DevR (DosR) mimetic peptides impair transcriptional regulation and survival of Mycobacterium tuberculosis under hypoxia by inhibiting the autokinase activity of DevS sensor kinase
© Kaur et al.; licensee BioMed Central Ltd. 2014
Received: 28 March 2014
Accepted: 11 July 2014
Published: 21 July 2014
Two-component systems have emerged as compelling targets for antibacterial drug design for a number of reasons including the distinct histidine phosphorylation property of their constituent sensor kinases. The DevR-DevS/DosT two component system of Mycobacterium tuberculosis (M. tb) is essential for survival under hypoxia, a stress associated with dormancy development in vivo. In the present study a combinatorial peptide phage display library was screened for DevS histidine kinase interacting peptides with the aim of isolating inhibitors of DevR-DevS signaling.
DevS binding peptides were identified from a phage display library after three rounds of panning using DevS as bait. The peptides showed sequence similarity with conserved residues in the N-terminal domain of DevR and suggested that they may represent interacting surfaces between DevS and DevR. Two DevR mimetic peptides were found to specifically inhibit DevR-dependent transcriptional activity and restrict the hypoxic survival of M. tb. The mechanism of peptide action is majorly attributed to an inhibition of DevS autokinase activity.
These findings demonstrate that DevR mimetic peptides impede DevS activation and that intercepting DevS activation at an early step in the signaling cascade impairs M. tb survival in a hypoxia persistence model.
The DevR dormancy regulon of Mycobacterium tuberculosis (M. tb) is a transcriptional program induced by low oxygen tension, nitric oxide (NO), carbon monoxide (CO) or vitamin C treatment that enables bacterial adaptation and survival during periods of non-replicating persistence in in vitro models of dormancy [1–4]. This regulon is believed to assist bacterial survival during latent tuberculosis (TB) infection, a chronic asymptomatic state that afflicts one-third of the global population . The expression of the ~48 gene regulon is coordinated by DevR (also known as DosR) which binds to target sites on DNA and induces transcription after its phosphorylation by DevS and/or DosT [3, 6–8]. DevS and DosT sensor kinases were therefore proposed as targets for the development of novel antibacterial compounds against dormant bacteria . DevS was also predicted to be a potential persistence target inhibiting dormant M. tb using an in silico approach .
Two component systems are considered as compelling targets for drug design due to a number of reasons including their absence in higher eukaryotes, the difference in bacterial two-component signaling as compared to signaling pathways in eukaryotes, and most importantly, the essential roles they play in bacterial viability, virulence and drug resistance. Inhibitors of bacterial histidine kinases have been reported in the literature [11–13] but most suffer from the drawback of being extremely hydrophobic. Most inhibitors exhibit multiple mechanisms of action including surfactant and membrane damaging properties that are independent of two-component system inhibition. The high hydrophobicity of these molecules makes formulation and delivery of the compounds extremely difficult. Furthermore, the compounds showed excessive plasma protein binding and minimal bioavailability and were ineffective in standard in vivo infection models. In contrast, peptides are believed to confer several advantages, such as high target specificity, lower accumulation in tissues and lower toxicity coupled with new efficient synthesis strategies and low monomer prices. A recent study identified potential PhoQ inhibitor compounds that inhibited autophosphorylation and also inhibited severe keratoconjunctival inflammation in mice inoculated with Shigella flexneri, suggesting that two-component systems are potential targets for the development of drugs against bacteria.
We had earlier described the properties of a DevR binding peptide DevRS1 identified through phage display technology that inhibited DevR function . Here we report the identification of peptides that majorly target the autokinase function of DevS sensor and inhibit DevR-mediated transcription and survival of M. tb under hypoxia. The ability to interfere with DevR-DevS function at more than one step demonstrates that this two-component system is a rich target for developing inhibitor(s) to effectively block M. tb adaptation to hypoxia, a potent dormancy signal.
Results and discussion
Isolation of DevS binding peptides from a phage display peptide library
Activity of 'Pep’ peptides
The 'Pep’ peptides identified by biopanning were tested for activity in DevS autokinase assays. Pep A, B and D peptides were found to inhibit DevS autokinase activity to varying degrees (see Additional file 1). However, the data showed a great variation and was not reproducible among the assay replicates for individual peptides. This could be attributed to protein aggregation that was observed upon addition of peptides to the assays. However, Pep C did not inhibit autokinase activity at any of the concentrations tested.
Similarity between PepA, PepB, PepC and PepD sequences and DevR catalytic centre
The 'Pep’ peptides identified by panning were aligned next with the protein sequence of DevR. Three peptides appeared to align near the Asp-54, Thr-82 and Lys-104 residues located in the N-terminal domain of DevR (Figure 2C). Asp-54 is the site of phosphorylation and Thr-82 and Lys-104 residues are involved in phosphorylation-induced conformational changes [7, 16]. The partial identity noted between 'Pep’ peptides and DevR protein sequences suggested that these peptides might represent surfaces on the N-terminal domain of DevR (DevRN) that could interact with DevS.
