unit, the amplicon-based microarray queries both the transcript and its reverse complement. Therefore, up-regulation of genes SAG_1548 to -1555 results in what is likely to be artifactual “up-regulation” of the inserted transposase. Further, the high nucleotide identity of all the transposases results in positive results for all chromosomal locations. Therefore, the IS1381 transposases were removed from subsequent analyses, as it is unlikely that this apparent up-regulation is biologically relevant.
Numbers of regulated genes in csrR and csrS mutants of GBS strain 2603 or 515 relative to their respective wild-type parent strainsa
Bioinformatics software was used to search for putative CsrR-binding motifs associated with CsrRS-regulated individual genes and operons in the genome sequences of strains 2603 and 515. Among a number of candidate motifs, we failed to identify any that were both overrepresented in intergenic regions and preferentially located upstream of CsrRS-regulated genes. Therefore, this analysis did not support the earlier suggestion of a distinct CsrR binding sequence (15
) but suggested rather that CsrR recognizes regions of DNA that are not readily identified by a canonical nucleotide sequence motif.
Validation of microarray results by qRT-PCR.
To confirm the changes in transcript abundance observed by microarray hybridization, we performed qRT-PCR for a subset of regulated genes using RNA samples from mutant and wild-type strains. Nine genes were selected for qRT-PCR testing using RNA samples from 515ΔcsrR, 515ΔcsrS, 2603ΔcsrR, and 2603ΔcsrS, and their respective wild-type parent strains. The change (fold) in gene expression by qRT-PCR correlated well with those calculated from the microarray experiments in each of the mutant strains (R2 = 0.94; see Fig. S1 and Table S3 in the supplemental material).
Characterization of the CsrRS regulon in GBS strains 2603 and 515.
Transcriptional analysis using genomic microarrays confirmed the previously reported regulation of known or putative virulence factors including the cyl operon (SAG_0662 to -0673) encoding the GBS β-hemolysin/cytolysin, scpB (SAG_1236; C5a peptidase), and cfb (SAG_2043, CAMP factor), as well as a second gene transcriptionally linked to cfb (SAG_2042) that is predicted to encode a rhodanese-like protein of unknown function (see Table S1 in the supplemental material). A second locus encoding a predicted protein with 67% amino acid identity to C5a peptidase was also regulated by CsrRS (SAG_0416). As reported previously, and in contrast to the earlier analysis of GBS strain NEM316, we did not find a consistent pattern of regulation of the cps capsular polysaccharide synthetic operon in strain 2603. In 515ΔcsrR, there was a trend of down-regulation of the cps operon, but this trend only reached the twofold threshold for cps1aJ (SAL_1283) and cps1aH (SAL_1285) among the 16 genes in the cps operon.
Regulated genes in one or both strains encoded proteins involved in a wide range of cell functions including known or predicted virulence factors, transporters of amino acids, peptides, sugars, and metals, and proteins that mediate adaptation to environmental stresses (see Table S1 in the supplemental material). Among the various functional categories of regulated genes, transporters of amino acids, peptides, and amines were most significantly overrepresented in the study (P < 10−7), followed by transport and binding proteins (P < 10−5) and pathogenesis genes (P < 10−5). We found evidence for regulation of expression of several proteins that are predicted to be secreted or surface associated, including SAG_0297 (aminopeptidase C), SAG_1002 (putative protease), SAG_1890 (putative endopeptidase O), and two operons predicted to encode membrane proteins (SAG_0364 to -365 and SAG_0798 to -799).
Genes encoding proteins involved in transport of a variety of substrates constituted a large group of regulated genes. These included the oppA1-F
operon (SAG_0148 to -0152), encoding an oligopeptide ABC transporter that has been implicated in modulating the attachment of GBS to host cells and the adc
operon (SAG_0154 to -0156), homologs of which encode a zinc/manganese transporter in Streptococcus pneumoniae
and a manganese acquisition and homeostasis system in Streptococcus gordonii
). Also regulated by CsrRS are homologs of a system involved in iron transport (SAG_1007-1010).
In keeping with the inferred role of CsrRS in adaptation to changing environments, the system regulates several stress response mechanisms in GBS. These included homologs of AphC and AphF (SAG_1833 to -1834), two components of the alkyl hydroperoxide reductase of S. pyogenes
. In that species, the alkyl hydroperoxide reductase system contributes to scavenging endogenous hydrogen peroxide and has been linked to virulence in a murine infection model (2
). Homologs of the enterococcal and lactococcal general stress protein Gls24 (SAG_1135 and SAG_1137) were also regulated by CsrRS in GBS. Gls24 has been implicated in stress response and virulence in Enterococcus faecalis
). The GBS CsrRS also regulates expression of two separate operons predicted to encode components of a glycine/betaine osmoregulation system (SAG_1796 to -1797 and SAG_0241 to -0244), a system that mediates adaptation to osmotic stress in Bacillus subtilis
and Lactococcus lactis
). Although their role in GBS is undefined, expression of two predicted transcriptional regulators is also controlled by CsrRS: SAG_0712 encoding a putative regulator of the OmpR family and SAG_0938 encoding a predicted GntR family transcriptional regulator (11
Comparison of the CsrRS regulons in three different GBS strains.
