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Bose Institute, Department of Microbiology, P1/12 CIT Scheme VIIM, Kolkata 700054, India
Correspondence
Sujoy K. Das Gupta
sujoy{at}boseinst.ernet.in
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
A substantial amount of information regarding stress tolerance pathways is available in the case of Escherichia coli, where it has been shown that the regulation is positively controlled by heat-shock sigma factors 
32 (Bukau, 1993; Georgopoulos et al., 1994; Yura et al., 1993) and E (Raina et al., 1995; Rouviere et al., 1995). Stress genes may also be negatively controlled by repressors. One such repressor, HrcA, which binds to a DNA sequence named CIRCE (controlling inverted repeat of chaperone expression) (Babst et al., 1996; Bahl et al., 1995; Schulz & Schumann, 1996; Segal & Ron, 1996; Zuber & Schumann, 1994), is the most widespread and controls the expression of dnaK and groE operons in several Gram-positive bacteria. Another well-known regulator is HspR, which binds to a sequence known as HAIR (HspR-associated inverted repeats) (Grandvalet et al., 1999). In Mycobacterium tuberculosis, HspR is expressed from the dnaK-grpE-dnaJ-hspR operon. HspR has been demonstrated to act as a repressor of this operon. Subsequent evidence has indicated that the expression of not only the dnaKJE genes, but also several other genes of M. tuberculosis, is repressed by HspR. One of these, acr2, encodes an 
-crystallin family protein which appears to be linked to virulence/pathogenesis (Stewart et al., 2005; Wilkinson et al., 2005). HspR in itself may be a virulence factor, since its knockout resulted in loss of persistence (Stewart et al., 2003). The mechanism by which this happens is not known in detail but it has been suggested that in hspR mutants, chaperones such as DnaK, which are highly antigenic, are overexpressed and this results in a better immune response in the host. However, since HspR controls several other virulence-related genes such as acr2 (Stewart et al., 2002), the overall mechanism of HspR action could be more complex than is presently thought. Binding of mycobacterial HspR to HAIR has been demonstrated using in vitro renatured protein (Stewart et al., 2001). However, this binding was not heat sensitive. It has been suggested that the presence of other factors could play a role in conferring heat sensitivity. In later studies, using a similar protein from Streptomyces coelicolor (Bucca et al., 2000, 2003), it was shown that DnaK acts as a co-repressor for HspR. Both these observations indicate that the activity of HspR-like regulators is possibly controlled by multiple factors, one of which is certainly DnaK.
Given the importance of HspR in regulating stress responses as well as virulence-related genes in M. tuberculosis, its DNA-binding activity was investigated with particular focus on the involvement of chaperones such as DnaK and GroEL in the process. The results show that whereas DnaK does indeed act as a co-repressor, as reported earlier (Bucca et al., 2000, 2003), its activity is positively modulated by GroEL and DnaJ, and negatively by GrpE.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Mycobacterial DnaK was purified after expression from plasmid pMRLB.6 dnak/RV0350 (acquired through ‘TB Research Materials & Vaccine Testing’, NIH, NIAID, based at Colorado State University) in E. coli BL21(DE3). Strains expressing mycobacterial GroELs 1 and 2 were obtained as gifts from Dr Sekhar Mande, CDFD, Hyderabad, India.
Construction of expression plasmid.
The M. tuberculosis HspR expression plasmid pTD-R30 was constructed by cloning the PCR-amplified 381 bp fragment of the hspR gene (RV0353) at the BamHI-HindIII site of pQE30 (Qiagen), using mycobacterial genomic DNA as template and 5'-CGGGATCCATGGCGAAGAACCC-3' and 5'-CCCAAGCTTTCACCGGCGCGGTTT-3' as forward and reverse primers, respectively. The protein expressed from the recombinant plasmid contains a stretch of six histidine residues at the N-terminus. Clones expressing M. tuberculosis DnaJ (RV0352) and GrpE (RV0351) were constructed in the same way by cloning the respective PCR amplicons in the expression vector pQE30.
Chemicals.
Chromatography material (Ni2+-NTA agarose), was purchased from Qiagen. ATP-
S was purchased from Calbiochem. Other chemicals for protein expression, purification and analysis were of the highest purity grade, obtained from SRL Laboratories, India. -32P-labelled ATP (12x1013 Bq mmol–1) was purchased from BRIT (Mumbai, India). Restriction enzymes and DNA-modifying enzymes, such as polynucleotide kinase and T4 DNA ligase, were obtained from New England Biolabs.
Purification of proteins.
