The Natural Expression of Genes Encoding Major Antigens of Rd1 and Rd9 in M. Tuberculosis and other Mycobacteria

The natural expression of genes of five major antigenic proteins present in the genomic region of difference (RD)1, i.e. pe35, ppeE68, esxA and esxB, and RD9, i.e. esxV, was determined, at mRNA and protein levels, in M. tuberculosis, M. bovis BCG, and environmental mycobacterial species of M.avium and M.vaccae. The expression of mRNA was determined using total mycobacterial RNA and gene-specific forward and reverse primers in reverse transcription-polymerase chain reaction (RT-PCR), and the expression of proteins was determined by Western immunoblot analysis of mycobacterial sonicates with anti-sera containing protein-specific antibodies raised in rabbits. Furthermore, the expression of each protein in various sub-cellular fractions of M.tuberculosis was studied by Western immunoblot analysis of cell walls, culture filtrates and sonicates of M.tuberculosis using the rabbit anti-sera. The results showed mRNA and protein expressions for PE35, PPE68, ESXA, and ESXB only in M. tuberculosis, whereas ESXV was expressed in M.tuberculosis and M.bovis BCG, but none of them were expressed in M.avium and M.vaccae. Furthermore, all of the proteins were detected in various sub-cellular fractions of M. tuberculosis. In conclusion, our study demonstrates that the five major antigenic proteins encoded by genes present in RD1 and RD9 regions are naturally expressed in M.tuberculosis, whereas only ESXV is expressed in M. bovis BCG and none of these proteins are expressed in the environmental mycobacterial species of M. avium and M. vaccae. As these proteins are major antigens recognized by Th1 cells of protective phenotype, they could be useful in specific diagnosis and/or development of new vaccines against tuberculosis.


Introduction
Tuberculosis (TB) is a major global health problem with 1/3 rd of the world population infected with Mycobacterium tuberculosis, about 9.3 million people developing active disease leading to 1.8 million deaths annually [1]. The threat posed by TB to human health is increasing due to emergence and world-wide spread of multi and excessive drug-resistant strains of M.tuberculosis and co-infections with human immunodeficiency virus (HIV) [1]. It is suggested that the effective control of TB may only be feasible if appropriate new tools become available for specific diagnosis and vaccination [2].
The current immunodiagnosis of TB is based on the use of purified protein derivative (PPD) of M. tuberculosis, as a tuberculin skin test. However, due to antigenic cross-reactivity with other mycobacteria, a positive tuberculin test may not differentiate between people with active/ latent TB, BCG vaccination, or cross-sensitization with environmental mycobacterial species [3]. The only available vaccine against TB is BCG, a live attenuated strain of pathogenic Mycobacterium bovis, developed by Calmette and Guerin in 1921 [4]. BCG has been widely used to vaccinate against TB, and has shown about 50% efficacy in children and against extra-pulmonary disease, but it has completely failed in adults against pulmonary disease, which is the major clinical manifestation of TB [5]. In addition, BCG vaccination can cause disseminated disease in immunocompromised individuals [6] and compromises the diagnostic efficacy of PPD due to the presence of crossreactive antigens [5]. These limitations of PPD and BCG have generated interest in the identification of species-specific and immunodominant antigens of M. tuberculosis for applications in diagnosis and development of new vaccines against TB [7,8].
The sequencing of complete genome of M. tuberculosis and its comparison with other mycobacteria has identified 11 genomic regions of difference (RD) that are deleted in all vaccine strains of M. bovis BCG i.e. RD1, RD4 to RD7, RD9 to RD13 and RD15 [9]. Testing of overlapping synthetic peptides corresponding to proteins encoded by genes predicted in these regions has identified RD1 and RD9 as regions having immunodominant antigens recognized by protective Th1 cells [10,11]. Further testing of individual proteins encoded by these regions identified PE35, PPE68, ESXA and ESXB of RD1 and ESXV of RD9 as major antigens in TB patients [12][13][14][15].However, PPE68 and ESXV were also recognized as major antigens in BCG-vaccinated healthy subjects [12,15].This could have been due to the presence of these genes or their homologs in BCG and/or environmental mycobacteria. The present study was conducted to determine if, in addition to M. tuberculosis, these genes are present and expressed in BCG and two species of environmental mycobacteria i.e. M. avium and M. vaccae at the level of mRNA and proteins by using gene-specific primers in reverse-transcription polymerase chain reaction (RT-PCR) and antiprotein antibodies in Western immunoblotting, respectively.
(obtained from the American Type Culture Collection, Rockville, MD, USA) were grown in Middlebrook 7H9 medium (Difco, Detroit, MI, USA) and sonicated as previously described [16]. M. tuberculosis culture filtrate (MT-CF) and cell walls (MT-CW) (provided by Dr. K. Dobos, Colorado State University, USA) were produced as part of NIH, NIAID Contract No. HHSN266200400091C entitled "Tuberculosis Vaccine Testing and Research Materials", which was awarded to Colorado State University.