The availability of genome-wide transcriptional profiling data in strains 2603 and 515 together with the previously described results in strain NEM316 provided the opportunity to compare the CsrRS regulons in independent GBS isolates representing the three most important capsular serotypes in human infection, types Ia (strain 515), III (strain NEM316), and V (strain 2603). This analysis revealed a core group of 39 genes whose expression was changed as a result of inactivation of csrR and/or csrS in all three strain backgrounds (Fig. and Table ). Two-way comparisons showed further overlap in the repertoire of CsrRS-regulated genes, with 16 genes regulated in both 2603 and 515, 18 in 2603 and NEM316, and 3 in 515 and NEM316. For each of the three strains, certain CsrRS-regulated genes were regulated in only one strain background. A higher number of uniquely regulated genes were identified in NEM316, but this may be a result of the differences in array platform and mutant design between the two studies. These combined results suggest that CsrRS regulates a conserved core group of genes in multiple GBS strains, including those coding for the important virulence factors β-hemolysin, C5a peptidase, and CAMP factor, as well as a large repertoire of genes whose regulation varies among GBS isolates.
FIG. 1. Conserved and strain-specific gene regulation by CsrRS. Numbers within the circle corresponding to each GBS strain represent the number of genes that are CsrRS regulated in one, two, or all three strains. The number of regulated genes shown here is lower (more ...)
Conserved genes of the CsrRS regulon in three GBS strains
Differential effects on gene regulation of inactivating CsrR versus CsrS.
Mutation of csrR in strain 2603 was associated with increased expression of 94 genes and reduced expression of 13 genes. This pattern suggests that CsrR acts predominantly as a transcriptional repressor but that it can also activate gene transcription, directly or indirectly. Inactivation of csrS resulted in expression changes of a smaller number of genes with 36 genes up-regulated in 2603ΔcsrS and 18 genes down-regulated (Table ). For 27 genes, we observed altered expression in both 2603ΔcsrR and in 2603ΔcsrS (Fig. ). In 23 of these 27 genes, gene expression was up-regulated in both 2603ΔcsrR and 2603ΔcsrS. For most regulated genes, mutation of csrR produced a greater effect than did mutation of csrS (see Fig. S2 in the supplemental material). We observed a change in expression in 2603ΔcsrR but not in 2603ΔcsrS for 80 genes (Fig. ). For the majority of these genes, the change in expression in 2603ΔcsrS was in the same direction as that in 2603ΔcsrR, but it did not reach the twofold threshold. For two linked genes, SAG_1706 (hypothetical protein) and SAG_1707 (putative drug resistance transporter), expression was increased in 2603ΔcsrR and decreased in 2603ΔcsrS.
FIG. 2. Differential regulation of gene expression in strain 2603ΔcsrR compared to 2603ΔcsrS. Pairs of bars represent the change (fold) in gene expression relative to wild type in strains 2603ΔcsrR (filled bars) and 2603ΔcsrS (open (more ...)
While the total number of CsrRS-regulated genes was somewhat lower in strain 515 than in 2603 (Table ), we observed a similar pattern with respect to the relative effects of mutation in csrR versus csrS. That is, the predominant pattern was a greater effect on target gene expression in 515ΔcsrR than in 515ΔcsrS, but for several genes, the effect was greater in 515ΔcsrS than in 515ΔcsrR (Fig. ).
FIG. 3. Differential regulation of gene expression in strain 515ΔcsrR compared to 515ΔcsrS. Pairs of bars represent the change (fold) in gene expression relative to wild type in strains 515ΔcsrR (filled bars) and 515ΔcsrS (open (more ...) CsrR binds directly to promoter regions of both activated and repressed genes.
An earlier investigation of GBS strain NEM316 used DNase I protection and EMSA to demonstrate direct binding of recombinant CsrR to a DNA segment upstream of the cyl
). DNase I protection experiments also suggested direct binding to the promoter regions of two other genes whose expression, like that of the cyl
operon, is repressed by CsrRS. To further characterize the interaction of CsrR with regulated promoters, we expressed CsrR as a His6
fusion in Escherichia coli
and purified the recombinant protein by Ni2+
-affinity chromatography. His6
-CsrR was used in EMSA with DNA probes from strain 2603 that corresponded to the promoter regions of the cyl
operon and scpB
(C5a peptidase), two genes whose expression is repressed by CsrRS; and cfb
(CAMP factor), a gene whose expression is activated by CsrRS. For all three promoters, band shifts were observed in the presence of His6
-CsrR, indicating direct binding of CsrR to DNA sequences upstream of both CsrRS-repressed and CsrRS-activated genes (Fig. ). No shift was observed after incubation of His6
-CsrR with a DNA segment corresponding to the promoter region of the cps
operon of strain 2603, a result that is consistent with the absence of CsrRS regulation of this locus in strain 2603 and that serves as a negative control for the specificity of CsrR binding to regulated promoters. Specificity of the binding interaction for each of the regulated promoters was also supported by competition with excess unlabeled probe, but not with excess unlabeled cps
promoter sequences. These results indicate that CsrR binds directly to both positively and negatively regulated promoter sequences.
