ABSTRACT
The Mycobacterium tuberculosis exported repetitive protein (RvErp) is a crucial virulence-associated factor as determined by its role in the survival and multiplication of mycobacteria in cultured macrophages and in vivo. Although attempts have been made to understand the function of Erp protein, its exact role in Mycobacterium pathogenesis is still elusive. One way to determine this is by searching for novel interactions of RvErp. Using a yeast two-hybrid assay, an adenylyl cyclase (AC), Rv2212, was found to interact with RvErp. The interaction between RvErp and Rv2212 is direct and occurs at the endogenous level. The Erp protein of Mycobacterium smegmatis (MSMEG_6405, or MsErp) interacts neither with Rv2212 nor with Ms_4279, the M. smegmatis homologue of Rv2212. Deletion mutants of Rv2212 revealed its adenylyl cyclase domain to be responsible for the interaction. RvErp enhances Rv2212-mediated cyclic AMP (cAMP) production. Also, the biological significance of the interaction between RvErp and Rv2212 was demonstrated by the enhanced survival of M. smegmatis within THP-1 macrophages. Taken together, these studies address a novel mechanism by which Erp executes its function.
IMPORTANCE RvErp is one of the important virulence factors of M. tuberculosis. This study describes a novel function of RvErp protein of M. tuberculosis by identifying Rv2212 as its interacting protein. Rv2212 is an adenylyl cyclase (AC) and produces cAMP, one of the prime second messengers that regulate the intracellular survival of mycobacteria. Therefore, the significance of investigating novel interactions of RvErp is paramount in unraveling the mechanisms governing the intracellular survival of mycobacteria.
INTRODUCTION
Discerning the molecular mechanisms used by specific mycobacterial proteins involved in infection and virulence requires an understanding of the protein-protein interaction network. The interactions of secretory proteins of Mycobacterium with the host machinery are vital for successful infection. One such secretory protein involved in virulence of Mycobacterium tuberculosis is Erp (Rv3810).
The erp gene of M. tuberculosis encodes an ∼28.0-kDa secretory protein that migrates as a 36.0-kDa protein and is present in all species of mycobacteria. Its disruption results in a marked decrease in virulence, with lower levels of survival not only in in vitro and cell culture assays but also under in vivo conditions (1, 2). It was recently shown that the nature of the erp allele strongly affects the number and the size of the lung lesions in infected animals (3). No homologue of Erp has been found in other bacterial species, making Erp a mycobacterial signature (4).
Erp has a composite structure made up of three domains. While the amino-terminal domain (amino acids 1 to 80) and the carboxy-terminal domain (amino acids 176 to 284) are conserved, the central domain, consisting of tandem repeats of 5 amino acids based on a PGLTS motif, is subjected to a high level of interspecies variability (1). A signal sequence is present in the amino terminus of Erp (5). Although the hydrophobic region present in the carboxy terminus anchors the Erp protein at the surface of the bacillus, it is not required for the complementation of the altered colony morphology of a Mycobacterium smegmatis erp deletion mutant and proved to be necessary for achieving resistance to detergent at wild-type levels (6). Knockout of the erp gene in M. tuberculosis causes the strain to fail to replicate intracellularly (7). Recently, the central and amino-terminal regions of Erp were found to interact with Rv1417 and Rv2617c in the cell envelope (8).
Although data on the interactions and indirect functions of Erp have started pouring in, detailed information on the mechanism by which Erp functions is still lacking. Therefore, in order to gain insights into the function of Erp, and also based on the premise that the function of unknown proteins may be discovered by analyzing their interaction with a protein target having a probable known function, the interactions of Erp were explored using a yeast two-hybrid (Y2H) assay. RvΔssErp protein (Erp devoid of signal sequence) of M. tuberculosis was used as a “bait” to fish out the “prey” proteins encoded by M. tuberculosis genomic DNA library. The Y2H assay pulled out the interaction of RvΔssErp with Rv2212, an adenylyl cyclase. A glutathione S-transferase (GST) pulldown assay under in vitro conditions and coimmunoprecipitation studies (Co-IPs) in M. smegmatis confirmed that RvΔssErp interacts with Rv2212. However, MsΔssErp, a homologue of ΔssErp in M. smegmatis, fails to interact with Rv2212 and Ms_4279. Coimmunoprecipitation studies with H37Rv lysate further established the interaction of Rv2212 with RvErp at the endogenous level. Moreover, the interaction of RvΔssErp with Rv2212 results in increased production of cyclic AMP (cAMP) under in vitro as well as in vivo conditions. Further, we have shown that the interaction of RvΔssErp with Rv2212 gives a survival advantage to M. smegmatis in THP-1 macrophages.
MATERIALS AND METHODS
Materials.
The GAL4 yeast two-hybrid phagemid vector kit was procured from Stratagene. All the reagents, including anti-FLAG M2 beads, FLAG peptide, ATP, cAMP, and anti-His and anti-GST antibodies, were purchased from Sigma. Anti-RpoB and anti-Ag85c antibodies were purchased from Abcam. Escherichia coli host strain BL21(DE3)pLysS, M15, plasmid vector pQE-30, and nickel-nitrilotriacetic acid (Ni-NTA) Superflow were procured from Qiagen. The 7H11 and 7H9 media and the oleic acid-albumin-dextrose-catalase (OADC) supplement were purchased from BD Difco. The cAMP Biotrak enzyme immunoassay (EIA) system, IPTG (isopropyl-β-d-thiogalactopyranoside), and imidazole were obtained from Wipro GE Healthcare, and glutathione-agarose was purchased from Thermo Scientific. Protein A beads were purchased from Roche.
Y2H assay.
Y2H was performed according to the protocol described in the instruction manual of Stratagene. The RvErp gene without its signal sequence, i.e., the RvΔssErp gene, was cloned into the pBD-GAL4 vector using SalI restriction enzyme (Table 1). RvΔssErp was tested for the transactivation and used as bait. M. tuberculosis H37Rv genomic DNA was prepared by the methods described by Belisle and Sonnenberg (9) and partially digested with EcoRI and XhoI enzymes. DNA fragments ranging from 0.1 to 2.5 kb were purified and ligated to EcoRI and XhoI sites of a predigested pAD-GAL4-2.1 vector. An M. tuberculosis genomic DNA library containing 1.3 × 105 recombinant clones of sizes ranging from ∼0.1 to 2.5 kb was used as the prey. In brief, the Y2H system of Stratagene is based on expression of the lacZ and HIS3 reporter genes. To rule out the leaky expression of HIS3 reporter gene, the colonies appearing on the triple selection plates were filter lifted twice and the LacZ assay was performed to check the expression of β-galactosidase according to the method of Breeden and Nasmyth (10). Only His+ LacZ+ YRG-2 colonies were selected for further evaluation.
TABLE 1.
