• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Microbiol. Author manuscript; available in PMC Dec 3, 2008.
Published in final edited form as:
PMCID: PMC2593684
NIHMSID: NIHMS79452

Regulation of cytotoxin expression by converging eukaryotic-type and two-component signalling mechanisms in Streptococcus agalactiae

Summary

Signal transducing mechanisms are essential for regulation of gene expression in both prokaryotic and eukaryotic organisms. Regulation of gene expression in eukaryotes is accomplished by serine/threonine and tyrosine kinases and cognate phosphatases. In contrast, gene expression in prokaryotes is controlled by two-component systems that comprise a sensor histidine kinase and a cognate DNA binding response regulator. Pathogenic bacteria utilize two-component systems to regulate expression of their virulence factors and for adaptive responses to the external environment. We have previously shown that the human pathogen Streptococcus agalactiae (Group B Streptococci, GBS) encodes a single eukaryotic-type serine/threonine kinase Stk1, which is important for virulence of the organism. In this study, we aimed to understand how Stk1 contributes to virulence of GBS. Our results indicate that Stk1 expression is important for resistance of GBS to human blood, neutrophils and oxidative stress. Consistent with these observations, Stk1 positively regulates transcription of a cytotoxin, β-haemolysin/cytolysin (β-H/C) that is critical for survival of GBS in the bloodstream and for resistance to oxidative stress. Interestingly, positive regulation of β-H/C by Stk1 requires the two-component regulator CovR. Further, we show that Stk1 can negatively regulate transcription of CAMP factor in a CovR-dependent manner. As Stk1 phosphorylates CovR in vitro, these data suggest that serine/threonine phosphorylation impacts CovR-mediated regulation of GBS gene expression. In summary, our studies provide novel information that a eukaryotic-type serine/threonine kinase regulates two-component-mediated expression of GBS cytotoxins.

Introduction

Signal transduction via phosphorylation of proteins is critical for organisms to respond to their dynamic internal and external environment. Protein kinases such as serine/threonine and tyrosine kinases are the principal signalling components in eukaryotes. Over 500 members of these protein kinases are present in humans (Manning et al., 2002) and aberration of normal phosphorylation events leads to altered cellular function and disease (for review, see Cohen, 2002). In contrast to eukaryotes, signal transduction in prokaryotes is accomplished predominantly by two-component systems. A typical two-component system comprises a sensor histidine kinase that phosphorylates a DNA binding response regulator to mediate gene expression (for review, see Foussard et al., 2001). Recently, a number of reports describe the existence of eukaryotic-type serine/threonine kinases in prokaryotes (for reviews, see Zhang, 1996; Kennelly, 2002). Further, pathogenic bacteria such as Yersinia, Mycobacteria and Streptococci that are deficient in expression of serine/threonine kinases are attenuated for virulence (Galyov et al., 1993; Rajagopal et al., 2003; Echenique et al., 2004; Walburger et al., 2004). However, not much is known about how serine/threonine kinases promote virulence of bacterial pathogens. To address this question, we analysed the role of the serine/threonine kinase (Stk1) in virulence of the human pathogen Streptococcus agalactiae or Group B Streptococcus (GBS).

Group B Streptococci are β-haemolytic, Gram-positive bacteria that cause a wide array of invasive infections in human newborns and adults (Baker and Edwards, 1995). GBS commonly resides as a commensal organism in maternal genital and lower gastrointestinal tracts. The organism can then transition into an aggressive neonatal pathogen that involves infiltration into the intrauterine compartment, invasion of the neonatal lung and dissemination into multiple neonatal organs (for reviews, see Baker and Edwards, 1995; Doran and Nizet, 2004). The diverse arrays of host niches encountered by GBS during its disease cycle indicate that the pathogen efficiently adapts to changing environments to survive and establish successful infections.

Although GBS is an important clinical pathogen, our understanding of the molecular events that mediate the transition of an otherwise commensal organism to an invasive pathogen is limited. A few factors that are important for virulence of GBS have been identified. The sialic acid-rich capsular polysaccharide (sia-CPS) belonging to one of nine serotypes Ia, Ib, II-VIII are essential for bacterial evasion of host immune defences (Edwards et al., 1982). A surface-associated toxin called β-haemolysin/cytolysin (β-H/C) promotes GBS invasion of host cells, induces host inflammatory responses, confers resistance to reactive oxygen species and is critical for GBS disease progression during sepsis, pneumonia, meningitis and arthritis (for review, see Nizet, 2002). CAMP (Christie, Atkins Munch-Peterson) factor is another cytotoxin that has been implied to be important for virulence of GBS (Jurgens et al., 1987). Despite the identification of critical virulence factors, not much is known about signalling mechanisms that regulate expression of these factors during GBS disease pathogenesis.

The presence of multiple signalling components in the GBS genome (Glaser et al., 2002; Tettelin et al., 2002) suggests that they may enable the pathogen to adapt, survive and infect various host compartments encountered during its disease cycle. Recently, a two-component system CovR/CovS (also known as CsrR/CsrS), comprising the sensor histidine kinase CovS (or CsrS) and the response regulator CovR (or CsrR), was shown to regulate expression of two GBS cytotoxins, β-H/C and CAMP factor (Lamy et al., 2004; Jiang et al., 2005). Notably, deletion of either or both covR and covS increased β-H/C and decreased CAMP factor synthesis in GBS (Lamy et al., 2004; Jiang et al., 2005).

The discovery of signalling systems in bacteria that are distinct from the classical two-component system mentioned above, such as the eukaryotic-type serine/threonine protein kinase and their cognate phosphatase has sparked an interest in understanding their function, particularly in pathogenic organisms. We have described the existence of a single pair of genes that encode a eukaryotic-type serine/threonine kinase (Stk1) and its cognate phosphatase (Stp1) in GBS (Rajagopal et al., 2003). Our previous results indicated that stk1 mutants are attenuated for virulence (Rajagopal et al., 2003). In this study, we present evidence that expression of a pluripotent GBS toxin β-H/C is significantly decreased in the stk1 mutants. Further, Stk1 can negatively regulate expression of another GBS cytotoxin called CAMP factor. Interestingly, both positive regulation of β-H/C and negative regulation of CAMP factor by Stk1 require the two-component regulator, CovR. As Stk1 phosphorylates CovR in vitro, we hypothesize that serine/threonine phosphorylation of CovR impacts CovR-mediated transcriptional regulation in GBS. Collectively, our studies provide novel evidence that discrete signalling components such as a eukaryotic-type serine/threonine kinase and a two-component response regulator are integrated for co-ordinate regulation of gene expression and consequently virulence of GBS.

Results

Stk1 expression is important for survival of GBS in human whole blood

We have described that GBS defective in expression of a eukaryotic-type kinase Stk1 (Δstk1, LR113), or both Stk1 and its cognate phosphatase Stp1 (Δstp1Δstk1, LR114) are attenuated for virulence (Rajagopal et al., 2003). The objective of this study was to understand how Stk1 contributes to virulence of GBS. As the stk1 mutants were attenuated for virulence in a sepsis model of infection, we assessed survival of these strains in non-immune human whole blood (NIHWB) as described (Jones et al., 2003). Our results indicate that both the Δstk1 (LR113) and Δstp1Δstk1 (LR114) mutants are significantly impaired for survival in human whole blood compared with the isogenic wild-type (WT) strain A909 (Fig. 1, compare survival index (SI) of A909, LR113 and LR114 in blood). As a control, we included a GBS strain A909ΔDcpsE that is deficient in sia-CPS synthesis. As sia-CPS prevents recognition of GBS by host innate immune defences (Marques et al., 1992), the A909ΔDcpsE strain is extremely sensitive to human whole blood (see Jones et al., 2003 and Fig. 1).

Fig. 1
Stk1 expression is important for survival of GBS in NIHWB. GBS were incubated in NIHWB and SI was calculated relative to the growth of each strain in human plasma over a 3 h period as described in Experimental procedures. Means were calculated from three ...