The DevRN domain interacts with the cytosolic domain of DevS
Having demonstrated that (1) 'Pep’ peptides exhibit a modest anti-DevR activity (Additional file 1), (2) 'Pep’ peptides bear sequence similarity with conserved sequences in the N-terminal domain of DevR (Figure 2C) and (3) DevRN interacts with DevS (Figure 3), we proceeded to synthesize 'DevR mimetic’ peptides that mapped in the same regions as the 'Pep’ peptides and were of identical sequence to DevR. The assumption was that DevR 'mimetic’ peptides would interact with greater affinity with DevS as compared to the 'Pep’ peptide sequences. The synthesized peptides were named as DevRS A (KDIKGME), DevRS B (ILTSYT), DevRS C (ELCRD) and DevRS D (VRLPD). Extended 10–16 mer versions of the DevRS peptides were also synthesized (peptides DevRS A-ext, C-ext, D-ext) assuming that they may afford better binding characteristics (Figure 2C). Due to the problem of protein aggregation observed in assays containing the 'Pep’ series of peptides, only extended 'DevRS’ and 'DevRS-ext’ peptides were characterized further. Moreover, these extended peptides were likely to afford better binding characteristics if indeed they represented interacting regions.
'DevRS’ peptides inhibit DevR-mediated transcription
'DevRS’ peptides specifically inhibit hypoxic survival of M. tuberculosis
'DevRS’ peptides inhibit DevS autokinase activity
Auto phosphorylation of the sensor kinase is the first step in the DevS/DosT-DevR signaling cascade. To decipher the mechanism of action of the peptides, DevRS A-ext and DevRS D were tested in an in vitro ATP-dependent autokinase assay as described . The addition of DevRS A-ext and DevRS D in the reaction inhibited DevS autokinase activity by 55% and 37% respectively (see Additional file 2). Our findings suggest that the inhibition of autokinase activity by DevRS A-ext and DevRS D is caused by peptide binding at/near the DevS phosphorylation site.
In addition to inhibiting autokinase activity (above), the peptides could in principle also inhibit transfer of the phosphosignal from DevS ~ P to DevR. However, neither DevRS A-ext nor DevRS D inhibited this reaction in an in vitro phosphotransfer assay (data not shown). Based on these findings, the observed defects in hypoxic adaptation of M. tb cultures are majorly attributed to a peptide-mediated block in the autokinase activity of DevS sensor kinase. Because (1) of the high sequence homology between DevS and DosT kinases, (2) DosT, in addition to DevS, can activate DevR through its autokinase activity under hypoxia [3, 6, 8, 24] and (3) exposure to DevRS A-ext and DevRS D peptides results in a severe loss of hypoxic viability (Figure 5), we further propose that these two peptides inhibit the activities of both DevS and DosT kinases.
Cytotoxicity of 'DevRS’ peptides
Screening of phage display library using cytoplasmic domain of DevS as bait
The recombinant cytosolic portion of DevS protein DevS201 (amino acids 377 to 578), was overexpressed and purified from E. coli BL21 harboring recombinant plasmid pSCS201 as described earlier . The Ph.D.-7 Phage display peptide library kit (New England Biolabs Inc., Beverely, MA, USA) was screened by biopanning using the manufacturer’s instructions with a few modifications as outlined in Figure 1. Briefly, three rounds of panning were performed on a polystyrene 96-well micro titer plate wherein duplicate wells were coated with purified protein DevS201 -His6 (10 μg/well) and the phage library (2 × 1011 phages) was incubated with the immobilized protein to allow binding of the phage particles. Decreasing the time of incubation with DevS201 protein and increasing the percentage of Tween-20 in the washing buffer in each successive round of panning achieved the stringent selection of high affinity binder phages. Three rounds of panning were performed in all and each time the phages in the glycine elution (0.2 M glycine, pH 2.2) were amplified and used as an input for the next round of panning. The titer of phages in the various elutions was determined according to the manufacturer’s instructions and the input phage number was maintained during each round of panning.
The high affinity binder phages were identified using ELISA. Briefly, individual phage plaques were picked from the glycine elution of the last round of panning, amplified and the culture supernatants (containing phages) were screened for binding to DevS201 or BSA or to plastic. Briefly, the plates were coated overnight with 10 μg/well DevS201 protein or left uncoated. The plates were then blocked overnight with BSA (5 mg/ml) followed by washing thrice with TBS (50 mM Tris–HCl, pH 7.5 and 150 mM NaCl). Thereafter, phage supernatants were added to coated wells and incubated for 1 hr. The plates were then washed vigorously with TBS containing 0.5% Tween 20. The bound phages were detected using HRP-conjugated anti-M13 antibody (Amersham Biosciences, UK) and o-phenylenediamine as a substrate and measurement of absorbance at A490. The extent of binding to DevS was calculated as A490 in DevS coated wells – A490 in control wells.