Genes, primers, plasmids, restriction enzyme sites, and strains used in this studya
| Gene product | Primer sequence (5′–3′) | RE site | Plasmid (antibiotic resistance) | Host strain |
|---|---|---|---|---|
| Full-length Erp (Rv3810) | FP-CCC AAG CTT GCC ACC ATG CCG AAC CGA CGC CG | HindIII | pQE-30 (Ampr) | M15 |
| RP-GAT TAA GCT TCT GCA GTT AGG CGA CCG GCA CG | ||||
| RvΔssErp (Rv3810) | FP-ACG CGT CGA CGG AGT CCT TGT GCA TAT TTT | SalI | pBD-GAl4 (CAMr) | YRG-2 |
| RP-GCT CGT CGA CTT AGG CGA CCG GCA CG | pQE-30 (Ampr) | M15 | ||
| Rv2212 | FP-GGA TCC CTG CAG ATG TAC GAT TCC TTG GAC TTC | BamHI | pGEX -KG (Ampr) | BL21(DE3)pLysS |
| RP-ATC GAT AAG CTT ATC ACT GGC GGC GGG GCT TGG | HindIII | pET-23a (Ampr) | M. smegmatis | |
| pVV16 (Kanr Hygr) | ||||
| RvΔssErp (Rv3810) | FP-GAT GGA TCC CTG CAG ATG gac gac tac aaa gat gac gac gat aag | PstI | p19Kpro (Hygr) | M. smegmatis |
| AGT CCT TGT GCA TAT TTT CTT GTC TAC | (FLAG-Tag) | |||
| RP-AAG CTT GAT ATC TTA GGC GAC CGG CAT GGA CAT | EcoRV | |||
| RvΔssErp (Rv3810) | FP-GAT GGA TCC CTG CAG ATG AGT CCT TGT GCA TAT TTT CTT GTC TAC | PstI | pSC301 (Hygr) | M. tuberculosis H37Ra |
| RP-AAG CTT GAT ATC TTA GGC GAC CGG CAT GGA CAT | EcoRV | |||
| ΔN-Rv2212 | FP-GCT GGA TCC ATG GGT ATC GGC TTT GCG GAT CTG | BamHI | pGEX-KG (Ampr) | BL21(DE3)pLysS |
| RP-ATC GAT AAG CTT ATC ACT GGC GGC GGG GCT TGG | HindIII | |||
| ΔAC-Rv2212 | FP-GGA TCC CTG CAG ATG TAC GAT TCC TTG GAC TTC | BamHI | pGEX-KG (Ampr) | BL21(DE3)pLysS |
| Fi-CAC GTC ACG CTT GCC GAC GTG GCG GCC GCA GCG CCA GGG | HindIII | pVV16 (Kanr Hygr) | M. smegmatis | |
| Ri-GCG GCC GCA GCG CCA GGG CAC GTC ACG CTT GCC GAC GTG | ||||
| RP- ATC GAT AAG CTT ATC ACT GGC GGC GGG GCT TGG | ||||
| ΔC-Rv2212 | FP-GGA TCC CTG CAG ATG TAC GAT TCC TTG GAC TTC | BamHI | pGEX-KG (Ampr) | BL21(DE3)pLysS |
| RP- GCT AAG CTT TAA CAC CAG GCG CAG CCA GGT TGA | HindIII | |||
| MsΔssErp (MSMEG_6405) | FP-GAA TTC GCA TGC ATG AGT CCA GTC GCC CTG ATT GCC GCA ACT | SphI PstI | pQE-30 (Ampr) | M15 |
| RP-AAG CTT CTG CAG TCA GGC AGG CGG CGG CAC GGG TGC | ||||
| MSMEG_4279 | FP-CTG CAG GGA TCC ATG GTC GAT TTC GAT GCG CTC GAA | BamHI | pGEX-KG (Ampr) | BL21(DE3)pLysS |
| RP-ATC GAT AAG CTT CTA GCC AGA CTC CCT GGG AGT GAG | HindIII |
FP, forward primer; RP, reverse primer; Fi, forward internal primer; RE, restriction enzyme; Ri, reverse internal primer; nucleotides in lowercase indicate the FLAG tag. Underlining indicates the restriction enzyme recognition sequence that was used for cloning with the respective primer.
Mycobacterium growth conditions.
M. smegmatis mc2155 and M. tuberculosis H37Ra and H37Rv were grown at 37°C in Middlebrook 7H9 broth supplemented with 10% OADC, 0.2% glycerol, Tween 80, and the appropriate antibiotic(s) when required. Transformants were selected on 7H11 agar medium supplemented with 10% OADC containing either kanamycin (20 μg/ml), hygromycin (50 μg/ml), or both. Growth was monitored at A600.
Overexpression and purification of RvErp, RvΔssErp, MsΔssErp, Rv2212, and Ms_4279 proteins.
The RvErp and RvΔssErp genes of M. tuberculosis H37Rv were amplified by PCR using primers detailed in Table 1 and cloned into the pQE30 vector at HindIII and SalI restriction enzyme sites, respectively. E. coli expression host M15 cells were transformed with the vector containing either the RvErp or the RvΔssErp gene of M. tuberculosis. The expression of proteins was induced with 0.5 mM IPTG for 6.0 h at 30°C. The clear supernatant obtained after sonication and by centrifugation at 14,972 × g, 4°C for 30 min, was loaded onto an Ni-NTA column, preequilibrated with lysis buffer containing 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole (pH 8.0). After washing the column with buffers containing 20 mM and 40 mM imidazole, the protein was eluted in elution buffer with 250 mM imidazole. The eluted fractions with ∼90% purity were pooled and dialyzed extensively to remove imidazole and to reduce the NaCl concentration from 300 mM to 20 mM. The dialyzed RvErp or RvΔssErp was further purified by passing it through an anion exchange column, Q Sepharose, preequilibrated with 20 mM NaH2PO4, 20 mM NaCl, and 1.0 mM EDTA buffer (pH 8.0). The column was washed with 10 ml of same buffer but with various concentrations of NaCl (20 mM, 50 mM, 100 mM, 200 mM, and 400 mM). Flowthrough and all the wash fractions were collected to check the presence and purity of RvErp or RvΔssErp proteins by electrophoresis on 10% SDS-PAGE. It was observed that the passage of dialyzed RvErp or RvΔssErp protein through Q Sepharose resulted in binding of only a small portion of RvErp or RvΔssErp protein and all the high-molecular-weight protein impurities to the column. Most of the purified RvErp or RvΔssErp proteins were recovered in the flowthrough. Washing the column with dialysis buffer containing 20 mM NaH2PO4 (pH 8.0) and 20 mM NaCl resulted in further elution of the desired Erp protein. Impurities and very small amounts of the desired proteins came out of the column when washed with buffer containing 50 mM, 100 mM, 200 mM, and 400 mM NaCl. Fractions of purified Erp proteins were pooled and concentrated. While the purity of RvErp was ∼90%, RvΔssErp was found to be more than 95% pure. The yields of the purified proteins were ∼2 to 3 mg/liter of culture. For measuring AC activity, the purified RvΔssErp protein was dialyzed extensively against buffer containing 20 mM Tris-Cl (pH 6.5) and 50 mM NaCl.
The MsΔssErp gene of M. smegmatis was amplified by PCR using the primers listed in Table 1 and cloned into pQE30 at SphI and PstI sites to have the MsΔssErp gene in frame with sequence for the His tag at the N terminus. The recombinant MsΔssErp protein was purified using an Ni-NTA column as described for M. tuberculosis RvΔssErp. MsΔssErp did not require Q Sepharose purification. The purity of the recombinant MsΔssErp protein was found to be ∼95%. The yield of the purified MsΔssErp protein was ∼1.5 mg/liter of the culture.
