EVALUATION OF DevR ANTIGEN (MSMEG_5244) AS A PUTATIVE BIOMARKER FOR THE SERODIAGNOSIS OF PERSISTANT Mycobacterium tuberculosis

- Tuberculosis (TB) is a major health problem in the developing world. 9 million new cases of TB and three million deaths are reported every year around the globe. The management of tuberculosis in the regions of developing countries urgently needs an efficient diagnostic test. DevR (also called DosR) is essential for promoting long-term survival of Mycobacterium tuberculosis under low oxygen conditions in a dormant state and may be responsible for latent tuberculosis in one-third of the world's population. The similarity in sequences of DevR in Mycobacterium smegmatis and Mycobacterium tuberculosis has been already proved and hence Mycobacterium smegmatis is used as a model in present study for studying and evaluating the immunogenic properties of antigen DevR. Elimi-nation of tuberculosis (TB) largely depends upon definitive rapid diagnosis and treatment. Widely used diagnostic methods do not qualify for use in a developing country due to lack of either desired accuracy and cost. In the present study ELISA was used to evaluate the diagnostic potential of an immuno-dominant antigen DevR from 36 BCG vaccinated and 12 non-vaccinated TB patients. 48 samples were collected from healthy control subjects. We have evaluated the diagnosis of tuberculosis, based on detection of anti-IgG antibodies in the TB patient’s sera. The mean IgG antibody levels in sera of both BCG vaccinated (n=36) and non-vaccinated (n=12) patients with tuberculosis were significantly higher compared to the healthy donors (p<0.05). The results suggest that DevR could be possible candidate biomarker for effective use in the serodiagnosis of persistant tuberculosis infections.

Generally, stimuli detected by these systems are transformed into a cellular signal by autophosphorylation of the sensor proteins at a conserved histidine residue. The phosphorylated histidine of these sensor proteins is the source for phosphorylation of an aspartic acid residue in the so-called 'receiver domain' of the transcription factor. Phosphorylation of the regulatory proteins induces a conformational change which alters their DNA-binding properties. A small number of TCSs are characterized by a complex phosphorelay between two histidine and two aspartic acid residues present in four signalling domains, which can either be independent proteins or be integrated into multidomain TCS proteins in various combinations [24]. Already in previous studies it has been reported that DevR is a stationaryphase regulator required for adaptation to oxygen starvation and resistance to heat stress in M. smegmatis [25]. Thus DevR is proposed to be an attractive target for the development of inhibitors against dormant organisms [26][27][28][29] and for developing vaccine against persistent organisms [30][31][32]. The present study aims to over express the recombinant protein (DevR) in E. coli BL21 (DE3) and purify it to determine the immunological reactiveness by testing sera from TB patients and healthy donors of south Indian population. This may help to identify and develop a novel anti-tuberculosis vaccine with higher efficiency than BCG by genetic engineering.

Materials and Methods Materials
Mycobacterium smegmatis was obtained from Tuberculosis research center, Chennai, India. Escherichia coli strains XL10 gold and BL21 (DE3) were obtained from Stratagene, USA. Plasmid pRSET-A was obtained from Invitrogen, USA. T4 DNA Ligase, SacI and KpnI were obtained from Fermentas, Thermo scientific, Inc. Spin Clean Chelating IDA Excellose column (1mg/mL) from Mbiotech, South Korea. Anti-Human IgG conjugated with HRP, TMB/ H2O2 were obtained from Merck, USA. All other chemicals were of molecular biology grade or higher.

Culturing of Mycobacterium smegmatis and E. coli
Mycobacterium smegmatis was inoculated in LJ slants incubated at 37°C. E. coli XL10 gold harboring pRSET-A was grown in LB broth with ampicillin (100 μg/mL) overnight at 37°C, in a shaker incubator (250 rpm). E. coli BL21 (DE3) and XL10 gold were grown in LB broth overnight at 37°C, in a shaker incubator.

