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J. Biol. Chem., Vol. 279, Issue 22, 23082-23087, May 28, 2004
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¶¶
From the
Department of Pathobiology, School of Public Health and Community Medicine, ¶Department of Biochemistry, ||Biomolecular Structure Center, **Biomolecular Structure and Design Graduate Program, and Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195
Received for publication, February 4, 2004 , and in revised form, March 12, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Although TB has been studied for centuries, the triggers that promote and maintain latent infections are still obscure. Two conditions frequently associated with latent TB in vivo are reduced oxygen tension and nitric oxide (NO) exposure (5, 6). Both of these stimuli can induce reversible bacterial stasis in vitro (7, 8), and both are encountered by bacilli in vivo (5, 9, 10). Further, although MTB requires oxygen for growth, it can survive without oxygen for surprisingly long periods of time (11, 12). Still the evidence linking hypoxia and NO to latent TB in vivo remains circumstantial. Analysis of the MTB response to these factors is needed to define the role they may play in promoting and maintaining TB latency in humans.
Previous reports identified a set of 47 MTB genes that are rapidly up-regulated in response to reduced oxygen tension or NO (8, 13). Among the MTB genes induced by hypoxia or exposure to NO is the putative two-component regulatory system dosR-dosS (also called devR-devS, Rv3133c/Rv3132c2) (8, 13). In bacteria, two-component response regulator systems are an important means by which a variety of environmental signals are transduced into a phenotypic response. These systems typically consist of a membrane-bound sensor kinase and soluble response regulator that is activated by a histidine-aspartate phosphorelay to bind upstream of specific genes and alter their expression (14). We hypothesized that dosR and dosS may form a signaling system involved in the initial adaptation of bacilli to conditions within the host (13). We showed previously that DosR binds upstream of hypoxic response genes (15). Further, nearly all MTB genes rapidly up-regulated in response to low doses of NO (8) or by hypoxia require DosR for their induction (15).
In this report, we demonstrate that DosS (Rv3132c) is a functional kinase of the two-component class and that it can transfer phosphate to DosR in vitro. We show that a second putative kinase, DosT (Rv2027c), also phosphorylates DosR. We demonstrate that mutants lacking both DosS and DosT can no longer activate DosR-dependent MTB gene expression in response to reduced oxygen tension.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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dosS. Homologous recombination and gene replacement was detected stepwise using hygromycin resistance followed by sacB counterselection and loss of hygromycin resistance as described previously (13). Gene replacement was confirmed by PCR using mutagenic and wild type dosT-specific primers along with primers specific for sequences flanking the site of integration (RPL12 and RPL13; see Table I) (data not shown). Digestion of the resulting amplicons with PacI also was performed to confirm the PCR screen results (data not shown).
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dosS, H37Rv:dosT, and H37Rv:dosS:dosT isolates were transformed with the plasmid pMH108 (16, 17) containing the firefly luciferase reporter gene (luc) under the control of the DosR-activated acr (hspX, Rv2031c) promoter. This plasmid integrates into the L5 phage attachment site within the Gly tRNA locus of the MTB chromosome (18). In three separate experiments transformants were grown in 7H9 medium in triplicate to an A600 of 0.3-0.4. 0.5 ml of each culture was removed to 13-ml tubes, which were sealed tightly with rubber septa. 0.2% O2 was infused through an 18-gauge needle for 30 s, and tubes then were incubated on a rotator for 2 h at 37 °C. Promoter activity was measured by combining a 100-µl aliquot of MTB isolate culture with 100 µl of luciferase assay reagent (Promega) in the wells of a 96-well plate, and incubation was continued for 5 min at room temperature. Luciferase activity was measured using a TD2020 luminometer.
dosT Complementation—For complementation studies, a pMH108-based construct was developed in which dosT is driven by mycobacterial optimal promoter (19). The dosT upstream region was amplified using primers DMR28 and DMR29 (see Table I). This construct was electroporated into H37Rv, H37Rv:dosS, H37Rv:dosT, and H37Rv:dosS: dosT with the DNA integrating into the MTB chromosome at the L5 phage attachment site. Luciferase reporter activity was measured as indicated above.