All the proteins used were N-terminally hexahistidine tagged. HspR was isolated by Ni2+-NTA agarose chromatography under denaturing conditions in the presence of 8 M urea via a standard protocol. Briefly, E. coli cells harbouring the HspR-expressing plasmid pTD-R30 were grown up to an OD600 of 0.6 at 37 °C in 500 ml Luria broth containing 100 µg ampicillin ml–1 as antibiotic. Expression of HspR was induced by the addition of 1 mM IPTG. Harvested cells were sonicated in lysis buffer (50 mM KH2PO4, 10 mM Tris/HCl, pH 8.0, 300 mM KCl, 8 M urea) and centrifuged at 10 000 g. The cleared lysate was loaded on to the affinity column. The column-bound HspR was washed with wash buffer (50 mM KH2PO4, 10 mM Tris/HCl, pH 6.3, 300 mM KCl, 8 M urea). The protein was then eluted using elution buffer (50 mM KH2PO4, 10 mM Tris/HCl, pH 4.5, 300 mM KCl, 8 M urea). In some experiments, HspR was also refolded spontaneously in-column without the aid of any folding-promoting agent. This was done by washing the column with 8 M urea in 50 mM Tris/HCl, 150 mM KCl, pH 8.0, followed by renaturation using a 100 ml gradient from 6 M urea in buffer A (50 mM Tris/HCl, 150 mM KCl, pH 8.0) to the buffer alone. The renatured protein was then eluted under native conditions using imidazole-containing buffer (50 mM KH2PO4, pH 7.5, 300 mM KCl, 250 mM imidazole). The resulting protein was dialysed against phosphate buffer (25 mM KH2PO4, pH 7.5, 200 mM KCl) containing 15 % (v/v) glycerol. DnaK, GroEL1, GroEL2, DnaJ and GrpE were isolated through Ni2+-NTA agarose chromatography under native conditions. In the case of GroEL1, the non-ionic detergent N-lauryol sarcosine was added at a concentration of 10 mM in the lysis buffer during sonication (Qamra et al., 2004).
Electrophoretic mobility shift assay (EMSA).
For EMSA a 110 bp DNA fragment derived from the DnaK promoter region (–128 to –38), encompassing two inverted HAIR repeats, was PCR amplified by using primers K-128F (5'-GCTCTAGAAGCGTTAGCATGCTCAGTAAGTTG-3') and K-38R (5'-GCTCTAGATCTTTCCTGCACTATGATGAG-3'). Ten picomoles of K-128F was 5' end-labelled with [-32P]ATP (12x1013 Bq mmol–1) (BRIT) and T4 polynucleotide kinase (New England Biolabs) and directly used in PCR. The PCR product was purified using a Qiagen column. The binding reactions contained (30 µl final volume) 3 µl 10x binding buffer (100 mM Tris/HCl, pH 8, 600 mM KCl, 30 mM MgCl2, 1 mM EDTA, 1 mM DTT, 20 % v/v glycerol), 1 µg salmon sperm DNA, 10 000 c.p.m (
3 ng) labelled DNA, and the required amount of purified proteins. The reaction mixtures were pre-incubated for 5 min with salmon sperm DNA and then for an additional 15 min on ice, after addition of the probe. The DNA–protein complexes were separated on a 4 %, 40 : 1, native PAGE by electrophoresis in 0.5x Tris/borate buffer (50 mM Tris/borate, 1 mM EDTA) at 150 V for 3–4 h at 4 °C after a pre-run at 100 V for 1 h. Following electrophoresis, the gel was dried, and the bands were visualized by autoradiography. In the case of binding in the presence of chaperones, HspR was pre-incubated without salmon sperm DNA for 30 min in the presence of desired chaperones at specified concentrations, followed by the addition of salmon sperm DNA and then the probe.
Antibody supershift EMSA experiments were performed using specific antisera raised against the affinity-purified recombinant proteins as described earlier (Basu et al., 2002). In each case 1 µl antiserum (either immune or pre-immune) was added directly into the binding mixture followed by incubation for an additional 10 min.
Refolding experiment.