Rv2212 and Ms_4279 genes were amplified by PCR using primers as listed in Table 1. The amplified PCR product of either the Rv2212 gene or the Ms_4279 gene was cloned at BamHI and HindIII sites of pGEX-KG to yield the respective protein in fusion with the GST tag at the N terminus. E. coli host strain BL21(DE3)pLysS cells were transformed with pGEX-KG-Rv2212 or pGEX-KG-Ms_4279. The expression of the proteins was induced with 0.5 mM IPTG for 4.0 h at 30°C. The clear supernatant obtained after sonication and removal of insoluble debris by centrifugation at 14,972 × g for 45 min was loaded onto a glutathione-agarose column preequilibrated with 50 mM Tris-Cl, 0.15 M NaCl, 2.7 mM KCl (pH 7.5). After washings with 1× phosphate-buffered saline (PBS) (pH 7.0), the proteins were eluted with buffer containing 50 mM Tris-HCl–10 mM reduced glutathione (pH 8.0). The eluted fractions with more than 95% purity were pooled, dialyzed extensively against 20 mM sodium phosphate buffer (pH 8.0), and concentrated. The yield of the purified GST-Rv2212 and GST-Ms_4279 was ∼2.0 mg/liter of culture.
For the hexahistidine tag at the C terminus, the Rv2212 gene was cloned into the pET-23a vector, expressed in BL21(DE3)pLysS cells, and induced at A600 of 0.6 with 0.5 mM IPTG for 6.0 h at 30°C. The clear supernatant obtained after sonication and removal of insoluble debris by centrifugation at 14,972 × g for 30 min was loaded onto an Ni-NTA column preequilibrated with lysis buffer containing 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole (pH 8.0). After washing the column with buffers containing 20 mM, 30 mM, 40 mM, and 50 mM imidazole, the protein was eluted in buffer containing 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole (pH 8.0). The elution fractions that were more than 95% pure were pooled and dialyzed extensively against 20 mM sodium phosphate buffer (pH 8.0). For measuring AC activity, the purified Rv2212 was dialyzed extensively against 20 mM Tris-Cl (pH 6.5) and 50 mM NaCl. The yield of the purified protein was ∼1.5 mg/liter of the culture.
The deletion mutants of Rv2212, namely, ΔN-Rv2212, ΔAC-Rv2212, and ΔC-Rv2212, were prepared either by normal PCR or by overlap extension PCR (Table 1; see also Fig. 7A). These deletion mutants were cloned into pGEX-KG at BamHI and HindIII restriction sites so as to keep the GST tag at the N terminus. The purification of these mutants was done in exactly the same way as that of wild-type Rv2212 using the glutathione-agarose column (see Fig. 7B). The yields of ΔN-Rv2212, ΔAC-Rv2212, and ΔC-Rv2212 were found to be ∼2.0 mg/liter of the culture.
FIG 7.
The adenylate cyclase domain of Rv2212 plays a role in its interaction with RvΔssErp. (A) Schematic representation of domain deletions of Rv2212. Rv2212 protein comprises 378 amino acids. N, amino-terminal domain (amino acids 1 to 206); AC, adenylate cyclase domain (amino acids 206 to 316); C, C-terminal domain (amino acids 316 to 378); domains were deleted using PCR. (B) Purification of GST-ΔN-Rv2212, GST-ΔAC-Rv2212, and GST-ΔC-Rv2212 using a glutathione-agarose column in Coomassie blue-stained gel. Lane M, protein marker; lanes ΔN, ΔAC, and ΔC, purified GST-ΔN-Rv2212, GST-ΔAC-Rv2212, and GST-ΔC-Rv2212 proteins, respectively. (C) Interaction of RvΔssErp with deletion mutants of Rv2212. A GST pulldown assay was performed using equimolar concentrations of purified RvΔssErp, GST, and GST-Rv2212 and its deletion mutants, namely, ΔN-Rv2212, ΔAC-Rv2212, and ΔCRv2212, as shown above the blot. Anti-GST antibody was used to probe GST-Rv2212 and its deletion mutants. We observed some degradation for GST-Rv2212 proteins in this experiment. Anti-His and anti-Erp antibodies detected GST and RvΔssErp proteins, respectively. Protein markers are shown on the left. (D) Far-UV CD spectra of Rv2212 and ΔAC-Rv2212 in an aqueous solution measured at 20°C.
While GST protein used in some assays (see Fig. 4A and 5B) was overexpressed and purified from the pGEX-KG vector, which has only the GST tag, other assays (see Fig. 4B and C, 5A, and 7C) included GST protein purified from the pET41a vector, which has a His tag in addition to a GST tag.
FIG 4.
Interaction of RvΔssErp with Rv2212 was performed in vitro and in vivo. (A) In vitro interaction of RvΔssErp with Rv2212. A GST pulldown assay was performed as shown on the top. RvΔssErp, GST-Rv2212, and GST proteins were detected with anti-RvΔssErp and anti-GST antibodies. (B) Dose-dependent interaction of full-length RvErp with Rv2212. Fixed concentrations of GST-Rv2212 with increasing concentrations of RvErp in the molar ratios of 1:1, 1:2, 1:4, and 1:8 were used for the GST pulldown assay as shown above the blot (lanes 5 to 8). RvErp, GST-Rv2212, and GST proteins were detected with anti-RvΔssErp, anti-GST, and anti-His antibodies, respectively. The input panel shows the presence of proteins used for the GST pulldown assay. (C) Rv2212 as a scavenger of RvErp. Fixed concentrations of RvΔssErp with increasing concentrations of GST-Rv2212 in the molar ratios of 1:1, 1:2, 1:4, 1:8, and 1:16 were used for the GST pulldown assay as shown above the blot (lanes 5 to 9). RvΔssErp, GST-Rv2212, and GST proteins were detected with anti-RvΔssErp, anti-GST, and anti-His antibodies, respectively. While the input panel shows the amount of proteins used in GST pulldown assay, the supernatant panel shows the amount of proteins left in solution after binding. (D) Interaction of RvΔssErp protein with Rv2212 under in vivo conditions. Lysates of M. smegmatis overexpressing either FLAG-RvΔssErp (lane 1) or His-Rv2212 (lane 2) or the two in combination (lane 3) as shown above the blot were used for coimmunoprecipitation using anti-FLAG-M2 beads. Coimmunoprecipitates (top panels) and lysates (bottom panels) were probed with anti-His antibody for Rv2212 and anti-Erp antibody for RvΔssErp proteins. The lysate panel shows the expression of respective proteins as indicated on the top of the figure. The names of the proteins detected in the immunoblots are listed on the right.
FIG 5.
MsΔssErp interacts neither with Rv2212 nor with Ms_4279. (A) MsΔssErp does not interact with Rv2212. A GST pulldown assay was performed as shown above the blot. Anti-GST antibody detected GST-Rv2212, anti-Erp antibody detected RvΔssErp and MsΔssErp, and anti-His antibody detected GST proteins. The input panel shows the amount of proteins used for GST pulldown. (B) MsΔssErp does not interact with Ms_4279. A GST pulldown assay was performed as indicated above the blot. Anti-GST antibodies detected Rv2212, GST-Ms_4279, and GST proteins. Anti-Erp antibodies detected RvΔssErp and MsΔssErp proteins. The input panel shows the amount of proteins used for GST pulldown. GST protein used was purified from pGEX-KG vector. Protein markers (in kilodaltons) are shown on the left.