Cloning and Expression of dosS and dosT Genes—Full-length dosS and dosT were PCR-amplified from H37Rv genomic DNA using the primers DMR005 to DMR008 (see Table I) and were cloned into the pET32 Ek/LIC vector (Novagen). In addition, dosS and dosT mutants encoding amino acid substitutions DosS (H395K/H397K) and DosT (H392K/H394K) were generated by mutagenic fusion PCR with primers DMR024 to DMR027 (see Table I) and were cloned into pET32 Ek/LIC. Recombinant proteins were overexpressed as S-Tag fusion proteins in Escherichia coli BL21(DE3) and localized within inclusion bodies. The proteins were extracted from the inclusion bodies using the protein refolding kit from Novagen. Briefly, E. coli cells were disrupted using the BugBuster protein extraction reagent (Novagen), and inclusion bodies were recovered by centrifugation. After several washes in dilute BugBuster reagent, the inclusion bodies were solubilized in 500 mM CAPS, pH 11.0, supplemented with 0.3% N-lauroylsarcosine. After centrifugation, the supernatant containing the proteins was dialyzed at 4 °C against 20 mM Tris-HCl, pH 8.5, and 0.1 mM dithiothreitol. After several dialysis buffer changes, dithiothreitol was omitted, and final overnight dialysis was performed in the presence of oxidized and reduced glutathione (1 and 0.2 mM, respectively) to promote proper protein folding. Samples were concentrated using Centricon Plus-20 spin filters (Amicon).
Cloning and Expression of DosR and DosR (D54E) Mutant—The plasmids pDosR and pDosR (D54E) were obtained by insertion of the wild type DosR and the DosR (D54E) mutant coding sequences (15) into pET-21d(+) vector (Novagen) via NcoI and XhoI sites. DNA was amplified with primers GW1 and GW2 (see Table I). The Rosetta (DE3) (Novagen) and the BL21-Gold(DE3) (Stratagene) E. coli strains were used as hosts for plasmids pDosR and pDosR (D54E), respectively. Cells were grown in LB media with 100 mg/liter ampicillin (with additional 34 mg/liter chloramphenicol for cells containing plasmid pDosR (D54E)) at 37 °C to A600 = 0.6 and then were induced with 1 mM isopropyl-1-thio-
-D-galactopyranoside. Growth was continued for 3 h at 30 °C after induction. The cells were harvested by centrifugation at 5,000 rpm for 20 min at 4 °C and stored at -80 °C before use.
DosR and DosR (D54E) Protein Purification—Cell pellets containing wild type DosR and DosR (D54E) mutant were resuspended in 300 mM NaCl, 10% glycerol, 1 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 20 mM Tris-HCl buffer, pH 8.0, and were lysed by French press. Protamine sulfate was added, and the lysate was incubated on ice for 30 min. The lysate was centrifuged at 20,000 x g for 40 min, and the supernatant was clarified by filtration and then was applied to a nickelnitrilotriacetic acid affinity column. The nonspecifically bound proteins were eluted with 30 mM imidazole in 300 mM NaCl, 10% glycerol, 20 mM Tris-HCl buffer, pH 8.0. DosR and DosR (D54E) mutant were eluted from the column with 200 mM imidazole in the same buffer. Protein fractions were pooled and dialyzed against a buffer containing 20 mM MES buffer, pH 6.0, 50 mM NaCl, 10% glycerol, 1 mM EDTA, and 1 mM tri[2-carboxyethyl]phosphine hydrochloride. The proteins then were purified by cation exchange chromatography using a 20HS column (PerSeptive Biosystems) and were eluted with 50 mM to 1 M NaCl gradient. Peak fractions were combined and concentrated using Amicon Centriplus spin concentrators.