Our previous studies indicated that Stk1 expression is important for GBS to sustain de novo purine biosynthesis (Rajagopal et al., 2005). Therefore, we hypothesized that the poor survival of the stk1 mutants (LR113 and LR114) in human whole blood may be due to growth defects caused by purine deficiencies. As LR113 and LR114 are not defective in the salvage of exogenous purines (Rajagopal et al., 2005), we predicted that addition of exogenous purines to human whole blood should circumvent any growth defects of the stk1 mutants. However, we noted that the poor survival of the Δstk1 (LR113) and Δstp1Δstk1 (LR114) mutants in human whole blood was not alleviated by the addition of exogenous purines (compare SI of A909, LR113, and LR114 in ‘Blood’ to ‘Blood + Purines’ in Fig. 1). We also noted that growth of the stk1 mutants was comparable to WT in both normal human plasma and human serum in the presence and absence of exogenous purines (data not shown). Collectively, these data suggest that Stk1 expression is important for survival of GBS in the bloodstream and is independent of its role in de novo purine biosynthesis.

GBS stk1 mutants are sensitive to phagocytic killing

GBS mutants that demonstrate decreased survival in human whole blood have also been associated with an increase in sensitivity to phagocytic killing (Harris et al., 2003; Jones et al., 2003). As polymorphonuclear leucocytes (PMN) are the primary host line of defence against GBS infections, the ability of GBS to escape host defences relies on its mechanisms to evade opsonization, phagocytic uptake and killing. To analyse the role of Stk1 in resistance of GBS to phagocytic killing, we examined the sensitivity of the Δstk1 (LR113) and Δstp1Δstk1 (LR114) mutants to PMN as described in Experimental procedures. Consistent with previous observations (Jones et al., 2003), we observed that the WT strain A909 is relatively resistant to phagocytic killing in the absence of serotype-specific antibodies (~15% killing, see Fig. 2). In contrast to WT, both the Δstk1 (LR113) and Δstp1Δstk1 (LR114) mutants demonstrate a substantial increase in sensitivity to phagocytic killing (~85% killing), similar to the A909ΔDcpsE strain (see Fig. 2). These results indicate that the attenuated virulence of the stk1 mutants is due to increased sensitivity to phagocytes.

Fig. 2
GBS stk1 mutants are sensitive to phagocytic killing. Bacteria were opsonized in normal human serum and incubated with PMN at a ratio of 1:3 (bacteria: PMN) for 1 h. The per cent kill for each strain was calculated relative to growth of the same strain ...

Two critical and interlinked factors that determine recognition, uptake and killing of GBS by professional phagocytes are sia-CPS expression and complement factor C3 deposition on the bacterial surface (Edwards et al., 1982). However, the increase in sensitivity of the stk1 mutants to phagocytic killing was not due to a decrease in sia-CPS synthesis or an increase in complement factor C3 deposition (data not shown).

GBS stk1 mutants are sensitive to H2O2

We next hypothesized that the stk1 mutants might be more susceptible to phagocytic killing due to increased sensitivity to oxidative burst within phagocytes. Phagocyte-derived reactive oxygen species are crucial determinants in host resistance to bacterial pathogens (Fang, 2004). Therefore, we evaluated sensitivity of the stk1 mutants to one of the principal oxidants produced by phagocytes, i.e. hydrogen peroxide (H2O2) as described in Experimental procedures. Our results indicate that both the Δstk1 (LR113) and Δstp1Δstk1 (LR114) mutants were approximately 50-fold more sensitive to H2O2 when compared with the WT strain A909 (Fig. 3). As a control, we included an isogenic GBS strain, IaΔcylE that is deficient in β-H/C expression and was shown to be extremely sensitive to H2O2 and other reactive oxygen species (Liu et al., 2004). The sensitivity of stk1 mutants LR113 and LR114 to H2O2 was similar to but less severe than IaΔcylE (Fig. 3). These results suggest that the attenuated virulence and increased susceptibility of the stk1 mutants to phagocytic killing is likely due to decreased resistance to host reactive oxygen species.

Fig. 3
Stk1 expression is important for resistance of GBS to reactive oxygen species. GBS strains were exposed to H2O2 (0.015% for 1 h). SI was calculated as (cfu at the end of assay)/(cfu at time 0). All experiments were repeated at least three times in triplicate. ...

β-Haemolysin expression is decreased in the GBS stk1 mutants

The importance of β-H/C and an associated carotenoid-like pigment in resistance of GBS to phagocytes and reactive oxygen species was recently described (Liu et al., 2004). The structural gene that encodes β-H/C in GBS was identified as cylE (Spellerberg et al., 2000; Pritzlaff et al., 2001). Interestingly, deletion of cylE also abolished production of an orange, carotenoid pigment in GBS that promotes resistance to reactive oxygen species (Spellerberg et al., 2000; Liu et al., 2004). To date, cylE is the only known link between β-H/C and pigment biosynthesis in GBS (Nizet, 2002; Liu et al., 2004).

We noted that the stk1 mutants displayed similar phenotypes of attenuated virulence, increased sensitivity to human whole blood, phagocytes and reactive oxygen species compared with the haemolysin and pigment-deficient GBS strain IaΔcylE (see Liu et al., 2004). This prompted us to re-examine both β-H/C and pigment synthesis in the stk1 mutants. Although we had previously stated that the stk1 mutants were not affected in β-H/C synthesis (Rajagopal et al., 2003), these tests were performed using the qualitative API 20 Strep (bioMérieux, NC, USA) that does not quantify the amount of β-H/C produced between GBS strains. Rather, this test is routinely used for identification of GBS in clinical samples. Therefore, we evaluated β-H/C activity in the stk1 mutants using a more sensitive assay based on lysis of sheep erythrocytes (Nizet et al., 1996). The results shown in Table 1 reflect β-H/C activity that is expressed as fold difference compared with WT A909. Of note, β-H/C activity assays were repeated to include all strains shown in Table 1 within a given experiment. Actual titers are not reported due to variations in susceptibility of sheep red blood erythrocytes (sRBC) in different batches of blood. Our results indicate that β-H/C activity in the stk1 mutants LR113 and LR114 were significantly lower (2–3%) when compared with WT A909 (see Table 1). Interestingly, β-H/C activity in the Δstk1 strain LR113 compared with the double Δstp1Δstk1 mutant LR114 was not significantly different (P = 0.3). Collectively, these results suggest that Stk1 regulates β-H/C activity in GBS.

Table 1
Stk1 positively regulates β-H/C activity.

To confirm that Stk1 expression can restore normal β-H/C expression to the mutants, we quantified haemolysin activity in complemented strains containing WT alleles of either stk1 or both stk1 and stp1 in LR113 and LR114 respectively. The complementing plasmids constitutively express Stk1 and/or Stp1 from the Ptet promoter (for details on complementing plasmids, see Rajagopal et al., 2003). We observed that β-H/C activity in LR113 and LR114 containing the WT stk1 allele (see LR113/pStk1 and LR114/pStk1 in Table 1) and LR114 containing both stp1 and stk1 (see LR114/pStp1Stk1 in Table 1) was restored to WT. These results confirm the link between Stk1 and β-H/C in GBS. Further, as the WT stp1 allele (pStp1) failed to increase β-H/C activity in LR114 (see LR114/pStp1 in Table 1), this indicates that Stp1 does not regulate β-H/C expression in GBS.

Stk1 positively regulates β-haemolysin expression in GBS serotype V

To corroborate our findings that Stk1 positively regulates β-H/C, we also examined the role of Stk1 on β-H/C expression in a GBS capsular serotype V strain NCTC10/84 (Wilkinson, 1977; Liu et al., 2004). Of note, both WT strains, i.e. A909 belonging to serotype Ia and NCTC10/84 belonging to serotype V, have been previously used to elucidate the role of β-H/C in GBS virulence (Nizet et al., 1996; Pritzlaff et al., 2001; Doran et al., 2002; Liu et al., 2004; Hensler et al., 2005). Interestingly, these studies have described that the NCTC10/84 GBS isolate is hyper-haemolytic and exhibits increased pigment production and even virulence when compared with A909 (Doran et al., 2002; Liu et al., 2004). While there is no correlation between β-H/C activity and GBS serotypes (V. Nizet, pers. comm.), the reason for the difference in haemolytic properties between these WT GBS strains is not yet understood.