FIG. 4. Binding of purified His6-CsrR to promoter regions of CsrRS-regulated genes. EMSA were performed using 32P-labeled DNA fragments corresponding to the promoter region of the indicated gene or operon and purified His6-CsrR protein. Lanes contain 32P-labeled (more ...) Phosphorylation of CsrR increases its affinity for promoter DNA.
Signaling through TCS typically is transduced by phosphorylation or dephosphorylation of the regulator component in response to interaction of the sensor with an environmental stimulus. In keeping with this general model, the phosphorylation state of the regulator has been shown to change its affinity for target DNA sequences in several TCS in other species, including the homologous CsrRS system in Streptococcus pyogenes
). To test the importance of CsrR phosphorylation for gene regulation in GBS, we incubated His6
-CsrR with acetyl phosphate to phosphorylate the CsrR protein. EMSA using a probe for the cyl
operon promoter revealed a minor increase in binding affinity (approximately twofold) for the phosphorylated compared to unphosphorylated His6
-CsrR (Fig. ). This modest effect of phosphorylation at the cyl
promoter is consistent with the results of Lamy et al., who found no significant effect of phosphorylation on binding of CsrR to the cyl
). In contrast, phosphorylation had a marked effect on binding of CsrR to the scpB
promoters, increasing binding affinity by approximately eightfold. Phosphorylation also appeared to enhance the formation of higher-molecular-size complexes, a result that suggests phosphorylation may promote oligomerization of CsrR. These results indicate that phosphorylation increases binding of CsrR to regulated promoters and that individual target promoters differ in their relative affinities for the phosphorylated versus unphosphorylated regulator protein.
FIG. 5. Phosphorylation of CsrR enhances binding to regulated promoters. EMSA were performed using 32P-labeled DNA fragments corresponding to the promoter region of the indicated gene or operon and increasing concentrations of purified His6-CsrR protein without (more ...) Inactivation of CsrR or CsrS has strain-specific differential effects on virulence.
The microarray analysis revealed both qualitative and quantitative differences in the relative effects on gene regulation of mutating csrR
in the background of strain 2603 compared to that in 515 (Fig. and and Table ). Furthermore, opposite regulatory effects were observed for 2603ΔcsrS
(e.g., with SAG_1706 to -1707), whereas such divergent effects were not observed in the background of strain 515. To investigate whether such differential effects of CsrR compared to CsrS might be reflected in the relative pathogenic potential of the mutant strains, we tested the virulence of csrR
mutants and wild-type strains 2603 and 515 in a murine model of systemic infection and septic arthritis. Mice were challenged intravenously with various doses of GBS and observed for development of signs of arthritis and for mortality. Wild-type strain 515 was the most virulent in these studies, with an LD50
of 7.2 × 104
. The LD50
was 320-fold higher at 2.3 × 107
, whereas 515ΔcsrS
had an intermediate level of virulence (LD50
of 8.5 × 106
). This hierarchy of relative virulence is the same as that reported previously for these strains in a murine intraperitoneal challenge model (12
). Similarly, strain 2603ΔcsrR
was attenuated in virulence (no deaths at challenge doses up to 108
CFU) relative to wild-type strain 2603 (LD50
of 2.8 × 107
CFU), as reported previously for the intraperitoneal challenge model (12
). In striking contrast, strain 2603ΔcsrS
was more virulent (LD50
of 2.4 × 106
CFU) than wild-type 2603. This virulence hierarchy was reflected not only in the relative lethality of the three strains in the 2603 background but also in severity of arthritis, whether scored by number of affected joints or by clinical severity index (Fig. ).
FIG. 6. Relative virulence of wild-type strain 2603, 2603ΔcsrS, or 2603ΔcsrR in a murine arthritis model. The values shown represent the clinical arthritis index after intravenous challenge with 1 × 107 CFU of the indicated GBS strain. (more ...)
These results demonstrate that differential patterns of regulation by CsrRS in strains 2603 and 515 are associated with striking differences in the overall relative virulence of csrS
mutants in the two strain backgrounds. In particular, we observed increased expression of certain genes in 2603ΔcsrS
, but not in 2603ΔcsrR
or in either mutant in the 515 background. Of these, possible virulence genes include SAG_1135 and SAG_1137 that encode homologs of Gls24, a stress response protein shown to contribute to virulence in experimental enterococcal infection (18
). A similar pattern of regulation was noted for SAG_1796 and SAG_1797, which are predicted to encode a glycine/betaine osmoregulation system implicated in adaptation to osmotic stress in other species (13
). Differential CsrS-dependent regulation of these loci in the 2603 strain background may account for the unexpectedly high virulence of strain 2603ΔcsrS