In Vitro Phosphorylation Assay—4 µg of the recombinant S-Tag fusion protein DosT or DosT (H392K/H394K) was assayed for the ability to autophosphorylate in a reaction mixture containing 50 µCi [
-32P]ATP (6000 Ci/mmol, PerkinElmer Life Sciences), 100 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 50 mM KCl2 in a final volume of 20 µl. The reaction was incubated at room temperature for 60 min. 4-µl aliquots were removed from the autophosphorylation reaction at 0 and 60 min and were stopped with the addition of an equal volume of SDS-PAGE (Laemmli) buffer and immediate immersion in a dry ice/EtOH bath. An additional 4-µl aliquot (800 ng) was removed and added to reactions containing 4 µg of either recombinant DosR or DosR (D54E) in the same reaction buffer as above (excluding isotope) for a final volume of 20 µl. 4-µl aliquots were removed for analysis at 0, 30 s, 1 min, 5 min, and 30 min. All aliquots were analyzed by SDS-PAGE and blotted to polyvinylidene difluoride membrane. The membranes were exposed to film overnight at -80 °C. Studies with DosS and DosS (H395K/H397K) were performed in the same manner except that a 10-µl aliquot (2 µg) was carried over from the autophosphorylation reaction to the reaction containing either wild type or mutant DosR, and membranes were exposed to film for 72 h at -80 °C.
Western Analysis—All blots were screened first with HRP-conjugated S-protein (Novagen), which binds the S-Tag with high affinity, at a 1:5000 dilution. ECL analysis was performed with exposure times varying from 1 to 10 s. Blots were then stripped in 200 mM glycine, pH 2.5, 0.2% Tween 20 followed by incubation in HRP color substrate to confirm loss of signal. The blots were rescreened with rabbit anti-rDosR polyclonal IgG antibody at a 1:5000 dilution followed by incubation with goat anti-rabbit IgG-HRP-conjugated secondary antibody (Pierce), and ECL analysis was performed again.
Electrophoretic Mobility Shift Assay—A double-stranded oligonucleotide containing the palindromic consensus promoter sequence of hypoxic response genes (15) was used as a DNA probe. The binding of DosR and DosR (D54E) was carried out by incubation at room temperature for 30 min in 10 µl of reaction mixtures composed of 12.5 µM DNA, 25 µM protein, 24 mM Tris-HCl buffer, pH 7.5, 20 mM MgCl2 in the presence or absence of the phosphate donor lithium potassium acetyl phosphate (20 mM). Following incubation the entire reaction volume was electrophoresed in a 15% non-denaturing Tris borate EDTA polyacrylamide gel (Bio-Rad). The gel was stained with 1 µg/ml ethidium bromide and visualized with a 312 nm transilluminator (Fisher Scientific).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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dosS. A mutated copy of dosT was inserted that contained premature stop codons, introduced a PacI restriction site, and lacked the sequence encoding the histidine-containing putative signaling region. Confirmation of the dosT gene replacement was determined by performing PCR on transformants and amplicon digestion with PacI (data not shown).
Effects of Sensor Kinase Gene Mutagenesis—One of the MTB genes powerfully induced under hypoxic conditions is acr (also called Rv2031c or hspX) encoding an 
-crystallin-like heat shock protein (13, 22). The acr promoter contains two high affinity DosR binding sequences (15, 23). Mutation of these sequences abolished DosR binding and resulted in dramatically reduced levels of expression (15). To determine the roles of DosS and DosT, both wild type and mutant bacteria were transformed with plasmid pMH108 in which the acr promoter is fused upstream of the firefly luciferase (luc) reporter gene. Isolates were subjected to hypoxic conditions, and levels of luciferase activity were measured (Fig. 1). In the wild type strain H37Rv, luc gene expression was induced about 115-fold (Fig. 1). In both the H37Rv:dosS and H37Rv:dosT mutants luc induction was reduced to 40-45% of wild type values. When both dosS and dosT genes were inactivated, reporter gene expression levels dropped to base-line values, indicating that DosR no longer is able to activate transcription (Fig. 1). These results demonstrate that both DosS and DosT are required for full activation of DosR under conditions of reduced oxygen tension.
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dosS, H37Rv:dosT, and H37Rv:dosS:dosT. The wild type dosT gene was PCR-amplified and cloned into the reporter construct pMH108 under the control of the constitutive mycobacterial optimal promoter. Each MTB isolate was transformed with the pMH108:dosT complementation constructs, incubated under hypoxic conditions, and tested for luciferase activity (Fig. 1). When dosT was reintroduced into each isolate, reporter gene expression was restored to levels comparable with or greater than those observed with wild type H37Rv, indicating that loss of hypoxic responsiveness in the mutants was caused by the lack of an appropriate sensor kinase.