Raising polyclonal antibodies against RvΔssErp and Rv2212 in rabbit.
Preimmune sera were collected from a rabbit 1 to 2 days before immunization. A primary dose of ∼125 μg purified recombinant proteins, either RvΔssErp or Rv2212, mixed with an equal volume of Freund's complete adjuvant was injected subcutaneously. The first booster dose was prepared by mixing equal volumes of RvΔssErp or Rv2212 and Freund's incomplete adjuvant and was administered 30 days after immunization. On the 11th day after the first booster dose, blood was collected and the antibody titer in the serum was determined by enzyme-linked immunosorbent assay (ELISA). A second booster dose was given 30 days after the first booster dose. Again, on the 11th day after the second booster dose, blood was collected and the antibody titer in the serum was determined by ELISA. Antiserum was used for detection of proteins in Western blot analysis (data not shown). Approval for raising polyclonal antibodies in rabbits was obtained from the Institutional Animal Ethics Committee (IAEC) of CSIR-IMTech. Approval for raising polyclonal antibodies in rabbits was obtained from the Institutional Animal Ethics Committee (IAEC) of CSIR-IMTech.
CD measurements.
All circular dichroism (CD) spectra were recorded at a protein concentration of 3 μM with a 1-mm cell on a Jasco J815 spectropolarimeter calibrated with ammonium (+)-10-camphorsulfonate at 20°C and fitted with a Peltier thermostat having an accuracy of ±0.1°C. Far UV-CD spectrum data collected from 250 nm to 190 nm represent an average from three individual scans. Data are expressed as the mean residual ellipticity ([MRE]) in degree square centimeter per decimole, which is defined as follows: [MRE] = θ × 100 × Mr/(c × d × NA), where θ is the observed ellipticity in degrees, c is the protein concentration in milligrams per milliliter, d is the path length in centimeters, Mr is the protein molecular weight, and NA is the number of amino acids.
Pulldown assay.
In the GST pulldown assay, the proteins were flipped, i.e., RvΔssErp acted as the prey, while GST or GST-Rv2212 was the bait. Equimolar concentrations of purified GST proteins, RvΔssErp, MsΔssErp, GST-Rv2212, and GST-Ms_4279 were added to the glutathione-agarose beads either alone or in combination and incubated for 2 h at 4°C with end-to-end mixing. The beads were washed three times with ice-cold PBS (pH 7.2) containing 1% Triton X-100, and the bound proteins were eluted in 20 mM reduced glutathione-containing buffer. The eluted fractions were electrophoresed on 10% SDS-PAGE. Western blotting was done with anti-His, anti-RvΔssErp, and anti-GST antibodies.
For probing endogenous interactions, M. tuberculosis H37Rv cells were suspended in ice-cold PBS (pH 7.2) and lysed using a bead beater. H37Rv lysate was centrifuged at 14,972 × g at 4°C for 15 min. The endogenous complex of Rv2212 and RvErp was immunoprecipitated using Rv2212 antibodies bound to protein A beads overnight at 4°C with end-to-end mixing followed by three washings with 1× PBS, heated in 1× Laemmli buffer at 100°C for 10 min, electrophoresed on 12% SDS-PAGE, and transferred to nitrocellulose membrane. Endogenous Rv2212 and RvErp were detected using Rv2212 and RvΔssErp antibodies, raised in our laboratory. The binding of lysate with protein A beads alone was kept as negative control.
Coimmunoprecipitation.
For in vivo coimmunoprecipitation, PCR-amplified Rv2212 and RvΔssErp genes were cloned into mycobacterium expression vectors pVV16 and p19Kpro using BamHI plus HindIII and PstI plus EcoRV restriction enzymes, respectively (Table 1).
M. smegmatis cells were transformed with either pVV16-Rv2212 or p19Kpro-RvΔssErp either alone or in combination using electroporation. Transformed cells were grown in 7H9 medium containing 10% OADC along with respective antibiotics at 37°C. After 48 h at an A600 of >1, cells were harvested, washed three times, and sonicated in 1× PBS buffer (pH 7.4) containing a protease inhibitor cocktail. Cleared lysates obtained after centrifugation at 14,972 × g at 4°C for 10 min were added to the anti-FLAG M2 beads and mixed end to end at 4°C for 2 h, followed by three washings with 1× PBS. The immunoprecipitated RvΔssErp along with coimmunoprecipitated proteins were eluted using FLAG peptide, electrophoresed on 10% SDS-PAGE, transferred onto nitrocellulose membrane, and probed with anti-RvΔssErp and anti-His antibodies.
Gel filtration.
Size exclusion chromatography (SEC) of either 4.0 μM recombinant Rv2212 protein or RvErp protein alone in 500 μl was performed on a Superose 6 Increase 10/300 GL filtration column (Wipro GE Healthcare). For stoichiometry of the Rv2212 and RvErp interaction, the complex was formed by incubating 4.0 μM (each) Rv2212 and RvErp (1:1 ratio) in 500 μl for 3.0 h at 4°C with end-to-end mixing. The complex was centrifuged at 10,000 rpm for 10 min at 4°C. Around 450 μl of the supernatant was loaded onto the column preequilibrated with 20 mM sodium phosphate buffer (pH 8.0) containing 150 mM NaCl and 2 mM EDTA. The flow rate was 0.1 ml/min. Fractions of 250 μl each collected from an elution volume of 13.75 ml to 19.50 ml were electrophoresed on 10% SDS-PAGE and transferred onto a nitrocellulose membrane, followed by probing with antibodies raised against either Rv2212 or RvErp. The gel filtration column was calibrated using molecular mass standards from Sigma: Blue Dextran 2000 (2,000 kDa), thyroglobulin (669 kDa), ferritin (440 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.9 kDa).
Rv2212 adenylyl cyclase-mediated cAMP production.
The RvΔssErp gene was cloned in mycobacterium shuttle vector pSC301 to measure cAMP production under in vivo conditions and intracellular survival within macrophages (Table 1). Adenylate cyclase activity of Rv2212 was checked in vitro by measuring cAMP production at 30°C for 30 min in a volume of 100 μl by the method described by Salomon et al. (11), with modifications. The reaction mixture contained 50 mM bis-Tris-HCl (pH 6.5), 22% glycerol, 3 mM MnCl2, and 500 μM ATP. Production of cAMP was measured according to the method of Haneda et al. (12) using the cAMP Biotrak enzyme immunoassay (EIA) kit (Wipro GE HealthCare). The increase in cAMP production upon increasing concentrations of Rv2212 is shown in terms of fold increases with respect to the cAMP levels corresponding to 0.1 μM Rv2212 (see Fig. 9A). The effects of RvΔssErp on Rv2212 activity were studied by measuring cAMP production with increasing concentrations of RvΔssErp from 0.5 μM to 3.0 μM at a concentration of 0.5 μM Rv2212. Fold differences were calculated with respect to cAMP production when 0.5 μM Rv2212 alone was used (see Fig. 9B). To study the effects of the interaction of RvErp with Rv2212 in vivo (see Fig. 9C), production of cAMP was measured in mixtures containing increasing amounts of either H37Ra lysate or H37Ra overexpressing RvΔssErp lysate, i.e., H37Ra/RvΔssErp. The results have been plotted as fold increases in cAMP levels relative to those obtained with 0.5 μg of wild-type H37Ra lysate.