pRSET-A Plasmid Isolation, Genomic DNA Isolation, PCR Amplification and Cloning of DevR Protein of Mycobacterium smegmatis
The pRSET-A plasmid was isolated from E. coli using SIGMA plasmid miniprep Kit. Genomic DNA of M. smegmatis was isolated by Phenol/Chloroform method and checked for purity by UV spectrophotometer and electrophoresis in agarose gel. The DevR gene is amplified using the primers, DevR-F-(5'-GATTCGAGCTCATGATCAGGGTTTTTCTGGT-3') and DevR-R-(5'-GTACCGGTACCCTAGTTGCGCCGGTCCAGTTTG-3').
PCR was performed for 34 cycles of denaturation at 94ºC for 30s, annealing at 55ºC for 30s, extension at 72ºC for 30s, followed by final extension for 5 min at 72ºC. The PCR product and pRSET-A plasmid were subjected to restriction digestion using SacI and KpnI. Then the PCR product and plasmid were ligated using T4 DNA Ligase. The ligated plasmids were transformed to E. coli BL21 (DE3) using calcium chloride mediated heat shock method as de-scribed by Chung and Miller with minor modifications [33]. The recombinants were selected by growing the transformed E. coli cells in ampicillin containing LB broth and confirmed by insert release from the recombinant plasmids.

DNA Sequence Analysis
The orientation of recombinant clone DevR: PRSETA, were commercially sequenced [Applied Biosystems (P) Ltd; Bangalore, India] to confirm inframe ligation. Oligonucleotide T7 forward and pRSET-A reverse primers were used for sequencing. The amplified products were preceded for Cycle sequencing PCR reactions. Extended products were precipitated and purified by using 3M sodium acetate and ethanol purification method and the samples were denatured at 94°C for 5minutes after adding hidiformamide and the sample plate was linked to 373-48 capillary array equipment, ABI (Applied Biosystems).

Expression and Purification of DevR Protein in E. coli BL21 (DE3)
The expression of DevR was induced by 1.5nM IsoPropyl-1-Thio-β-D-Galactopyranoside (IPTG) in mid-log phase (OD600 nm of 0.6-0.7) at 25ºC for 5 hours. The expressed recombinant protein was isolated and purified by capturing the DevR using Spin Clean Chelating IDA Excellose column (1mg/mL). The quality of the expressed and purified DevR was checked by Tricine-SDS-PAGE followed by staining with Coomassie brilliant blue R-250 stain.

Immunoscreening of the Recombinant Protein Antigen in TB Patients Study Population
After informed consent, 5 mL of venous blood samples were obtained from 36 BCG vaccinated and 12 non-vaccinated TB patients (20-60yrs old patients from Govt. Hospital of Thoracic Medicine, Tambaram sanatorium, Chennai and Govt. Hospitals at Erode and Salem, Tamilnadu, India). The sera were separated by standard procedure and the decomplementation was done at 56ºC for 30 minutes. Finally the sera were stored at -20ºC with appropriate labeling. A total of 48 samples were collected from healthy control subjects.

ELISA
The recombinant protein was screened for antigenic properties. Enzyme linked immunosorbant assay (ELISA) was done to detect the IgG antibodies to the purified protein in the serum of both M. tuberculosis infected patients and healthy control subjects. For ELI-SA, 96-well polystyrene microtiter plates were coated with antigen (DevR) at 1µg/mL (0.1mL per well) in carbonate-bicarbonate buffer (PH 9.0) overnight at 4ºC. The plates were washed thrice with washing buffer. Plates were blocked with phosphate buffered saline (pH 7.4) containing 1% BSA for 2 Hrs. at 37ºC and washed extensively with PBS. Serum samples were diluted at 1:500 in sample dilution buffer and 0.1 mL of diluted serum was added to antigencoated wells in duplicate and incubated for 30 min at room temperature. Plates were washed extensively with PBS-T and then incubat-

Statistical Methods
The ELISA results are expressed as percentages wherever appropriate. The means and standard deviations (SD) were calculated for the sera of uninfected healthy donors (n=48) and the cut off for positive response was calculated as greater than the mean plus 3 SD, and for high level responses, as the mean plus 6 SD. The mean of control group (N=48) was compared with the mean of TB patients (BCG vaccinated and non-vaccinated) group (n=48) for the significance by using students T-test [34].