We hypothesized that if Stk1 positively regulates β-H/C, then Δstk1 mutants derived from NCTC10/84 should also be less haemolytic compared with the isogenic WT. To test this hypothesis, we derived a Δstk1 mutant (strain LR122) and a double Δstp1Δstk1 mutant (strain LR123) from NCTC10/84 as described in Experimental procedures. Using in vitro phosphorylation of GBS membrane fractions (Rajagopal et al., 2003), we confirmed that Stk1 is expressed in NCTC10/84 and was absent in the isogenic stk1 mutants LR122 and LR123 (see Fig. S1). Importantly, β-H/C activity in the serotype V Δstk1 (LR122) and Δstp1Δstk1 (LR123) strains were fourfold lower compared with the isogenic WT NCTC10/84 (see Table 1). Collectively, these data confirm that Stk1 positively regulates β-H/C activity in two different GBS serotypes.

Attenuated pigment production in GBS stk1 mutants

β-H/C is associated with production of a pigment in GBS that confers resistance to reactive oxygen species (Spellerberg et al., 2000; Liu et al., 2004). We hypothesized that the decrease in β-H/C and increase in sensitivity to reactive oxygen species observed in the stk1 mutants including serotype V (data not shown) should correlate to a decrease in pigment production. Hence, we analysed pigment production in all stk1 mutants as described (Liu et al., 2004). The results shown in Fig. 4A and B indicate a relative decrease in pigment production in the Δstk1 (LR113 and LR122) and Δstp1Δstk1 (LR114 and LR123) mutants compared with the isogenic WT (A909 and NCTC10/84) respectively. As the biochemical nature and molecular structure of the GBS pigment are unknown, we are unable to quantify the amount of pigment produced by each strain. Collectively, these results indicate that Stk1 positively regulates both β-H/C and pigment synthesis in GBS.

Fig. 4
Attenuated pigment production in GBS stk1 mutants

Transcription of cylE is decreased in the stk1 mutants

We then examined if the decrease in haemolysin and pigment synthesis in the stk1 mutants was due to decreased transcription of the gene encoding β-H/C (cylE). The structural gene for β-H/C, i.e. cylE is linked to pigment production and encodes a 78 kDa surface associated protein with no homology to any known proteins in the database (Spellerberg et al., 1999; Pritzlaff et al., 2001; Nizet, 2002). We compared transcription of cylE between WT and isogenic stk1 mutants using RNase protection assays (RPA) as described in Experimental procedures. An increase or decrease in transcription of ≥ 2.0 compared with WT was considered significant (Lamy et al., 2004). Interestingly, we observed a 2.5-fold decrease in cylE transcription in all stk1 mutants compared with the isogenic WT (Fig. 5A, compare LR113 and LR114 with A909 and LR122 and LR123 with NCTC10/84). As expected, a cylE-specific transcript was not detected in the control strain lacking cylE (see IaΔcylE in Fig. 5A). Collectively, these data suggest that Stk1 is required for normal transcription of cylE (β-H/C) in GBS. We also noted that cylE transcription in NCTC10/84 was 3.4-fold greater than A909 (compare NCTC10/84 with A909 in Fig. 5A), suggesting that differences in β-H/C activity between these isolates can be attributed to altered cylE transcription. Hence, β-H/C activity was greater in the NCTC10/84 stk1 mutants compared with A909 stk1 mutants although the decrease in cylE transcription due to the lack of Stk1 is similar.

Fig. 5
Stk1 positively regulates cylE transcription

Analysis of cylE transcription in the complemented strains revealed that introduction of the WT stk1 allele into LR113 and LR114 restored cylE transcription to WT levels (see LR113/pStk1 and LR114/pStk1 in Fig. 5B). As expected, introduction of only the WT stp1 allele into the double Δstp1Δstk1 mutant LR114 did not restore β-H/C expression to WT levels (see LR114/pStp1 in Fig. 5B). Similar results were obtained with complemented stk1 mutants derived from NCTC10/84 (data not shown). These data confirm that Stk1 positively regulates transcription of β-H/C (cylE) in GBS.

Positive regulation of β-H/C by Stk1 requires CovR

Although our results indicated that transcription of cylE was decreased in the stk1 mutants, it is noteworthy that neither Stk1 nor Stp1 contain consensus DNA binding domains (see Rajagopal et al., 2003 for sequence). Hence, it is unlikely that Stk1 directly binds DNA to regulate transcription. We hypothesized that Stk1 may regulate β-H/C expression by affecting the activity of a DNA binding regulator in GBS. Recently, a two-component response regulator CovR was shown to bind to the promoter region of cylE (PcylX) and to repress transcription of β-H/C in GBS (Lamy et al., 2004). Further, the cognate sensor of CovR, i.e. CovS was shown to augment but not be essential for CovR-mediated repression of β-H/C in GBS (Jiang et al., 2005). As CovR represses transcription of β-H/C, CovR-deficient GBS strains exhibit enhanced β-H/C synthesis when compared with WT (Lamy et al., 2004; Jiang et al., 2005). We speculated that positive regulation of β-H/C by Stk1 may be due to its ability to impact CovR-mediated repression of β-H/C. To test the possibility of a link between Stk1 and CovR, we constructed GBS mutants deficient for CovR expression in WT and isogenic stk1 mutants and compared haemolysin activity between these strains (see Experimental procedures). We reasoned that if Stk1 impedes CovR-mediated repression of β-H/C, positive regulation of β-H/C by Stk1 should be abolished in the absence of CovR. Consistent with this hypothesis, we observed that β-H/C transcription and activity were identical in A909ΔcovR, LR113ΔcovR and LR114ΔcovR (see Table 1 and Fig. 5C). Likewise, β-H/C activity and transcription were similar in all ΔcovR strains derived from NCTC10/84 (see Table 1 and Fig. 5C). Consistent with the role of CovR as a repressor of β-H/C (Lamy et al., 2004; Jiang et al., 2005), cylE transcription was 16-fold higher in A909ΔcovR compared with WT A909 (data not shown). Taken together, these results confirm that positive regulation of β-H/C by Stk1 requires the response regulator, CovR.

Stk1 negatively regulates CAMP factor expression in GBS serotype V

Our results indicate that Stk1 impacts CovR-mediated repression of β-H/C. Interestingly, CovR has also been described to activate expression of a secreted GBS cytotoxin called CAMP factor (Lamy et al., 2004; Jiang et al., 2005). Therefore, we hypothesized that Stk1 should also impact CovR-mediated activation of CAMP factor in GBS. Consequently, CAMP factor expression should be increased in the absence of Stk1. To test this hypothesis, we examined CAMP factor expression between WT and stk1 mutants as described (Ross et al., 1999). Surprisingly, we observed that CAMP factor expression in the serotype Ia stk1 mutants LR113 and LR114 was comparable to the isogenic WT A909 (Fig. 6A). In contrast, we observed that CAMP factor expression in the serotype V stk1 mutants LR122 and LR123 was fourfold greater than their isogenic WT, NCTC10/84 (see Fig. 6A). We also noted that the hyper-haemolytic NCTC10/84 produced less CAMP factor than the moderately haemolytic A909 (see Fig. 6A). We speculate that this difference in β-H/C and CAMP factor expression between WT A909 and NCTC10/84 may correlate to differences in CovR levels or CovR activity. This conclusion is supported by the observation of Jiang et al. (2005) that expression of β-H/C and CAMP factor due to CovR varied among WT GBS strains used in their study. Collectively, these results indicate that Stk1 negatively regulates CAMP factor expression in the GBS serotype V strain NCTC10/84.

Fig. 6
Stk1 negatively regulates CAMP factor expression in GBS serotype V

Transcription of cfb (CAMP factor) is decreased in the serotype V stk1 mutants

We then analysed if the increase in CAMP factor expression in the serotype V stk1 mutants was due to increased transcription of the cfb gene. The monocistronic cfb gene was previously shown to encode CAMP factor in GBS (Podbielski et al., 1994). We performed RPA to examine cfb transcription in WT and isogenic stk1 mutants. The results shown in Fig. 6B indicate that transcription of cfb is similar in A909 and isogenic stk1 mutants LR113 and LR114 (Fig. 6B, compare A909, LR113 and LR114, fold difference < 2.0, P &ge;0.1, Student’s t-test) and is consistent with CAMP factor expression in these strains. In contrast, cfb transcription was 2.0-fold greater in the sero-type V stk1 mutants LR122 and LR123 compared with their isogenic WT (Fig. 6B, compare LR122 and LR123 to NCTC10/84, P-value ≤0.01). These results indicate that Stk1 negatively regulates cfb transcription in GBS NCTC10/84. As cfb transcription is sevenfold higher in A909 compared with NCTC10/84 (see Fig. 6B), we speculate that CAMP factor transcription may be at a maximum in A909. Therefore, any further increase in CovR activity due to the absence of Stk1 may not contribute to an increase in transcription of cfb in the A909 stk1 mutants. Collectively, our results indicate that Stk1 positively regulates transcription of β-H/C and can negatively regulate CAMP factor expression in GBS.