FIG 9.
RvΔssErp protein increases the Rv2212-mediated cAMP production in a dose-dependent manner. (A) Production of cAMP when 0.1 μM to 5.0 μM purified Rv2212 protein was used. (B) Effects of RvΔssErp on Rv2212-mediated cAMP production under in vitro conditions: 0.5 μM Rv2212 protein was treated with 0.5 μM to 3 μM purified RvΔssErp protein, and levels of cAMP were monitored. cAMP production by 0.5 μM Rv2212 alone was used to calculate the fold difference. (C) Overexpressed RvΔssErp enhances the activity of endogenous MRA_2228 under in vivo conditions: 0.5 μg, 1.0 μg, and 2.0 μg lysates of H37Ra alone or H37Ra overexpressing RvΔssErp (H37Ra/RvΔssErp) were used to monitor the cAMP production. While the anti-Erp immunoblot shows levels of RvErp in respective lysates, the anti-GroEL immunoblot indicates equal loading of lysates in lanes 1 and 2, in lanes 3 and 4, and in lanes 5 and 6. Graphs were generated using Microsoft Excel software. Data are means ± standard deviations from three independent experiments. Graph Pad was used to determine P values: *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005.
Intracellular survival assay.
THP-1 monocytes were cultured at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 10% (vol/vol) fetal bovine serum. THP-1 monocytes seeded at a density of 106 cells/well were differentiated with 30 ng/ml phorbol-12-myristate-13-acetate (PMA) for 24 h and infected after 24 h with log phase cultures of M. smegmatis harboring either RvΔssErp, Rv2212, or ΔAC-Rv2212 gene alone or in combination. M. smegmatis transformed with vector alone was included in every experiment as a negative control. The intracellular survival assay was performed as described earlier (13).
Statistical analysis.
Student's t test in Graph Pad software was used to analyze the adenylate cyclase activity of Rv2212 (see Fig. 9) and the CFU at 24.0 h and 48.0 h postinfection in the intracellular survival assay (see Fig. 10). P values of ≤0.05 were considered statistically significant.
FIG 10.
Intracellular survival of M. smegmatis harboring either the RvΔssErp, Rv2212, or ΔAC-Rv2212 gene of M. tuberculosis either alone or in combination in THP-1 cells. Differentiated THP-1 macrophages were infected with M. smegmatis harboring vector or the RvΔssErp, Rv2212, or ΔAC-Rv2212 gene of M. tuberculosis either alone or in combination. Survival of intracellular bacteria was quantified at 0 h, 24 h, and 48 h postinfection. The graph was generated using Origin 9.1 software. Data are means ± standard deviations from three independent experiments. Graph Pad was used to determine P values: *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005.
RESULTS
RvΔssErp interacts with Rv2212 in yeast two-hybrid screening.
The erp gene devoid of a 22-amino-acid-long signal sequence was cloned into bait vector pBD-GAL4 Cam having the DNA binding domain (BD) of GAL4. The signal sequence of Erp was deleted to abrogate its secretion. The possibility of RvΔssErp acting as a transcription factor was ruled out by a transactivation test in which RvΔssErp was unable to autoactivate the expression of either HIS3 or lacZ reporter genes (data not shown). Thus, RvΔssErp qualified as biological bait for a Y2H assay. An M. tuberculosis partial genomic DNA library was cloned into prey vector pAD-GAL4 having the activation domain (AD) of GAL4. Approximately 1.3 × 105 independent clones were screened for interaction with RvΔssErp. Clones were initially selected for histidine prototrophy. The HIS3 reporter gene has a leaky expression, so the results were further confirmed with a LacZ assay. By BLAST searches, we found that one clone with high β-galactosidase activity encoded Rv2212 protein of M. tuberculosis (Fig. 1). Rv2212 has been reported earlier as a soluble adenylate cyclase (14).
FIG 1.
Interaction of RvΔssErp with Rv2212 using a LacZ assay in a yeast two-hybrid system. In the yeast two-hybrid assay, RvΔssErp and an M. tuberculosis library were used as bait and prey, respectively. (Left) Filter lift assay using β-galactosidase activity. (Right) Schematic details of the left panel. pBD-WT + pAD-WT and pBD-WT + pLaminC are positive and negative controls, respectively.
Purification of RvErp, RvΔssErp, MsΔssErp, Rv2212, and Ms_4279 proteins.
Overexpressed recombinant proteins, i.e., RvErp, RvΔssErp, MsΔssErp, Rv2212, and Ms_4279 proteins were purified to homogeneity using either an Ni-NTA Superflow or a glutathione-agarose column, as mentioned in Materials and Methods. A single band on SDS-PAGE confirmed the homogeneity of the proteins (Fig. 2A to G). Anomalous electrophoretic mobility for RvErp is known (3); therefore, recombinant RvErp and RvΔssErp did not migrate according to their calculated molecular weights.
FIG 2.
Purification of RvErp, RvΔssErp, GST-Rv2212, MsΔssErp, His-Rv2212, and GST-Ms_4279 proteins was performed as described in Materials and Methods. (A) Purification of RvErp protein using an Ni-NTA column is shown in a Coomassie blue-stained gel. (Left) Lane 1, load; lane 2, flowthrough; lanes 3 and 4, washings with buffer containing 20 mM and 40 mM imidazole, respectively; lanes 5 to 14, various elution fractions of RvErp protein. (B) Purification of RvErp protein using a Q Sepharose column is shown by Coomassie blue-stained gel. (Left) Lane 1, Ni-NTA-purified RvErp dialyzed in dialysis buffer with 20 mM NaCl, i.e., load; lane 2, flowthrough; lanes 3 to 6, elution with 20 mM, 100 mM, 200 mM, and 400 mM NaCl buffer. (Right) Purified RvErp protein after final purification and concentration. (C and D) Coomassie blue-stained gels showing purified and concentrated RvΔssErp and MsΔssErp proteins, respectively. (E) Coomassie blue-stained gel showing the purification of His-Rv2212 protein using an Ni-NTA column. Lane M, protein marker; lane 1, uninduced; lane 2, induced; lane 3, flowthrough; lanes 4 to 7, various fractions eluted with 250 mM imidazole. (F) Coomassie blue-stained gel showing purification of GST-Rv2212 protein using a glutathione-agarose column. Lane M, protein molecular mass marker (in kilodaltons); lane 1, uninduced cell lysate; lane 2, 0.5 mM IPTG-induced cell lysate; lane 3, flowthrough; lane 4, washing with PBS; lanes 5 to 8, elution fractions of GST-Rv2212 protein eluted with buffer containing 10 mM reduced glutathione. (G) Purification of GST-Ms_4279 protein using glutathione-agarose column is shown in a Coomassie blue-stained gel. Lane 1, uninduced; lane 2, induced; lanes 3 and 4, washing with PBS; lanes 5 to 9, elution fractions of GST-Ms_4279 with buffer containing 10 mM glutathione. All the panels show protein markers on the left.