HspR (1 µg) isolated under denaturing conditions was refolded in a final volume of 100 µl by dilution in renaturation buffer containing 20 mM Tris/HCl, pH 7.5, 1 mM DTT, 10 mM KCl, 1 mM MgCl2 and 0.5 mM ATP. In some experiments ATP was omitted or replaced by the non-hydrolysable analogue ATP-S. Renaturation was done in either the absence or presence of the chaperones for 1 h. After this time, aliquots (25 µl) were removed and added to the DNA-binding assays. All DNA-binding assays were performed in a final volume of 30 µl. After 5 min pre-incubation in the presence of salmon sperm DNA, probe binding was done for 15 min as mentioned above. Binding was then monitored by EMSA. For a kinetic analysis, the refolding period was divided into four time intervals of 15 min each.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). That the observed binding did not arise out of direct interaction of DnaK with the target was confirmed by performing a control experiment in which HspR was omitted (Fig. 1b, lanes 1 and 2). In contrast to DnaK, no stimulatory effect was encountered in the case of either of the mycobacterial GroELs, GroEL1 and GroEL2 (Fig. 1c and d, respectively). In order to investigate whether these GroELs play a role in the activation process in the presence of DnaK, a binding assay was set up in which the HspR concentration was lowered fourfold relative to that used in Fig. 1(a) and the DnaK concentration was kept at the lowest level. Under these conditions, neither HspR nor the DnaK-HspR combination showed any binding (Fig. 1e, lanes 2 and 6). GroEL1 and GroEL2 were then added either under refolding conditions (in the presence of ATP and pre-incubation in the presence of chaperones for 1 h) or directly into the binding assay. The results (Fig. 1e, lanes 3, 4, 7 and 8) show significant stimulation by both the GroELs irrespective of the manner in which they were used. GroEL requirement, however, appeared to be essential only under limiting conditions. This was evident from the activity observed using a higher concentration of HspR (Fig. 1e, lanes 10–12). In this case, no additional stimulatory effect was observed with the GroELs over and above that obtained in the presence of DnaK.
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) showed that the presence of GroEL1 aided the refolding of heat-denatured HspR in a dose-dependent manner. Maximum activity was encountered when the molar ratio between DnaK and GroEL1 was approximately 1 : 1. The reactivation kinetics of heat-treated HspR was then investigated either with DnaK alone or with DnaK in the presence of GroEL1 or GroEL2. The results showed that within the stipulated time of 1 h, DnaK failed to reactivate HspR, but in the presence of either of the GroELs, a time-dependent increase in activity was observed (Fig. 2b, lanes 5–8 and 9–12). These results confirm that the presence of GroEL is essential for efficient refolding of HspR.
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, respectively) was allowed to refold in the presence of DnaK alone or in combination with one of the two GroELs. Activity of the refolded protein was then judged by binding assays. The results showed that in the absence of chaperones there was little or no activity (Fig. 3a and b, lanes 1). If renatured in the presence of DnaK alone, a low level of reactivation was observed, particularly in the case of the heat-denatured protein (Fig. 3b, lane 2). This low level of DnaK-mediated reactivation was ATP dependent (Fig. 3b, lane 2 compared to lane 3). The level of reactivation was, however, markedly increased in the presence of either of the two GroELs (Fig. 3a and b, lanes 8–11). This reactivation appeared to be substantially, though perhaps not absolutely, dependent on ATP (Fig. 3a and b, lanes 9 and 11 compared to 8 and 10). ATP hydrolysis seems to be an important factor, since in the presence of the non-hydrolysable ATP analogue ATP-S, renaturation achieved (Fig. 3c, lanes 3 and 6) was at the basal level, similar to that obtained in the absence of ATP (Fig. 3c, lanes 2 and 5). To test whether DnaK becomes part of the final complex, an antibody supershift EMSA was attempted. The results showed that the anti-DnaK immune serum (but not pre-immune serum) produced a distinct supershift (Fig. 3d, lane 3 compared to 2). Anti-GroEL serum failed to produce similar supershifts (Fig. 3d, lanes 6 and 9 compared to 5 and 8). These results indicated that DnaK, but not GroEL, becomes part of the final complex.
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, lanes 6–9 compared to 2–5). It may be added that, in the absence of DnaK, DnaJ did not stimulate HspR activity (data not shown). Addition of GrpE to the refolding mixture did not show any stimulation. In fact, there were indications that the presence of GrpE was inhibitory. To obtain a clear idea of the extent of inhibition, a renaturation experiment was performed in which GrpE was present along with DnaK. In this assay the amount of HspR was doubled, so that the initial binding activity was strong enough to allow detection of inhibition by GrpE. The results showed that when HspR was renatured in the presence of GrpE, its DnaK-dependent binding activity decreased sharply (Fig. 4b, lanes 2–5 compared to 1). This indicated that GrpE inhibits the DNA-binding capacity of the DnaK–HspR combination.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Previous investigations into mycobacterial HspR appear to be limited to two studies (Stewart et al., 2001, 2002) where the consequences of the deletion of hspR were examined. Some idea about how mycobacterial HspR might function may be gathered from an investigation done on a similar protein from S. coelicolor (Bucca et al., 2000, 2003). One of the points made categorically in the case of S. coelicolor was that DnaK is capable of acting as a co-repressor in HspR-mediated binding to the operator DNA sequence known as HAIR.