Negative regulation of CAMP factor by Stk1 requires CovR

We reasoned that if Stk1 negatively impacts CovR-mediated activation of CAMP factor, then the increase in CAMP factor observed in the NCTC10/84 stk1 mutants should be abolished in the absence of CovR. To test this hypothesis, we compared CAMP factor expression between WT, Δstk1 and isogenic Δstk1ΔcovR mutants as described in Experimental procedures. Consistent with our hypothesis, we observed that the increase in CAMP factor expression observed in LR122 and LR123 is abolished in isogenic LR122ΔcovR and LR123ΔcovR mutants (see Fig. 7). CAMP factor titers of all ΔcovR strains derived from A909 and NCTC10/84 was < 1 and transcription of cfb in all isogenic ΔcovR strains was similar (data not shown). These results confirm the link between Stk1 and CovR in CAMP factor expression in GBS. Taken together with our results on Stk1 regulation of β-H/C, our studies provide evidence of a novel link between eukaryotic and two-component signalling mechanisms in regulation of cytotoxin expression in GBS.

Fig. 7
Increased CAMP factor expression in serotype V stk1 mutants requires CovR. Upper panel indicates CAMP factor expression in WT serotype Ia strain A909 and ΔcovR strains derived from A909 and isogenic stk1 mutants LR113 and LR114. Lower panel indicates ...

Stk1 does not regulate covR transcription

One explanation for the decrease in β-H/C and increase in CAMP factor expression in the stk1 mutants is increased transcription of covR/S. An increase in covR/S transcription could lead to enhanced repression of β-H/C and activation of CAMP factor (i.e. Δstk1 phenotype). As covR and covS are co-transcribed (Lamy et al., 2004; Jiang et al., 2005), we analysed covR transcription in WT and isogenic stk1 mutants. The RPA shown in Fig. 8 indicates that covR transcription is similar (fold difference < 2.0) in all GBS strains including IaΔcylE. These results indicate that Stk1 does not regulate covR transcription. Surprisingly, we also observed that transcription of covR was similar between the two GBS isolates A909 and NCTC10/84. These data suggest that the differences in cytotoxin expression in these isolates may not be due to altered CovR/S levels. Further analysis of the activity of CovR and CovS will provide important clues on differences in cytotoxin expression between WT GBS isolates.

Fig. 8
Transcription of covR is similar in WT and stk1 mutants. DNA-free RNA was isolated from WT GBS A909 and NCTC10/84, Δstk1 mutants LR113 and LR122, Δstp1Δstk1 mutants LR114 and LR123. RPA were performed on 25 mg total RNA from each ...

Stk1 phosphorylates CovR in vitro

We next hypothesized that Stk1 may phosphorylate either CovR or its cognate sensor CovS and hence impact CovR-mediated repression of β-H/C and activation of CAMP factor. Therefore, we performed in vitro phosphorylation assays to gain insight into the link between Stk1 and CovR. To this end, we derived C-terminal His-tag fusion constructs to the GBS covR and covS genes and recombinant fusion proteins were purified as described in Experimental procedures. Approximately 500 ng of GST-Stk1 (Glutathione S-transferase fusion), 1.5 mg CovR and 1 mg CovS fusion proteins were analysed on 10% SDS-PAGE and stained with Coomassie (see Fig. 9A).

Fig. 9
Stk1 phosphorylates CovR in vitro

Subsequently, in vitro phosphorylation reactions were performed on equal amounts of either CovR or CovS in the presence and absence of Stk1 as described in Experimental procedures. The reaction products were heated to 100°C, analysed on a 10% SDS-PAGE, stained with Coo-massie and exposed to autoradiography (see Fig. 9B). A 96 kDa protein corresponding to autophosphorylated Stk1 (GST-Stk1; Rajagopal et al., 2003) and a previously observed break-down product of Stk1 (Rajagopal et al., 2005) are seen in reactions containing Stk1 (Fig. 9B, lanes 2, 3 and 4). We did not observe autophosphorylation of either CovR or CovS in the presence of [γ-32P]ATP (Fig. 9B, lanes 1 and 5). While CovS can autophosphorylate at histidine residues, phosphohistidines are not heat stable (Rosenberg, 1996; Klumpp and Krieglstein, 2002; Ritte et al., 2002). The presence of a phosphorylated band corresponding to the 27 kDa CovR in the presence of Stk1 (Fig. 9B, lane 2) indicates that Stk1 phosphorylates CovR. The absence of a phosphorylation product corresponding to the CovS fusion protein in the presence of Stk1 (Fig. 9B, lane 4) suggests that Stk1 may not phosphorylate CovS. To further confirm that Stk1 phosphorylated CovR, we repeated the in vitro phosphorylation assays on CovR in the presence of either GST-Stk1 or equal amounts of GST protein that was purified identical to the Stk1 fusion protein. We also included significantly higher concentrations of radiolabelled ATP to enhance detection of phosphorylation (see Fig. 9C). Under these conditions, we noted that Stk1 phosphorylated CovR unlike the control GST protein. As expected, due to the high concentration of radiolabelled ATP, autophosphorylation of Stk1 was significantly greater than the intensity of phosphorylated CovR. The difference in intensity of phosphorylation of CovR compared with autophosphorylation of Stk1 may in part be due to fewer S/T residues in CovR that are phosphorylated by Stk1. These data confirm that Stk1 phosphorylates CovR in vitro. Given that the phenotypes of the stk1 mutants of GBS are indicative of increased CovR activity, we hypothesize that serine/threonine phosphorylation of CovR relieves CovR repression of β-H/C and activation of CAMP factor. The amino acid sequence of CovR reveals the presence of 14 threonine and eight serine residues. Further studies to identify the serine/threonine-phosphorylated residues of CovR and their role on CovR activity are in progress in our laboratory and will provide insight into the mechanism of interaction between discrete signalling families that regulate cytotoxin expression and adaptive responses in GBS.

Discussion

Since the discovery of eukaryotic-type serine/threonine kinases in prokaryotic organisms, a few homologues have been described to be important for virulence of bacterial pathogens. Notably, serine/threonine kinases are important for virulence of Yersinia, Mycobacteria, Streptococcus agalactiae, Streptococcus pneumoniae and possibly in Streptococcus pyogenes (Galyov et al., 1993; Rajagopal et al., 2003; Echenique et al., 2004; Walburger et al., 2004; Jin and Pancholi, 2006). However, unlike the serine/threonine kinases encoded by Yersinia (YpkA) and Mycobacteria (PknG) that are secreted into host cells (Juris et al., 2000; Walburger et al., 2004), serine/threonine kinases of Streptococci lack a secretion signal and are associated with the bacterial membrane (Rajagopal et al., 2003; Echenique et al., 2004; Jin and Pancholi, 2006). Membrane-associated serine/threonine kinases are also present in other Gram-positive pathogens such as Staphylococcus and Listeria (see Rajagopal et al., 2003 for homology alignments). These membrane-associated kinases have a predicted extracellular sensory domain and an intracellular catalytic domain (Zhang, 1996; Avenue-Gay and Everett, 2000), suggesting that they may play a role in adaptive responses of these bacteria.

In this study, we investigated the role of Stk1 in GBS virulence. Consistent with their attenuated virulence (Rajagopal et al., 2003), we show that GBS strains defective in Stk1 expression demonstrate decreased survival in human blood and increased sensitivity to phagocytic killing and oxidative stress. Further, we demonstrate that expression of β-H/C and its associated pigment that are critical for resistance of GBS to human blood, phagocytes and oxidative stress (Liu et al., 2004) is significantly lower in the stk1 mutants. Therefore, it is possible that the decrease in β-H/C and pigment synthesis may in part contribute to the attenuated virulence of the stk1 mutants.