Cloning of DevR Gene and Recombinant Selection
The genomic DNA was isolated from M. smegmatis [ Fig-1] and PCR amplification of DevR gene was obtained with no non-specific amplification [Fig-2]. The ligated recombinant plasmid with DevR gene was successfully screened [Fig-3]. The insert was released by digesting with SacI and KpnI [Fig-4] and sequenced to find the DevR gene is in-frame with the plasmid [Fig-5]. The recombinant plasmids were over expressed by IPTG induction for 4 hours [Fig-6] and the DevR protein product (30 KDa) was purified using Spin Clean Chelating IDA Excellose column [Fig-7].         tion-based signal transduction mechanism itself, surprisingly little information is available about the molecular basis for its contribution to bacterial virulence. Future research should be aimed at understanding the targets of TCS mediated regulation, and the regulatory networks into which the TCSs are integrated to control the expression of such a multifarious phenotype as bacterial virulence [35][36][37].
The search for structurally unique antibiotics that inhibit new molecular targets has led researchers to prokaryotic two-component systems. Two-component systems are attractive for several reasons [38]. First of all, they are widespread in bacteria and, so far, absent in mammals. Therefore, general HK (Histidine kinase) or RR (Response regulators) inhibitors could potentially be broadspectrum antibiotics. Alternatively, by targeting specific HKs or RRs, selective inhibition may be achieved. The problem is that most twocomponent Systems are nonessential. However, there is interdependence not only among the proteins in these systems, but among the systems themselves. Cessation or slowing down of these intracellular networks may be a way to affect a cellular shutdown. Perhaps the most attractive reason for targeting two-component systems is that they are used by pathogenic bacteria to control the expression of virulence factors required for infectivity. Thus DevR could serve as a potential target for antimicrobial therapy.
In search for rapid and cost-effective diagnostic methods for TB, immunodiagnosis is considered an attractive option, which uses the specific humoral and cellular immune responses of the host to infer the presence of infection or disease. Recently, the antigen-specific ex vivo induction of interferon gamma (IFN-γ) had been used to detect infection with M. tuberculosis. However, the IFN-γ release assay could not differentiate the latent tuberculosis infection and active tuberculosis efficiently and cannot be recommended for the diagnosis of tuberculosis in developing countries, as large proportions of the populations in such countries are likely to harbor latent infection with M. tuberculosis [39][40][41]. Historically speaking, serology for the diagnosis of TB has been explored since 1898, when crude cell preparations containing carbohydrates, lipids, and proteins from M. tuberculosis or M. bovis BCG were used as antigen preparations showing high sensitivity but low specificity [42]. Modern developments in the purification of antigens, generation of monoclonal antibodies and chromatographic techniques have led to a considerable improvement in specificity [43][44][45][46][47][48][49] The serum IgG antibodies against these proteins were measured from healthy donors (negative control, group of 48 individuals) and TB patients (n=48).The mean and standard deviation were calculated from the healthy donors. Comparatively, the mean IgG antibody levels in the sera of both BCG vaccinated (n=36) and nonvaccinated (n=12) patients with tuberculosis were appreciably higher than the healthy donors (p<0.05).The above findings presented here suggest that DevR (M. smegmatis) could be excellent diagnostic marker and very good candidate for vaccine against tuberculosis. Improved serodiagnostic test for TB can be developed only by rational design of carefully selected antigen covering broad spectrum of antibodies. Use of additional sero-reactive antigens of Mycobacterium tuberculosis will result in the detection of specific antibodies in almost all patients with active TB. To obtain high diagnostic specificity and accuracy, ideal candidates of serologically active antigens specific for Mycobacterium tuberculosis complex should be selected. The new generation of immunodiagnostic assays using immunodominant antigen could be a milestone in the diagnosis of tuberculosis and in the control of TB.
In conclusion, the recombinant protein DevR of Mycobacterium smegmatis was found to be immunodominant with apparent diagnostic potential. A large scale evaluation of this recombinant Mycobacterial protein in different geographic locations of India/Asia/ Africa will definitely help to develop serodiagnostic tests. Identification of more such immunogenic antigens of Mycobacterium tuberculosis in various populations will assist in the development of novel vaccine against Tuberculosis.