Disruption of signal sequence of RvErp does not affect its structure.
To eliminate the possibility of any structural changes due to the deletion of the signal sequence, structural features of RvΔssErp were compared with those of RvErp. The far-UV CD spectrum of RvΔssErp exhibited no difference from that of RvErp (Fig. 3). The CD spectra of RvErp or RvΔssErp proteins in aqueous solution exhibited a strong negative peak at 198 nm with a small shoulder at 215 nm, indicating that Erp protein has random coils under experimental conditions.
FIG 3.
Far-UV CD spectra of RvErp and RvΔssErp were measured in an aqueous solution at 20°C to show the structural differences between RvErp and RvΔssErp.
Confirmation of interaction of RvΔssErp with Rv2212 using a GST pulldown assay.
Equimolar concentrations of purified RvΔssErp, GST-Rv2212, and GST proteins were used either alone or in combination. Glutathione-agarose beads precipitated GST and GST-Rv2212 proteins but failed to precipitate RvΔssErp, ruling out the nonspecific association of RvΔssErp not only to glutathione-agarose beads (Fig. 4A, lane 2) but also to GST proteins (Fig. 4A, lane 4). Rv2212 specifically coprecipitated RvΔssErp (Fig. 4A, lane 5) which shows the interaction between Rv2212 and RvΔssErp to be direct and not mediated by any third protein partner.
RvErp interacts with Rv2212 in a dose-dependent manner.
Next, we checked whether full-length RvErp with its intact signal sequence interacts with Rv2212. A fixed amount of GST-Rv2212 was added to increasing concentrations of purified RvErp in the molar ratios of 1:1, 1:2, 1:4, and 1:8. An increase in the interaction of RvErp with Rv2212 was observed in a dose-dependent manner in the GST pulldown assay (Fig. 4B, lanes 5 to 8).
To establish the specificity of the interaction of RvErp and Rv2212, a GST pulldown assay was set up with a constant amount of RvΔssErp and increasing concentrations of Rv2212 in the molar ratios of 1:1, 1:2, 1:4, 1:8, and 1:16. As shown in the top panel of the pulldown section in Fig. 4C, there is an increased interaction between Rv2212 and RvΔssErp (lanes 5 to 9) with increasing concentrations of Rv2212. In the upper panel of the supernatant section, the corresponding depletion of RvΔssErp with increasing amounts of Rv2212 (lanes 5 to 9) clearly established that Rv2212 may act as an efficient scavenger of Erp.
The RvΔssErp protein interacts with Rv2212 under in vivo conditions.
The interaction of Erp and Rv2212 was investigated under in vivo conditions. FLAG-RvΔssErp and His-Rv2212 either alone or in combination were electroporated in M. smegmatis. While anti-FLAG M2 beads did not immunoprecipitate Rv2212 protein, the FLAG-RvΔssErp protein specifically coimmunoprecipitated Rv2212 protein (Fig. 4D, lane 2 versus lane 3, upper panel in Co-IPs). This result clearly demonstrated that Rv2212 specifically interacts with RvΔssErp in vivo.
M. smegmatis Erp does not interact with Rv2212.
M. smegmatis Erp (MSMEG_6405 or MsErp) shares 55% identity with M. tuberculosis Erp (https://blast.ncbi.nlm.nih.gov/). Therefore, the interaction of M. smegmatis Erp with Rv2212 was checked to determine whether the Erp orthologue from nonpathogenic M. smegmatis was functionally equivalent or not. RvΔssErp was taken as a positive control for the interaction. For in vitro binding assays, equimolar concentrations of purified recombinant RvΔssErp, MsΔssErp, GST-Rv2212, and GST were used either alone or in combination. The GST-Rv2212 was able to bind to the RvΔssErp but failed to interact with MsΔssErp (Fig. 5A, lanes 7 and 8).
M. smegmatis Erp does not interact with the M. smegmatis homologue of Rv2212.
Bearing the fact that identity between Rv2212 and Ms_4279 is only 66% (https://blast.ncbi.nlm.nih.gov/), we next examined whether MsErp interacts with Ms_4279. Equimolar concentrations of purified recombinant RvΔssErp, MsΔssErp, GST-Rv2212, GST-Ms_4279, and GST were used either alone or in combination in the GST pulldown assay. While GST-Rv2212 was able to bind to the RvΔssErp, GST-Ms_4279 failed to interact with MsΔssErp (Fig. 5B, lanes 4 versus 5), confirming the specific interaction of Rv2212 with RvErp.
Localization of RvErp and Rv2212 in M. tuberculosis.
The subcellular fractions of M. tuberculosis HN878 were obtained from BEI Resources and electrophoresed on SDS-PAGE followed by transfer onto nitrocellulose membrane. Upon Western analysis, it was found that Rv2212 was present in whole-cell lysate, cytosol, and cell wall at ∼40 kDa (Fig. 6A, top panel, lanes 1, 2, and 4), whereas RvErp was detected in all four fractions, i.e., whole-cell lysate, cytosol, cell membrane, and cell wall (Fig. 6A, middle panel, lanes 1 to 4). In the cell wall fraction, RvErp was found to electrophorese at a higher speed (Fig. 6A, middle panel, lane 4), probably due to the loss of signal sequence in the process of secretion. Besides being present in the cell wall, the truncated RvErp was also detected in the whole-cell lysate and cell membrane fractions. The different mobility of the truncated form of RvErp in cell membrane fraction might be due to the partial cleavage of signal sequence during its translocation through membrane. The purity of the cytosolic and cell wall fractions was checked by probing the fractions with antibodies against RpoB and Ag85c, respectively. The localization of RvErp and Rv2212 in cytoplasm and cell wall clearly established that these two proteins are present in the same compartment of the cell.
FIG 6.
Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis HN878. Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed on SDS-PAGE, transferred onto nitrocellulose membranes, and probed with either anti-Rv2212 antibody or anti-Erp antibodies. Anti-RpoB and anti-Ag85c antibodies were used to establish the purity of cytosolic and cell wall fractions, respectively. Lane 1, whole-cell lysate (WCL); lane 2, cytosol (CYT); lane 3, cell membrane (CM); lane 4, cell wall (CW). (Bottom) Coomassie blue-stained gel showing the equal amount of proteins in cell fractions used in the above panels. Molecular mass markers (in kilodaltons) are shown on the left. (B) RvErp interacts with Rv2212 at the endogenous level. Lysates of M. tuberculosis H37Rv were prepared and subjected to an immunoprecipitation assay as described in Materials and Methods. Lane 1 in the IP panel shows the interaction of endogenous Rv2212 with endogenous H37RvErp; lane 2 in the IP panel depicts the nonspecific association and serves as a negative control. Anti-Erp and anti-Rv2212 antibodies detected RvErp and Rv2212, respectively. Inputs indicate the levels of Rv2212 and RvErp present in the H37Rv lysate. Markers are shown on the left.
RvErp interacts with endogenous Rv2212.