Our studies also reveal that positive regulation of β-H/C by Stk1 is dependent on the two-component response regulator, CovR. Previous studies have described that the GBS two-component system CovR/CovS is a transcriptional repressor of β-H/C (Lamy et al., 2004; Jiang et al., 2005). We observed that Stk1 positively regulates transcription of the gene encoding β-H/C (cylE). However, as Stk1 lacks canonical DNA binding domains to directly bind DNA to regulate transcription, we tested the hypothesis that that Stk1 may negatively impact CovR repression of β-H/C. Consistent with this hypothesis, we observed that Stk1-mediated upregulation of β-H/C is abolished in the absence of CovR. These data provide evidence of a link between a eukaryotic-type kinase (Stk1) and a two-component system (CovR/CovS) in expression of the pluripotent GBS toxin, β-H/C.

The CovR/CovS system has also been described to activate transcription of another GBS cytotoxin known as CAMP factor (Lamy et al., 2004; Jiang et al., 2005). The role of CAMP factor as a virulence determinant of GBS stems from previous observations that co-administration of CAMP factor with a sublethal dose of GBS induced septicaemia and death in a mice model of GBS infection (Jurgens et al., 1987). However, to our knowledge, the virulence of a GBS strain defective only in CAMP factor biosynthesis has not been tested in animal models of infection. Therefore, the exact role of CAMP factor in GBS disease pathogenesis remains uncertain. In order to confirm the link between Stk1 and CovR in regulation of GBS gene expression, we examined CAMP factor transcription and expression in WT and isogenic stk1 mutants. Our studies reveal that stk1 mutants derived from serotype V demonstrate a fourfold increase in CAMP factor transcription and expression. This increase in CAMP factor expression observed in the serotype V stk1 mutants requires CovR. Unexpectedly, the stk1 mutants LR113 and LR114 derived from serotype Ia did not demonstrate any significant increase in transcription or expression of CAMP factor compared with the isogenic WT A909. As transcription of CAMP factor in A909 is sevenfold greater than NCTC10/84, we speculate that CAMP factor transcription may already be at a maximum in A909. Consequently, an increase in CovR activity in the A909 stk1 mutants did not further enhance CAMP factor transcription.

Our results suggest that the link between Stk1 and CovR is post-translational. Although an increase in CovR/CovS expression can contribute to enhanced repression of β-H/C and activation of CAMP factor (i.e. stk1 mutant phenotype), transcription of covR was similar between WT and the stk1 mutants in both GBS serotypes (Ia and V). These data indicate that Stk1 does not directly or indirectly regulate covR/covS transcription. Our observation that Stk1 phosphorylates CovR in vitro suggests that serine/threonine phosphorylation impacts CovR-mediated repression of β-H/C and activation of CAMP factor. Whether Stk1 impacts the ability of CovR to bind DNA and/or regulate downstream transcription or alternatively affect the stability of CovR in GBS is currently being investigated. The role of CovR as a transcriptional repressor of virulence factors in Group A Streptococcus (GAS) is well described (Federle et al., 1999; Heath et al., 1999; Dalton and Scott, 2004). However, both CovR and RocA have been shown to regulate CovR expression in GAS (Biswas and Scott, 2003; Gusa and Scott, 2005). Unlike its homologue in GAS, CovR does not autoregulate its expression in GBS (Lamy et al., 2004). Therefore, it can be expected that transcription of CovR is not affected in the GBS stk1 mutants even though Stk1 can phosphorylate and impact CovR activity.

Co-ordinate regulation of gene expression for adaptive responses in bacterial pathogens are critical for disease pathogenesis (for recent review, see Beier and Gross, 2006). However, this was thought to be primarily under the control of two-component signalling systems that sense extracellular signals and activate or repress transcription of genes important for virulence. The extracellular signals that trigger CovS-mediated activation of CovR in GBS are not known (Lamy et al., 2004; Jiang et al., 2005). Furthermore, CovS is not essential for CovR-mediated repression of β-H/C or activation of CAMP factor (Jiang et al., 2005). Previous studies (Jiang et al., 2005) and our results indicate that the sensor kinases CovS and Stk1 have a moderate effect on CovR regulation of cytotoxin expression. This might be due to the fact that both CovS and Stk1 may regulate CovR activity particularly during GBS growth in laboratory media. Further, as Stk1 is constitutively expressed during GBS growth in laboratory media (Rajagopal et al., 2005) these data suggest that its activity may not be modulated under these conditions. However, we predict that the sensor kinases CovS and Stk1 may preferentially respond to certain extracellular signals encountered during GBS infection that dictate whether Stk1 or CovS regulate CovR activity for regulation of adaptive gene expression. Such co-ordinate regulation of gene expression/virulence factor expression would be beneficial to the pathogen in the diverse environments encountered during its disease cycle. In the present study, we have described the link between Stk1 and CovR in regulation of GBS cytotoxins. However, CovR has been shown to repress transcription of 76 genes and activate transcription of 63 genes in GBS that includes β-H/C, CAMP factor, cell wall and metabolic components as well as other putative transcriptional factors (Lamy et al., 2004). These studies indicate the importance of CovR as a global regulator of GBS gene expression. Analysis of the role of Stk1 and CovS on expression of other CovR regulated genes and identification of signals that activate CovS or Stk1 will delineate converging signalling mechanisms that regulate adaptive responses and virulence of GBS.

A key discovery in our analysis of eukaryotic-type signalling in GBS is that regulation of gene expression is not exclusively determined by two-component systems in organisms that also harbour eukaryotic-type signalling enzymes. Although the coexistence of eukaryotic-type kinases and phosphatases in prokaryotes (Kennelly, 2002) and bacterial two-component systems in eukaryotes (Loomis et al., 1997) has been described, coupling of these phosphotransfer families in a single signalling pathway was established in a few organisms. Responses to ethylene and osmotic stress in Arabidopsis thaliana and Saccharomyces cerevisiae involve two-component systems that sense extracellular signals and regulate downstream MAP (serine/threonine) kinase signalling pathways (Maeda et al., 1994; Sato et al., 2003; Stepanova and Alonso, 2005). Recently, expression of the transcriptional regulator (MrpC) was shown to be independently regulated by a eukaryotic-type kinase (Pkn14) and a two-component system (MrpA/B, see Nariya and Inouye, 2005) in the non-pathogenic Myxococcus xanthus. A mycobacterial serine/threonine kinase (PknH) was described to phospho-rylate a transcriptional regulator EmbR, the eukaryotic-like, forkhead-associated (FHA) domain in EmbR was shown to be essential for PknH phosphorylation (Molle et al., 2003; Sharma et al., 2006). It is indeed interesting that FHA domains are not present in Gram-positive pathogens such as GBS, GAS, Listeria or Staphylococcus (Pallen et al., 2002; Madera et al., 2004) that contain serine/threonine kinase homologues. These data imply that FHA domains are not the target for serine/threonine protein kinase in these bacterial pathogens. Our studies indicate that the eukaryotic-type serine/threonine kinase Stk1 directly phosphorylates a classical two-component response regulator CovR and affects CovR-mediated regulation of GBS cytotoxins. These observations suggest that pathogenic organisms such as GBS harbour multiple signalling mechanisms to fine tune their ability to regulate expression of virulence factors and adaptive responses. Identification of the target amino acids of CovR that are phosphorylated by Stk1 and the role of the sensor kinases Stk1 and CovS on CovR-mediated gene expression will delineate complex and intertwined signalling mechanisms that regulate GBS virulence.

Experimental procedures

Bacterial strains and plasmids

Bacterial strains, plasmids and primers used in this study are listed in Table 2. All chemicals were purchased from Sigma-Aldrich, USA, unless mentioned otherwise. Routine recombinant DNA techniques were performed as described (Sambrook et al., 1989). Open reading frame (ORF) and BLAST homology searches were performed using the NCBI Internet server (http://www.ncbi.nlm.nih.gov). Molecular biology reagents were purchased from New England Biolabs (NEB), USA.

Table 2
Bacterial strains, plasmids and primers.