In Vitro Phosphorylation Assay—The first steps in two-component signal transduction are generally signal recognition by the sensor kinase followed by dimerization and autophosphorylation at a specific His residue (14). Once autophosphorylated, the kinase transfers the phosphate to a second protein, the response regulator. We utilized recombinant DosS, DosT, and DosR in an in vitro phosphorylation assay to determine whether they functioned as a two-component system. In addition to testing wild type proteins, mutant DosS and DosT were generated via mutagenic fusion PCR. The mutations encoded amino acid substitutions intended to disrupt the histidine kinase phosphorylation motifs, giving rise to DosS (H395K/H397K) and DosT (H392K/H394K). Recombinant proteins were overexpressed in E. coli as S-Tag fusion proteins. The production of the recombinant S-Tag proteins and their purification from inclusion bodies was verified via SDS-PAGE Coomassie Blue staining and Western analysis with S-protein HRP conjugate (data not shown). In both instances a protein slightly larger than the 75-kDa marker was detected, corresponding to the molecular mass of both DosS and DosT (
62 kDa) fused to the 16-kDa S-Tag.
First we analyzed whether DosT had the ability to autophosphorylate and then transfer the phosphate to DosR. DosT was incubated at room temperature with 50 µCi [-32P]ATP, and aliquots of the reaction were removed for analysis at 0 and 60 min. An additional aliquot from the autophosphorylation reaction was removed at 60 min and put into a relay reaction containing purified DosR. Aliquots of the relay reaction were removed at 0, 0.5, 1, 5, and 30 min. The aliquots from both reactions were fractionated by SDS-PAGE, blotted, and visualized by autoradiography (Fig. 2). The DosT (H392K/H394K) mutant also was analyzed for its ability to autophosphorylate in the same manner.
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78-kDa DosT fusion protein size. Upon transfer to the relay reaction, this same protein was observed at time 0, but the radiolabel in this band waned with time until it was barely detectable at 30 min. In contrast, a radiolabeled protein consistent with the 26-kDa size of DosR was barely detectable at time 0 of the phosphorelay reaction yet became more intense as the 30-min mark approached, indicating that phosphotransfer is occurring between the two proteins. To confirm the identity of the differentially labeled proteins, immunoblot analyses were performed with an S-protein HRP conjugate to detect DosT (Fig. 2B) and after stripping with an anti-DosR antibody (Fig. 2C). Reactivity with the anti-DosR antibody was observed only in the phosphorelay lanes with the radiolabeled 26-kDa protein confirming its identity as DosR, whereas reactivity with the S-protein HRP conjugate was observed with the radiolabeled protein at 78 kDa confirming it as DosT. The additional bands in Fig. 2 also reacted with the S-protein HRP conjugate indicating that they are truncated forms of DosT that no longer have the ability to transfer phosphate (data not shown). When the DosT (H392K/H394K) mutant replaced the wild type DosT in the assay, there was no detectable autophosphorylation after 60 min and thus no subsequent phosphate transfer to DosR (data not shown). In addition, no labeling was observed in control phosphotransfer reactions from which DosT was omitted (data not shown), demonstrating that labeling of DosR was caused by phosphotransfer from DosT as opposed to DosR autophosphorylation. To determine whether the phosphorelay proceeded in a manner consistent with two-component response regulators, we performed the same reaction with a DosR mutant harboring an amino acid substitution at position 54, which replaces the putative phosphorylation site aspartate residue with a glutamate incapable of receiving the donor phosphate. The autoradiograph of the reaction again showed that DosT undergoes autophosphorylation, but when DosT was removed to the relay reaction containing DosR (D54E) there was no observable decrease in its label throughout the 30-min time course (Fig. 2D). Similarly, there was no protein consistent with DosR showing an increase in radiolabel, unlike when wild type DosR is the substrate. Immunoblot analyses confirm the presence and identities of both DosT (Fig. 2E) and DosR (D54E) (Fig. 2F). These results demonstrate that the phosphotransfer observed between DosT and DosR occurs at a specific site consistent with their function as a two-component signal transduction system.