DNA sequencing of phages
DNA of thirty high affinity binder phages was sequenced to determine the DevS binding peptide sequences. The desired peptides were then synthesized commercially with a purity level of > 98%.
Activity of 'DevRS’ peptides in M. tb cultures
M. tb H37Rv was grown to logarithmic phase in Dubos broth containing 0.05% Tween 80 supplemented with 10% albumin dextrose complex (DTA medium) and sub-cultured with shaking at 220 rpm to A595 ~ 0.5. The culture was diluted in DTA medium (without Tween 80) to A595 ~ 0.025 for aerobic viability assays (REMA) and A595 ~ 0.005 for hypoxic assays (HyRRA) and used as described earlier .
The REMA assay was performed in black, clear-bottomed, 96-well micro plates as described earlier . Peptides were diluted in DMSO, and subsequent 2-fold serial dilutions were performed in 0.1 mL of Dubos medium supplemented with 0.05% glycerol in the micro plates. Approximately 5 × 104 cfu M. tb bacteria were added per well. Appropriate control wells were included to calculate percentage inhibition of viability. The plates were incubated at 37°C for 64 h after which Resazurin (0.003% final concentration) and Tween-80 (1.25% final concentration) were added. The wells were observed after 24 and 48 h for a colour change from blue to pink and fluorescence of control wells >50,000 relative fluorescence units (RFU). Fluorescence was measured by excitation at 530 nm and emission at 590 nm using Gemini XS spectrofluorimeter in bottom reading mode. Percentage inhibition of viability was defined as: 1- (test well fluorescence/mean fluorescence of triplicate wells containing only bacteria) × 100.
The HyRRA assay was performed as described earlier . Briefly, 3 mL culture aliquots (A595 ~ 0.003 containing ~1.5 × 106 cfu/mL M. tb H37Rv) were injected into 5 mL uncoated Vacutainer tubes and kept stationary at 37°C to allow for self-generation of hypoxia in the cultures. The peptides were injected into anoxic cultures (as judged by decolorization of methylene blue) at different concentrations on day 30 and the tubes were further incubated for 5 days at 37°C. Resazurin (0.0023% final concentration) and Tween 80 (0.66%/well final concentration) were injected into each tube and the tubes were incubated overnight. Culture aliquots (200 μl) were transferred to a 96-well black micro plate and fluorescence determined as described above.
GFP Reporter assays
For the reporter assays, 200 μl M. tb culture aliquots harboring p3134c-1/pSigA reporter plasmids were used to measure GFP fluorescence in a spectrofluorimeter in bottom reading mode using an excitation wavelength of 483 nm and an emission wavelength of 515 nm as described .
Auto phosphorylation of DevS201
DevS201 (15 μM final concentration) was incubated with 5 μCi of γ32P ATP (5000 Ci mmol-1, BRIT, India) in a 10 μl reaction mixture (containing 50 μM Tris HCl pH 8.0, 50 mM KCl, 10 mM MgCl2, 50 μM ATP) at 25°C for 60 mins. The reactions were stopped by dilution with 200 μl of cold phosphate-buffered saline (PBS). The diluted contents were promptly transferred to a 96-well MultiScreen HA-plate prewetted with PBS and processed by high-throughput filtration using a vacuum manifold device to remove the reaction components and trap the proteins on the membrane . The wells were washed thrice with 300 μl PBS using the vacuum manifold. The filters were subsequently dried on the vacuum for 2 mins at room temperature and individual filters were cut using the filter removal device. The radioactivity that was retained on each filter was quantitated using liquid scintillation counting. To monitor the effect of peptides on DevS201 autokinase activity, the peptides at various concentrations were preincubated with DevS201 in the reaction mixture for 30 mins at 25°C. ATP was then added to this reaction cocktail and tubes were incubated at 25°C for 1 h. The reaction was terminated by dilution with 200 μl of cold PBS and the samples were analyzed as described above.
Phosphorylation of DevR/DevRN by phosphotransfer from DevS201
To study the phosphotransfer reaction, DevS201 (15 μM final concentration) was phosphorylated for 60 mins as described above. Subsequently, full-length DevR or DevRN protein (1–144 amino acid residues, 20 μM final concentration) was added to the phosphorylated DevS201 mix, and the reaction was incubated at 25°C for 2 mins. The reaction was terminated with 10 μl of 2X stop buffer (100 mM Tris-Cl, pH 6.8, 20% glycerol, 2% SDS, 280 mM β-mercaptoethanol, 0.01% bromophenol blue) and the samples were resolved on SDS-PAGE. The gel was rinsed in water and subjected to autoradiography.
This study was financially supported by a grant to J.S.T. from CSIR, Government of India. We acknowledge the valuable suggestions of Dr Deepak Saini and Dr Deepti Saini during phage library screening. We thank Dr. Santosh Chauhan for providing DevRN protein. J.S.T. is thankful to the DST for the J.C. Bose National Fellowship. K.K., N.K.T. and S.D. are thankful to UGC, CSIR and DBT respectively, for their Research Fellowships.
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