Group B Streptococci were cultured in Todd Hewitt Broth (THB, Difco) in 5% CO2 at 37°C. The WT GBS strains used in this study are A909 (Madoff et al., 1991) and NCTC10/84 (Wilkinson, 1977). A909 is a clinical isolate of GBS that belongs to the capsular polysaccharide serotype Ia (Madoff et al., 1991). LR113 is a derivative of A909 and has a WKm-2 insertion cassette within the coding region [amino acid 13 (aa13)] of stk1; this strain is deficient for Stk1 expression, as demonstrated previously (Rajagopal et al., 2003). LR114 is isogenic to A909; insertion of the ΩKm-2 cassette at aa13 of stk1 abolished Stk1 expression, identical to LR113. In addition, the coding sequence of the gene encoding stp1 was allelically replaced with a gene conferring resistance to chloramphenicol (cat) in LR114 (Rajagopal et al., 2003). Control GBS strains used in this study are the non-haemolytic Type IaΔcylE (Pritzlaff et al., 2001) and sia-CPS-deficient A909ΔDcpsE (Jones et al., 2003) and are isogenic to A909. Complementing plasmids used in this study are described in Rajagopal et al. (2003) and are listed in Table 2. Of note, as both the Δstk1 and Δstk1Δstp1 strains display similar phenotypes (see Results) and are defective in Stk1 expression, for convenience, we have often referred to these strains as stk1 mutants.

Construction of serotype V stk1 mutants

To support conservation of the role of Stk1 in GBS, we also constructed Stk1-deficient strains from the WT GBS strain NCTC10/84. NCTC10/84 is a clinical isolate of GBS that belongs to the capsular polysaccharide serotype V (Pritzlaff et al., 2001). LR122 is a Δstk1 mutant and LR123 is a double Δstp1Δstk1 mutant derived from NCTC10/84 as described (Rajagopal et al., 2003). Briefly, the temperature-sensitive plasmid pLR5 containing a kanamycin insertion in aa13 of stk1 was introduced into WT NCTC10/84 and selection for the Δstk1 mutant was performed as described (Rajagopal et al., 2003). To obtain the double Δstp1Δstk1 mutant, the plasmid pLR8 containing both the kanamycin insertion in aa13 of stk1 and an allelic replacement of stp1 with cat was introduced into NCTC10/84. Subsequent selection and screening for the Δstp1Δstk1 mutant was performed as described (Rajagopal et al., 2003). Autophosphorylation of membrane fractions of GBS confirmed the absence of Stk1 activity in these strains (see Fig. S1). RT-PCR was used to confirm Stp1 expression in LR122 and its absence in LR123 as described (Rajagopal et al., 2003).

Construction of covR mutants

GBS strains defective in CovR expression (ΔcovR) were derived from A909, LR113, LR114, NCTC10/84, LR122 and LR123 for this study. Selection for ΔcovR mutants was performed using the plasmid pJR233csrRD::Sp (csrR is also known as covR) as described (Jiang et al., 2005).

Whole blood bactericidal assays

Bactericidal assays in NIHWB were performed as described in Jones et al. (2003). Briefly, approximately 103 colony forming units (cfu) of each GBS strain were mixed with 400 ml of freshly drawn NIHWB. Blood was obtained following consent from non-immune individuals as per institutional guidelines. As controls, 400 ml of human plasma (isolated from blood of the same donor) was mixed with 103 cfu for each strain. All samples were incubated for 3 h at 37°C and were plated in triplicate on THA plates for enumeration of cfu. SI was calculated using the formula: (cfu in NIHWB)/(cfu in human plasma). To test if addition of exogenous purines to human blood or plasma can stimulate growth of the stk1 mutants, adenine, guanine, xanthine and the pyrimidine uracil were added at a final concentration of 0.1 mM (Rajagopal et al., 2005). SI was calculated relative to the growth of each strain in human blood and plasma containing purines.

Phagocytic killing assays

Opsonophagocytic killing assays were performed and analysed as described (Jones et al., 2003). Briefly, GBS strains were opsonized in 10% normal human serum and incubated with human PMN at a ratio of 1:3 [bacteria: PMN] for 1 h. For each GBS strain, controls included samples containing PMN and heat-inactivated sera and samples lacking PMN but containing human sera. The per cent kill for each strain was calculated relative to the growth of the same strain in the absence of PMN.

H2O2 killing assay

H2O2 killing assays were performed as described in Liu et al. (2004). Exponential phase GBS cultures (OD = 0.6) wer exposed to H2O2 at a final concentration of 0.015% for 1 h. Subsequently, 1000 units of catalase (Sigma, USA) was added to quench remaining H2O2 and the bacteria were plated on THA medium for enumeration of cfu. SI was calculated using the formula: (cfu at the end of assay)/(cfu at time 0). All experiments were performed in triplicate and repeated at least three times.

Estimation of β-haemolysin (β-H/C) titers

β-H/C titers were estimated from overnight GBS cultures using methods described in Nizet et al. (1996). Briefly, approximately 109 cfu for each GBS strain was centrifuged and resuspended in 1/10 original volume of PBSGS (PBS + 1% glucose + 2% starch) and incubated at 37°C for 1 h. Subsequently, the GBS cells were centrifuged and supernatants containing starch bound β-H/C was extracted. Twofold serial dilutions of the β-H/C extract from each strain were incubated with an equal volume of 1% sRBC in 96 well plates at 37°C for 1 h. Following that, the plates were spun at 3000 g for 10 min to pellet unlysed sRBC. The supernatants were transferred to a replica 96 well plate and haemoglobin release was measured by recording the absorbance at 420 nm. Positive and negative controls included wells that contained sRBC with 0.1% SDS or PBSG respectively. Haemolytic titers for a given strain was determined as the reciprocal of the greatest dilution producing 50% haemoglobin release compared with the SDS control. Pigment extracts were prepared and analysed as described (Liu et al., 2004).

CAMP factor expression and titers

CAMP factor expression on blood agar plates were performed by cross streaking a β-lysin producing Staphylococcus aureus strain with GBS as described (Tapsall and Phillips, 1987; Jiang et al., 2005). Estimation of CAMP factor titers were performed as described (Ross et al., 1999). Briefly, sRBC were presensitized with a sphingomyelinase solution prepared by soaking one Beta Lysin disk (Remel, USA) in 0.5 ml of Tris Buffer as described (Ross et al., 1999). Supernatants obtained from overnight GBS cultures containing approximately equal cell numbers were filtered through a 0.22-mm-pore-size membrane. Twofold serial dilutions of culture supernatants that contain CAMP factor were incubated with an equal volume of presensitized sRBCs for 30 min at 30°C as described (Ross et al., 1999). Fifty microlitres of Tris buffer or THB media was used as a negative control and 0.1% SDS was used as a positive control. Unlysed sRBC was centrifuged and the absorbance was recorded at 490 nm and the titer was defined as the reciprocal of the greatest dilution producing 50% haemoglobin release compared with the SDS control.

RNase protection assays

Isolation of total RNA

Total RNA was isolated from exponential phase (OD = 0.6) GBS cultures using the Qiagen RNA isolation kit (Qiagen, USA). The isolated RNA was treated with DNase (Promega, USA) as described by the manufacturer and control PCR reactions were performed to confirm the absence of DNA in the RNA samples.

Construction and labelling of probes

Internal coding regions to cylE, cfb and covR were amplified from A909 chromosomal DNA using high-fidelity PCR (Invitrogen, USA). The following primer pairs were used to make specific probes: for cylE, RPAcylEF and RPAcylER; for cfb, RPAcfbF and RPAcfbR; and for covR, RPAcovRF and RPAcovRR. The reverse primers were engineered to incorporate a T7 promoter such that the transcript was generated in the antisense orientation. A MAXIscript® T7 in vitro transcription kit (Ambion, USA) was used as directed by the manufacturer to create [α32P]UTP-labelled antisense RNA from the resulting PCR products.

RNase protection assays

RPA were performed on 25 mg total RNA with antisense mRNA probe to cylE (250 bp), covR (250 bp) and cfb (150 bp) using the HybspeedRPA kit (Ambion, USA) as described by the manufacturer. Antisense RNA probes had a specific activity ranging from 1 × 109 μg−1 to 2 × 109μg−1 and fourfold molar excess probe was used in each assay as described by the manufacturer. Visualization and quantification of mRNA transcripts were performed using the Typhoon 9710 Phosphoimager and ImageQuant 5.2. RPA were repeated at least four times. Fold changes in transcription were measured relative to the isogenic WT strain and statistical significance was evaluated using paired two-tailed t-tests.