DosS was shown previously to undergo autophosphorylation (24), but its ability to transfer phosphate to DosR was not established. To address this issue, a second set of in vitro phosphorylation experiments was conducted using S-Tagged DosS and DosS (H395K/H397K). As shown in Fig. 3, DosS underwent autophosphorylation and was able to transfer the phosphate to DosR (Fig. 3A) but not to DosR (D54E) (Fig. 3B). The mutant DosS (H395K/H397K) was unable to autophosphorylate (data not shown). The identities of DosS and DosR were confirmed by immunoblotting as above (data not shown). Results with DosS were strikingly similar to those with DosT (Fig. 2). However, under identical experimental conditions DosT was more efficient at autophosphorylation and phosphotransfer than DosS. To visualize DosS phosphoactivity comparable with that observed with DosT, we needed to transfer more of the autophosphorylation reaction to the relay reaction and expose the blots for longer time (see "Experimental Procedures").
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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That hypoxia and NO activate the same set of MTB genes suggests the DosS and DosT kinases may be able to recognize multiple inputs. Alternatively these stimuli may each alter a common factor that can then interact with the kinases. Consistent with this possibility, hypoxia and NO are additive in their effects on MTB growth and gene expression (8). Analogous sensor systems have been described in other microbes. For example, the E. coli sensor kinase ArcB detects changes in oxygen levels through interaction with the pool of cytosolic quinones (26). Oxidized quinones generated during oxygen availability inhibit ArcB autophosphorylation and subsequent ArcA regulator activation. In contrast, reduced quinones generated under oxygen limitation do not inhibit ArcB autophosphorylation, leading to phosphorylated ArcA and differential gene expression. Similarly, the Rex repressor of Streptomyces coelicolor alters gene expression in response to changes in cellular oxygen levels through competitive binding of NAD+ and NADH (27). Finally, cytochrome oxidase, a hemeprotein that can bind both oxygen and NO, has already been proposed as a mediator of DosR signal transduction (8).
The DosR/DosS/DosT two-component system has been implicated in the adaptation of mycobacteria other than MTB to hypoxic conditions. Mutant Mycobacterium smegmatis lacking dosR no longer could activate a universal stress response gene required for survival under hypoxia (28). Similarly, when Mycobacterium bovis BCG was tested in a hypoxic model of nonreplicating persistence, survival of a mutant lacking dosR was reduced 1500-fold by day 40 (21). Our experiments to define phenotypes for the dosS, dosT, and dosS/dosT mutants of MTB under hypoxic conditions in culture have been highly variable thus far, but none of our experiments (n = 4) have yielded a phenotype nearly as dramatic as that reported in M. bovis BCG by Boon and Dick (21). We currently are investigating whether the difference in these results stems from genetic differences in MTB and M. bovis BCG.
In addition to the data from culture models, the DosR/DosS/DosT two-component system also has been studied in animals. In a recent study, a dosR mutant of MTB was reported to be hypervirulent in both severe combined immunodeficient and DBA mice (29). In contrast, another recent report indicated that a dosR mutant of MTB was attenuated for virulence in guinea pigs (30). Thus, although both studies implicate the DosR response in MTB virulence, the precise role of this regulon in vivo still must be determined.
Detailed analysis of the DosR/DosS/DosT two-component signaling system is an important step in characterizing the MTB response to hypoxia and NO, two stimuli that promote nonreplicating persistence in culture. By determining the molecular processes involved in the adaptation of MTB to non-replicating persistence, we hope to provide insight into the etiology of TB latency. Data are accumulating that DosR response genes are induced during infections (8, 29, 31-33). Careful dissection of the DosR response should help to establish whether it plays a role in TB latency in vivo and may facilitate a more focused approach to the discovery of new MTB targets for drug and vaccine development.
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FOOTNOTES |
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Supported by National Institutes of Health Training Grant AI07509 awarded to the University of Washington Pathobiology Department.
Supported by National Institutes of Health Grant CA65656.
¶¶ Supported by National Institutes of Health Grant AI47744. To whom correspondence should be addressed. Tel.: 206-221-5381; Fax: 206-543-3873; E-mail: dsherman{at}u.washington.edu.
1 The abbreviations used are: TB, tuberculosis; MTB, Mycobacterium tuberculosis; CAPS, 3-(cyclohexylamino)propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; HRP, horseradish peroxidase.
2 Genes are denoted both by name and the designation assigned by the TubercuList Web site: genolist.pasteur.fr/TubercuList/.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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0.0012).