To establish the interaction at the physiological level, the endogenous complex of Rv2212 and native full-length RvErp was immunoprecipitated from H37Rv lysate using Rv2212 antibody. Rv2212 antibody successfully immunoprecipitated endogenous Rv2212 protein (Fig. 6B, lane 1, lower panel of IPs). The presence of RvErp protein in the immunoprecipitates of Rv2212 clearly established that endogenous Rv2212 interacts with full-length, native, endogenous RvErp (Fig. 6B, lane 1, upper panel of IPs).
The AC domain of Rv2212 is important for its interaction with RvErp.
Protein sequence analysis of Rv2212 by InterProScan and ScanProsite revealed a specific adenylate cyclase domain flanked by N-terminal and C-terminal domains. To investigate the domains of Rv2212 involved in the interaction with RvΔssErp, three deletion mutants of Rv2212, namely, ΔN-Rv2212, ΔAC-Rv2212, and ΔC-Rv2212 were constructed (Table 1 and Fig. 7A) and purified to homogeneity using an N terminus GST tag (Fig. 7B). For in vitro GST binding assays, equimolar concentrations of purified recombinant His-RvΔssErp, GST-Rv2212, GST-ΔN-Rv2212, GST-ΔAC-Rv2212, GST-ΔC-Rv2212, and GST proteins were used either alone or in combination. While GST-Rv2212, GST-ΔN-Rv2212, and GST-ΔC-Rv2212 were able to interact with RvΔssErp (Fig. 7C, lanes 8, 9, and 11), GST-ΔAC-Rv2212 failed to interact with RvΔssErp (Fig. 7C, lane 10), indicating a critical role of the adenylate cyclase domain in the interaction.
Any anomaly in the folding of Rv2212 due to the deletion of adenylate cyclase domain was checked by comparing the far UV-CD spectrum of full-length Rv2212 with that of ΔAC-Rv2212. Similar structural features for ΔAC-Rv2212 and Rv2212 proteins were observed, which consist mainly of α-helices and β-sheets (Fig. 7D). The similar structural features of ΔAC-Rv2212 and Rv2212 confirmed that deletion of the adenylate cyclase domain of Rv2212 does not alter the folding of protein.
Stoichiometry of the complex containing Rv2212 and RvErp.
To determine the stoichiometry of the complex of Rv2212 and RvΔssErp, we first examined the individual oligomeric status of purified recombinant Rv2212 and RvErp by size exclusion chromatography (SEC). The various fractions collected were electrophoresed on SDS-PAGE, transferred onto nitrocellulose membrane, and probed with either anti-Rv2212 or anti-RvErp antibodies. Purified RvErp protein on a calibrated Superose 6 Increase 10/300 GL column eluted as a single discrete peak at a retention volume of ∼16.50 ml (Fig. 8A) with a molecular mass of about ∼170 kDa. When the elution volumes of the marker proteins were plotted as a function of log molecular weight, the results of the SEC studies along with the predicted molecular weight based on the actual number of amino acids reported for RvErp (5) demonstrate that RvErp proteins exist as homohexamers. The absence of another peak(s) in the gel filtration chromatogram confirmed the homogeneity of purified protein. Also, Western blot analysis with anti-Erp antibodies showed the presence of RvErp protein in fractions F9 to F15 with a peak at fraction F12 corresponding to hexamers of a molecular mass of ∼170 kDa (Fig. 8A, lanes 9 to 12 in the left panel and lanes 1 to 3 in the right panel).
FIG 8.
Stoichiometry of the interaction between Rv2212 and RvErp was studied using size exclusion chromatography (SEC). (A and B) SEC profiles of purified recombinant RvErp and Rv2212, respectively, in 20 mM phosphate buffer (pH 8.0) at 4°C. The inset shows the standard molecular weight markers (in thousands) run under the same conditions for molecular weight determination. (C) Stoichiometric determination of the interaction of RvErp with Rv2212 using SEC. The complex consisting of purified RvErp and Rv2212 was fractionated by gel filtration chromatography. Fractions from F1 to F23 were collected, electrophoresed on SDS-PAGE, transferred onto nitrocellulose membrane, and immunoblotted with anti-Rv2212 (top panels) and anti-RvErp (lower panels) antibodies. Molecular mass markers (in kilodaltons) electrophoresed on SDS-PAGE are shown on the left and right sides of the panels. Cyt-C, cytochrome c (12.9 kDa); CAH, carbonic anhydrase (29 kDa); ADH, alcohol dehydrogenase (150 kDa); BAE, β-amylase (200 kDa); FER, ferritin (440 kDa); TG, thyroglobulin (669 kDa).
Purified recombinant Rv2212 protein on a calibrated Superose 6 Increase 10/300 GL gel filtration column eluted as a discrete sharp peak at a retention volume of ∼17.24 ml (Fig. 8B). The results of the SEC studies in conjunction with the subunit mass determined by SDS-PAGE demonstrate that Rv2212 exist as homodimers of ∼80 kDa under experimental conditions. Dimers of Rv2212 were verified in Western blot analysis by the presence of Rv2212 in fractions F12 to F18 with a peak at fraction F15 (Fig. 8B, lane 12 in the left panel and lanes 1 to 6 in the right panel).
For determining the stoichiometry of the complex of Rv2212 and RvErp, the complex was subjected to fractionation by gel filtration on a calibrated Superose 6 Increase 10/300 GL column. The various collected fractions were separated on SDS-PAGE and transferred onto nitrocellulose membrane, followed by Western blotting. Cofractionation of RvErp and Rv2212 in fractions F6 to F14 (Fig. 8C, lanes 6 to 12 in the left panel and lanes 1 and 2 in the right panel of the complex) with a peak at fraction F10 (Fig. 8C, lane 10, left panel of the complex) of ∼245 kDa confirmed that the complex consists of one dimer of Rv2212 and one hexamer of RvErp.
Interaction of RvΔssErp with Rv2212 modulates cAMP production.
Rv2212 is an adenylate cyclase (14). Activity of Rv2212 was measured as cAMP production and found to be increased with increases in the concentration of Rv2212 in a dose-dependent manner (Fig. 9A). Next, the effects of the interaction of RvΔssErp with Rv2212 on the cAMP production were checked by titrating increasing concentrations of RvΔssErp against 0.5 μM Rv2212. The Rv2212-mediated cAMP production was found to be increased significantly with increases in the concentration of RvΔssErp and was ∼2.0-fold higher with 3.0 μM RvΔssErp than with 0.5 μM RvΔssErp (Fig. 9B).
RvΔssErp enhances the cAMP production under in vivo conditions.
H37Ra and H37Rv being the avirulent and virulent counterparts of a laboratory strain of M. tuberculosis share 100% identity. M. tuberculosis H37RvErp (Rv3810) is 100% identical to M. tuberculosis H37RaErp (MRA_3850), and the identity level between Rv2212 from M. tuberculosis H37Rv and M. tuberculosis H37Ra (MRA_2228) is also 100%. Therefore, overexpression of H37RvΔssErp into H37Ra would result in a modulation of cAMP levels. An increase in cAMP levels was observed upon increasing the amount of lysates from 0.5 μg to 2.0 μg. A 2.0-μg amount of lysates of M. tuberculosis H37Ra overexpressing H37RvΔssErp (H37Ra/RvΔssErp) exhibited a ∼2.5-fold increase in cAMP levels over those of M. tuberculosis H37Ra control lysate (Fig. 9C).