Purification of recombinant proteins and in vitro phosphorylation

DNA fragments containing covR and covS were PCR amplified using high-fidelity PCR from WT A909 and cloned in frame into pET32ck (Seepersaud et al., 2006) to obtain C-terminal His-tag fusion proteins respectively. The following primer pair’s CovRFNcoI & CovRR and CovSF & CovSR were used for amplification of full-length covR and covS respectively. The PCR products were digested with the enzymes for which, restriction sites were engineered in the primers, cloned in frame into the MCS of pET32ck to obtain C-terminal His6 fusion proteins respectively. The plasmid pLR136 encodes CovR-His6 and plasmid pLR137 encodes CovS-His6 fusion proteins on IPTG induction respectively. The His-tagged CovR fusion protein is soluble and was purified using Ni-NTA Superflow as described by the manufacturer (Qiagen, USA). The His-tagged CovS fusion protein was purified under denaturing conditions as described (Seepersaud et al., 2005). Briefly, induced cell pellets were resuspended in denaturation buffer containing 8 M urea, 0.1 M sodium phosphate [pH 7.5], 300 mM NaCl, 10% glycerol and incubated overnight at 4°C. The clarified lysate was mixed with Ni-NTA Superflow for 2 h at 4°C. The resin was washed with a gradient of 8–0 M urea in 0.1 M sodium phosphate (pH 7.5)-300 mM NaCl-10% glycerol, and recombinant protein was eluted in elution buffer as described (Seepersaud et al., 2005). Purification of the full-length Stk1 fusion protein (GST-Stk1) and in vitro phosphorylation reactions were performed as described (Rajagopal et al., 2003). Of note, the GST-Stk1 protein is relatively insoluble due to the presence of its transmembrane domain and hence a purification protocol for insoluble GST fusion proteins was used to purify the GST-Stk1 as described (Rajagopal et al., 2003). Due to its insoluble nature, the GST-Stk1 fusion protein is usually obtained at a final concentration of 0.1 mg ml−1. Approximately 500 ng of purified Stk1 (GST-Stk1) was incubated with equal amounts of either CovR or CovS in kinase buffer containing 10 μCi [γ-32P] ATP for 15 min. Controls included reactions not containing Stk1 and those containing Stk1 alone. Subsequently, all reactions were heated to 100°C for 5 min as phosphoserine and phosphothreonine are heat stable unlike phosphohistidine (Rosenberg, 1996; Ritte et al., 2002). The samples were then analysed on 10% SDS-PAGE, stained with Coomassie and exposed to autoradiography.

Acknowledgments

We thank Dr Michael R. Wessels for the kind gift of the plasmid pJR233csrRD::Sp and Dr Victor Nizet for the strain IaΔcylE. We are grateful to Dr George Liu for advice on β-H/C assays. We acknowledge Meriyana Fnu and Hang Tran for technical assistance. We are grateful to Kristy Seidel for biostatistical assistance. This work was supported by funding from the National Institutes of Health, Grant # RO1 AI056073 to CER and CHRMC Basic Science Steering Committee Award to L.R.