In the present study, it was shown that, as in the case of the S. coelicolor HspR, in this case too, DnaK acts as a co-repressor. However, the novel aspect presented here is the stimulatory role of the GroEL class chaperones of M. tuberculosis, GroEL1 and GroEL2, in the process. It could be demonstrated that whereas the presence of DnaK was necessary, it was not sufficient for HspR activation. The mycobacterial GroELs, GroEL1 and 2, had a major role to play in the activation processes. This co-stimulatory role of the GroELs was particularly evident when limiting amounts of HspR were used. ATP also seems to play a role, particularly in cases where the completely denatured HspR protein was refolded. Specifically, ATP hydrolysis appears to be a key event since the non-hydrolysable counterpart failed to support activation. There appears to be a degree of redundancy in the choice of co-chaperones, since DnaJ was found to be as effective as GroEL. Although the end result is the same, it is most likely that GroEL and DnaJ assist DnaK through different mechanisms. GroEL is generally known to act as a ‘cage’ within which a protein can fold efficiently (Mayhew et al., 1996), whereas DnaJ is known to facilitate interactions between DnaK and its substrates (Liberek et al., 1995). The positive role played by DnaJ is on expected lines but the inhibitory effect of GrpE was unexpected. GrpE is considered to be a nucleotide exchange factor that replaces DnaK-bound ADP with ATP (Brehmer et al., 2004). ATP-bound DnaK has lower affinity for the substrate compared to the ADP-bound protein and therefore exchange of ADP with ATP by GrpE could possibly result in the release of HspR, although this has not been specifically verified. Nevertheless, this result suggests that following its successful folding, HspR must continue to be associated with DnaK in order to be active. That DnaK is indeed retained in the final DNA–protein complex was demonstrated by the antibody supershift experiment. The GroELs do not seem to be part of the complex. No specific supershift was observed with the respective antiserum although their titres and specificities were comparable to that of anti-DnaK serum.
The observations presented are novel, since in the case of S. coelicolor HspR, activation depended solely on DnaK: neither GroEL nor ATP nor its co-chaperones had any role to play (Bucca et al., 2000, 2003). The differences in observations could be due to the simple fact that these proteins are after all derived from different systems. Alternatively, it could be due to the fact that in the previous study with the S. coelicor HspR, the DnaK and GroELs used were from the unrelated bacterium E. coli, whereas in this study all the proteins used were derived from the same system, M. tuberculosis. However, the most likely reason appears to be the concentrations used. As this study shows, the stimulatory effect of the GroELs was discernible only when the HspR-binding activity was maintained at a suboptimal level.
How the mycobacterial GroELs, GroEL1 and GroEL2, assist DnaK is not clear. As mentioned earlier, GroELs are generally known to act as cages for protein folding (Mayhew et al., 1996). A complex cycle of ATP-dependent events ensures that an unfolded protein is captured and released as a completely or partly folded protein. Most of the information regarding GroEL function is derived from studies using E. coli GroEL. The role of mycobacterial GroELs in promoting protein folding in general is still not clearly understood. It has been observed that these GroELs do not require either ATP or GroES to promote protein folding (Goyal et al., 2006). Hence, in this particular scenerio the ATP dependence is unlikely to be due to the GroELs; rather it is most likely that it is DnaK that utilizes the ATP. That this is possibly the case is evident from the observation that, in some of the experiments where HspR activity was limiting, DnaK alone could activate HspR, albeit at a low level, in an ATP-dependent manner.
Since the present set of experiments strongly suggests a role for protein folding, the question may be raised as to how this phenomenon is relevant in the cellular context. To find a rationale it must be taken into account that HspR is a regulator of the heat-shock response. In response to heat or equivalent stress, HspR is expected to be denatured and released from the HAIR operator. However, the cell must get back quickly to its ground state. For this, HspR must attain its native structure as soon as possible, and in this context the presence of GroEL along with DnaK could be an important factor. Whether this control has anything to do with pathogenesis is a matter of conjecture, but since several virulence genes are regulated by HspR (Stewart et al., 2002), some relationship with pathogenesis is likely to emerge in the future.
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ACKNOWLEDGEMENTS |
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Edited by: T. Parish
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REFERENCES |
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Received 7 August 2007;
revised 16 October 2007;
accepted 1 November 2007.
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