M. smegmatis strains expressing RvΔssErp and Rv2212 show enhanced survival within macrophages.
Given the fact that MsErp does not interact with Rv2212 (Fig. 5A) and the identity between the adenylate cyclase domain of Rv2212 and its homologue in M. smegmatis, i.e., Ms_4279, is only 66%, it is not far-fetched to speculate that RvΔssErp would not interact with Ms_4279. Therefore, M. smegmatis is an appropriate choice to examine the effects of the interaction of RvErp with Rv2212 on the intracellular survival of a mycobacterial pathogen within macrophages. M. smegmatis strains overexpressing RvΔssErp, Rv2212, and ΔAC-Rv2212, either alone or in combination, were used for an intracellular survival assay in THP-1 macrophages. The CFU at zero hour indicated bacillary uptake by the macrophages. M. smegmatis overexpressing either RvΔssErp or Rv2212 alone exhibited ∼1.5 and ∼1.75-fold increases, respectively, in survival at 24 h postinfection in comparison to M. smegmatis harboring vector alone. The respective fold differences were augmented to ∼2.0- and ∼6.5-fold at 48.0 h postinfection. Furthermore, M. smegmatis harboring a combination of RvΔssErp and Rv2212 exhibited a ∼2.0-fold increase in survival at 24 h postinfection, which was increased to ∼8.0-fold at 48 h postinfection. Survival of M. smegmatis overexpressing RvΔssErp and ΔAC-Rv2212 was compromised ∼1.7-fold in comparison to M. smegmatis overexpressing RvΔssErp and Rv2212 at 24 h postinfection. This fold difference was further compounded to ∼3.5-fold at 48 h postinfection (Fig. 10). This result confirmed that the interaction of Rv2212 with RvErp is important for the enhanced intracellular survival of M. smegmatis.
DISCUSSION
RvErp is an important virulence determinant of M. tuberculosis, but studies deciphering its exact role and regulation are lacking. Therefore, we decided to explore the interacting proteins of RvErp to unravel its novel functions using a yeast two-hybrid assay. Two hybrid approaches have been used successfully in Mycobacterium species (8, 15, 16) to identify protein-protein interactions. In a yeast two-hybrid assay, Rv2212, an adenylyl cyclase, was found to interact with RvErp. Rv2212 is known to be regulated by fatty acids and operates as an ATP sensor by integrating the signals sensing fatty acids and intracellular pH with the cAMP as the output (14). Rv2212 is also shown to modify the proteome and infectivity of Mycobacterium bovis BCG (17).
M. tuberculosis possesses 17 putative adenylyl cyclases (18) that regulate distinct signaling pathways. Of these 17, only few are important for the physiology of Mycobacterium (19–21). We were able to capture the specific interaction of Rv2212 with RvErp.
An important prerequisite for two proteins to interact is that they should be present in the same fractions of the cell. The localization of RvErp has been studied earlier (6) but the localization of Rv2212 was not known. Therefore, we first checked the presence of Rv2212 in various subcellular fractions of M. tuberculosis. Rv2212 was found to be present in cytosol and cell wall. To establish whether Rv2212 and RvErp are present in the same fractions under our experimental conditions, we checked the localization of RvErp. Interestingly, RvErp was found to be present in the cytosol, cell membrane, and cell wall of M. tuberculosis. Although Erp has been reported earlier as a secretory protein and found to be present in the cell wall and cell membrane (6), a cytoplasmic localization of Erp has not been reported so far. This is the first report on the cytoplasmic localization of RvErp. Despite having the signal sequence, the presence of Erp in significant amounts in the cytosol of M. tuberculosis indicates that it may have novel functions in cytosol.
The presence of RvErp and Rv2212 in the same fractions, i.e., cytosol and cell wall, of M. tuberculosis along with their interaction at the endogenous level confirms that these interactions are important and have physiological relevance. The increased interaction of Erp with increasing concentrations of Rv2212 confirms that Rv2212 acts as an efficient scavenger of Erp. RvErp not only interacts with Rv2212 under in vitro and in vivo conditions but also increases the production of cAMP by modulating its activity.
cAMP being a second messenger is an important signaling molecule that regulates biofilm formation, virulence, and a wide variety of cellular functions (19–27). Elevated cAMP levels in macrophages affect the fusion of phagosomes with lysosomes during mycobacterial infection (28) with a resultant increase in the survival of mycobacteria inside host macrophages. cAMP plays a role in the persistence of M. tuberculosis infection by regulating the glyoxylate shunt metabolism (29). Also, cAMP regulates gene expression in mycobacteria under the low-oxygen and CO2-enriched growth conditions faced by Mycobacterium during infection within the host (29). The cAMP produced by Mycobacterium in the macrophage cytoplasm modifies the host environment to facilitate the long-term survival of the pathogen (30). In sum, an increased level of cAMP provides a survival advantage to the Mycobacterium within macrophages.
We have shown that the interaction of RvErp with Rv2212 enhances the survival of M. smegmatis within macrophages, which was more evident at 48.0 h than at 24.0 h postinfection. This result also indicates the delayed clearance of M. smegmatis harboring either RvErp or Rv2212. Similar results have also been reported earlier for Eis protein, a thermostable aminoglycoside acetyltransferase, which enhances the survival of Mycobacterium within macrophages (13, 31). A recent report by Pedroza-Roldan et al. (17) has also shown that the overexpression of Rv2212 caused the enhanced replication and survival of Mycobacterium bovis BCG Pasteur 1173P2.
Rv2212 specifically interacts with RvErp protein of M. tuberculosis and not with Erp orthologues from M. smegmatis. de Mendonça-Lima et al. have reported that Erp from nonpathogenic species is functionally inequivalent to Erp from pathogenic species (3), thus giving the latter the benefit of efficiently colonizing the lungs at the early time points of infection. This indicates that the virulence due to Erp proteins from different species may be attributed to their specific interactions.
In this study, increased cAMP production due to the interaction of RvErp with Rv2212 and the survival profile indicate that this interaction may play an important role in enhancing the survival of M. smegmatis within macrophages. This study adds crucial information in understanding the mechanism(s) by which Erp functions and also opens up the field for the discovery of many unexplored roles of Erp in the cytoplasm of M. tuberculosis.
ACKNOWLEDGMENTS
We gratefully acknowledge the kind gift of expression vectors pSC301 from Youssef Av Gay and p19Kpro from D. B. Young. BEI resources are acknowledged for the pVV16 vector, whole-cell lysate, and cytosol, cell membrane, and cell wall fractions of M. tuberculosis HN878.
We report no potential conflicts of interest.
This work was supported by funding from the Council of Scientific and Industrial Research (grant numbers BSC0210-CSIR-INFECT and OLP0057), New Delhi, India. Fellowships from the Council of Scientific and Industrial Research (CSIR), New Delhi, to A.A.G., G.T., S.S.J., S.A., and A.S., from the Department of Biotechnology (DBT), New Delhi, to A.K., and S.K., and from DST-INSPIRE, New Delhi, to V.R. are acknowledged.
The funding source had no role in the design of the study, data analysis, decision to publish, or writing of the manuscript.
Funding Statement
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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