References

  • Av-Gay Y, Everett M. The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis. Trends Microbiol. 2000;8:238–244. [PubMed]
  • Baker CJ, Edwards MW. Group B streptococcal infections. In: Remington JS, Klein JO, editors. Infectious Diseases of the Fetus and Newborn Infant. Philadelphia, PA: W.B. Saunders; 1995. pp. 980–1054.
  • Beier D, Gross R. Regulation of bacterial virulence by two-component systems. Curr Opin Microbiol. 2006;9:143–152. [PubMed]
  • Biswas I, Scott JR. Identification of rocA, a positive regulator of covR expression in the group A streptococcus. J Bacteriol. 2003;185:3081–3090. [PMC free article] [PubMed]
  • Chaffin DO, Rubens CE. Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression. Gene. 1998;219:91–99. [PubMed]
  • Cohen P. Protein kinases – the major drug targets of the twenty-first century? Nat Rev Drug Discov. 2002;1:309–315. [PubMed]
  • Dalton TL, Scott JR. CovS inactivates CovR and is required for growth under conditions of general stress in Streptococcus pyogenes. J Bacteriol. 2004;186:3928–3937. [PMC free article] [PubMed]
  • Doran KS, Nizet V. Molecular pathogenesis of neonatal group B streptococcal infection: no longer in its infancy. Mol Microbiol. 2004;54:23–31. [PubMed]
  • Doran KS, Chang JC, Benoit VM, Eckmann L, Nizet V. Group B streptococcal beta-hemolysin/cytolysin promotes invasion of human lung epithelial cells and the release of interleukin-8. J Infect Dis. 2002;185:196–203. [PubMed]
  • Echenique J, Kadioglu A, Romao S, Andrew PW, Trombe MC. Protein serine/threonine kinase StkP positively controls virulence and competence in Streptococcus pneumoniae. Infect Immun. 2004;72:2434–2437. [PMC free article] [PubMed]
  • Edwards MS, Kasper DL, Jennings HJ, Baker CJ, Nicholson-Weller A. Capsular sialic acid prevents activation of the alternative complement pathway by type III, group B streptococci. J Immunol. 1982;128:1278–1283. [PubMed]
  • Fang FC. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat Rev Microbiol. 2004;2:820–832. [PubMed]
  • Federle MJ, McIver KS, Scott JR. A response regulator that represses transcription of several virulence operons in the group A streptococcus. J Bacteriol. 1999;181:3649–3657. [PMC free article] [PubMed]
  • Foussard M, Cabantous S, Pedelacq J, Guillet V, Tranier S, Mourey L, et al. The molecular puzzle of two-component signaling cascades. Microbes Infect. 2001;3:417–424. [PubMed]
  • Galyov EE, Hakansson S, Forsberg A, Wolf-Watz H. A secreted protein kinase of Yersinia pseudotu-berculosis is an indispensable virulence determinant. Nature. 1993;361:730–732. [PubMed]
  • Glaser P, Rusniok C, Buchrieser C, Chevalier F, Frangeul L, Msadek T, et al. Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease. Mol Microbiol. 2002;45:1499–1513. [PubMed]
  • Gusa AA, Scott JR. The CovR response regulator of group A streptococcus (GAS) acts directly to repress its own promoter. Mol Microbiol. 2005;56:1195–1207. [PubMed]
  • Harris TO, Shelver DW, Bohnsack JF, Rubens CE. A novel streptococcal surface protease promotes virulence, resistance to opsonophagocytosis, and cleavage of human fibrinogen. J Clin Invest. 2003;111:61–70. [PMC free article] [PubMed]
  • Heath A, DiRita VJ, Barg NL, Engleberg NC. A two-component regulatory system, CsrR-CsrS, represses expression of three Streptococcus pyogenes virulence factors, hyaluronic acid capsule, streptolysin S, and pyrogenic exotoxin B. Infect Immun. 1999;67:5298–5305. [PMC free article] [PubMed]
  • Hensler ME, Liu GY, Sobczak S, Benirschke K, Nizet V, Heldt GP. Virulence role of group B Streptococcus beta-hemolysin/cytolysin in a neonatal rabbit model of early-onset pulmonary infection. J Infect Dis. 2005;191:1287–1291. [PubMed]
  • Jiang SM, Cieslewicz MJ, Kasper DL, Wessels MR. Regulation of virulence by a two-component system in group B streptococcus. J Bacteriol. 2005;187:1105–1113. [PMC free article] [PubMed]
  • Jin H, Pancholi V. Identification and biochemical characterization of a eukaryotic-type serine/threonine kinase and its cognate phosphatase in Streptococcus pyogenes: their biological functions and substrate identification. J Mol Biol. 2006;357:1351–1372. [PubMed]
  • Jones AL, Needham RH, Clancy A, Knoll KM, Rubens CE. Penicillin-binding proteins in Streptococcus agalactiae: a novel mechanism for evasion of immune clearance. Mol Microbiol. 2003;47:247–256. [PubMed]
  • Jurgens D, Sterzik B, Fehrenbach FJ. Unspecific binding of group B streptococcal cocytolysin (CAMP factor) to immunoglobulins and its possible role in pathogenicity. J Exp Med. 1987;165:720–732. [PMC free article] [PubMed]
  • Juris SJ, Rudolph AE, Huddler D, Orth K, Dixon JE. A distinctive role for the Yersinia protein kinase: actin binding, kinase activation, and cytoskeleton disruption. Proc Natl Acad Sci USA. 2000;97:9431–9436. [PMC free article] [PubMed]
  • Kennelly PJ. Protein kinases and protein phosphatases in prokaryotes: a genomic perspective. FEMS Microbiol Lett. 2002;206:1–8. [PubMed]
  • Klumpp S, Krieglstein J. Phosphorylation and dephosphorylation of histidine residues in proteins. Eur J Biochem. 2002;269:1067–1071. [PubMed]
  • Lamy MC, Zouine M, Fert J, Vergassola M, Couve E, Pellegrini E, et al. CovS/CovR of group B streptococcus: a two-component global regulatory system involved in virulence. Mol Microbiol. 2004;54:1250–1268. [PubMed]
  • Liu GY, Doran KS, Lawrence T, Turkson N, Puliti M, Tissi L, Nizet V. Sword and shield: linked group B streptococcal beta-hemolysin/cytolysin and carotenoid pigment function to subvert host phagocyte defense. Proc Natl Acad Sci USA. 2004;101:14491–14496. [PMC free article] [PubMed]
  • Loomis WF, Shaulsky G, Wang N. Histidine kinases in signal transduction pathways of eukaryotes. J Cell Sci. 1997;110(Pt 10):1141–1145. [PubMed]
  • Madera M, Vogel C, Kummerfeld SK, Chothia C, Gough J. The SUPERFAMILY database in 2004: additions and improvements. Nucleic Acids Res. 2004;32:D235–D239. [PMC free article] [PubMed]
  • Madoff LC, Michel JL, Kasper DL. A mono-clonal antibody identifies a protective C-protein alpha-antigen epitope in group B streptococci. Infect Immun. 1991;59:204–210. [PMC free article] [PubMed]
  • Maeda T, Wurgler-Murphy SM, Saito H. A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature. 1994;369:242–245. [PubMed]
  • Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science. 2002;298:1912–1934. [PubMed]
  • Marques MB, Kasper DL, Pangburn MK, Wessels MR. Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci. Infect Immun. 1992;60:3986–3993. [PMC free article] [PubMed]
  • Molle V, Kremer L, Girard-Blanc C, Besra GS, Cozzone AJ, Prost JF. An FHA phosphoprotein recognition domain mediates protein EmbR phosphorylation by PknH, a Ser/Thr protein kinase from Mycobacterium tuberculosis. Biochemistry. 2003;42:15300–15309. [PubMed]
  • Nariya H, Inouye S. Identification of a protein Ser/Thr kinase cascade that regulates essential transcriptional activators in Myxococcus xanthus development. Mol Microbiol. 2005;58:367–379. [PubMed]
  • Nizet V. Streptococcal beta-hemolysins: genetics and role in disease pathogenesis. Trends Microbiol. 2002;10:575–580. [PubMed]
  • Nizet V, Gibson RL, Chi EY, Framson PE, Hulse M, Rubens CE. Group B streptococcal beta-hemolysin expression is associated with injury of lung epithelial cells. Infect Immun. 1996;64:3818–3826. [PMC free article] [PubMed]
  • Pallen M, Chaudhuri R, Khan A. Bacterial FHA domains: neglected players in the phospho-threonine signalling game? Trends Microbiol. 2002;10:556–563. [PubMed]
  • Podbielski A, Blankenstein O, Lutticken R. Molecular characterization of the cfb gene encoding group B streptococcal CAMP-factor. Med Microbiol Immunol (Berl) 1994;183:239–256. [PubMed]
  • Pritzlaff CA, Chang JC, Kuo SP, Tamura GS, Rubens CE, Nizet V. Genetic basis for the beta-haemolytic/cytolytic activity of group B Streptococcus. Mol Microbiol. 2001;39:236–247. [PubMed]
  • Rajagopal L, Clancy A, Rubens CE. A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth, cell segregation, and virulence. J Biol Chem. 2003;278:14429–14441. [PubMed]
  • Rajagopal L, Vo A, Silvestroni A, Rubens CE. Regulation of purine biosynthesis by a eukaryotic-type kinase in Streptococcus agalactiae. Mol Microbiol. 2005;56:1329–1346. [PMC free article] [PubMed]
  • Ritte G, Lloyd JR, Eckermann N, Rottmann A, Koss-mann J, Steup M. The starch-related R1 protein is an alpha-glucan, water dikinase. Proc Natl Acad Sci USA. 2002;99:7166–7171. [PMC free article] [PubMed]
  • Rosenberg IM. Protein Analysis and Purification. Boston, MA: Birkhäuser; 1996.
  • Ross RA, Madoff LC, Paoletti LC. Regulation of cell component production by growth rate in the group B Streptococcus. J Bacteriol. 1999;181:5389–5394. [PMC free article] [PubMed]
  • Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning; A Laboratory Manual. Plainview, NY: Cold Spring Harbor Laboratory Press; 1989.
  • Sato N, Kawahara H, Toh-e A, Maeda T. Phosphorelay-regulated degradation of the yeast Ssk1p response regulator by the ubiquitin-proteasome system. Mol Cell Biol. 2003;23:6662–6671. [PMC free article] [PubMed]
  • Seepersaud R, Hanniffy SB, Mayne P, Sizer P, Le Page R, Wells JM. Characterization of a novel leucine-rich repeat protein antigen from group B streptococci that elicits protective immunity. Infect Immun. 2005;73:1671–1683. [PMC free article] [PubMed]
  • Seepersaud R, Needham RH, Kim CS, Jones AL. Abundance of the {delta} subunit of RNA poly-merase is linked to the virulence of Streptococcus agalactiae. J Bacteriol. 2006;188:2096–2105. [PMC free article] [PubMed]
  • Sharma K, Gupta M, Pathak M, Gupta N, Koul A, Sarangi S, et al. Transcriptional control of the mycobacterial embCAB operon by PknH through a regulatory protein, EmbR, in vivo. J Bacteriol. 2006;188:2936–2944. [PMC free article] [PubMed]
  • Spellerberg B, Pohl B, Haase G, Martin S, Weber-Heynemann J, Lutticken R. Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition. J Bacteriol. 1999;181:3212–3219. [PMC free article] [PubMed]
  • Spellerberg B, Martin S, Brandt C, Lutticken R. The cyl genes of Streptococcus agalactiae are involved in the production of pigment. FEMS Microbiol Lett. 2000;188:125–128. [PubMed]
  • Stepanova AN, Alonso JM. Arabidopsis ethylene signaling pathway. Sci STKE 2005. 2005:cm4. [PubMed]
  • Tapsall JW, Phillips EA. Presumptive identifi-cation of group B streptococci by rapid detection of CAMP factor and pigment production. Diagn Microbiol Infect Dis. 1987;7:225–228. [PubMed]
  • Tettelin H, Masignani V, Cieslewicz MJ, Eisen JA, Peterson S, Wessels MR, et al. Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc Natl Acad Sci USA. 2002;99:12391–12396. [PMC free article] [PubMed]
  • Walburger A, Koul A, Ferrari G, Nguyen L, Prescianotto-Baschong C, Huygen K, et al. Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science. 2004;304:1800–1804. [PubMed]
  • Wertman KF, Wyman AR, Botstein D. Host/vector interactions which affect the viability of recombinant phage lambda clones. Gene. 1986;49:253–262. [PubMed]
  • Wilkinson HW. Nontypable group B streptococci isolated from human sources. J Clin Microbiol. 1977;6:183–184. [PMC free article] [PubMed]
  • Wilkinson HW, Eagon RG. Type-specific antigens of group B type Ic streptococci. Infect Immun. 1971;4:596–604. [PMC free article] [PubMed]
  • Zhang CC. Bacterial signalling involving eukaryotic-type protein kinases. Mol Microbiol. 1996;20:9–15. [PubMed]

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...