Synthetic peptides
Synthetic peptides (25-mers overlapping neighbouring peptides by 10 amino acids) covering the sequence of RD1 and RD9 proteins were designed based on their amino acid sequence deduced from the nucleotide sequence of the respective genes [10,17]. These peptides were commercially synthesized by Thermo Hybaid GmBH (Ulm, Germany) using fluonerylmethoxycarbonyl chemistry, as described previously [18][19][20]. The stock concentrations (5 mg/ml) of the peptides were prepared in normal saline (0.9%) by vigorous pipetting, and the working concentrations were prepared by further dilution in phosphate buffered saline (PBS, pH 7.0) as described previously [21,22].

Oligonucleotide primers
The DNA sequences of forward (F) and reverse (R) primers for the amplification of pe35, ppe68, esxA, esxB and esxV genes, based on the N-and C-terminals of these genes in M. tuberculosis H37Rv [17] are shown below: o facilitate gene cloning in plasmid vectors, additional sequences were added at the 5' ends of F and R primers, which contained BamH I and Hind III restriction sites (bold face nucleotides), respectively.

Isolation of RNA from mycobacteria and RT-PCR
Total cellular RNA were isolated from 14 days old cultures of mycobacterial cells and RT-PCR was performed according to procedures described previously [23].In brief, the bacterial pellets were lysed and total nucleic acids were isolated using the MasterPure RNA purification kit (Epicenter Technologies, USA) according to the manufacturer's instructions. The contaminating DNA was digested with 5µl of RNase-free DNase I solution (Epicenter Technologies, USA) for 30 min at 37°C. The reaction was stopped by adding 1µl of 25 mM ethylenediaminertetraacetic acid (EDTA) and the enzyme was inactivated at 75°C for 10 min. The isolated RNA was used to synthesize first-strand cDNA by reverse transcription using Not I-d(T)18 primer and other components included in the first-strand cDNA synthesis kit (Amersham Biosciences, UK) according to the manufacturer's instructions. The synthesized cDNA were amplified by PCR in a thermal cycler (Perkin Elmer System 9600) by using forward and reverse primers described above for each gene, according to standard procedures [23]. The same primers were used in PCR with genomic DNA and DNA-free RNA isolated from each mycobacterial species as positive and negative controls, respectively. A common mycobacterial target of 162 bp DNA was amplified by using primers MD1/MD2 to confirm the synthesis of cDNA from RNA of the tested mycobacterial species, as described previously [23]. To determine the amplification of the targets, aliquots (20µl) of the amplified products were analyzed by agarose gel electrophoresis, as described previously [24].

Gene cloning, expression and protein purification
Full-length DNA corresponding to pe35, pe68, esxA, esxB and esxV were amplified by PCR using forward and reverse primers specific for each gene, as given above, and genomic DNA of M.tuberculosis H 37 Rv according to standard procedures [25]. The amplified DNA were cloned into pGEM-T Easy vector (Promega Corporation, Madison. WI, USA) and subcloned subsequently into pGES-TH-1 plasmid for the expression in E.coli according to procedures described previously [26]. Recombinant proteins were expressed in E.coli cells as fusion proteins with Glutathione-S-Transferase (GST) as the fusion partner, and purified to near homogeneity by affinity chromatography and treatment with thrombin protease, as described previously [27].

Raising polyclonal antibodies against recombinant proteins in rabbits
Polyclonal antibodies were raised in rabbits against the purified and GST-free PE35, PPE68, ESXA, ESXB and ESXV recombinant proteins according to standard procedures [28]. In brief, purified proteins (50 µg/ml) were emulsified with an equal volume of incomplete Freund's adjuvant (IFA, SIGMA, USA) and injected intramuscularly in the right and left thigh. The rabbits were boosted twice with the same amount of protein at two weeks intervals. The animals were bled from the ear before each immunization and two weeks after the last immunization. The sera were stored frozen in aliquots at -20°C.

Enzyme linked immunosorbent assay (ELISA)
ELISA was performed to detect antibodies in rabbit sera against full-length purified recombinant proteins and overlapping synthetic peptides corresponding to each protein using standard procedures [29]. In brief, wells of 96 well PolySorb plates (Nunc, USA) were coated with antigens/peptides (10 µg/ml), blocked with the blocking buffer, incubated with the primary antibody (rabbit sera at 1:100), followed by secondary antibody (alkaline phosphatase conjugated anti-rabbit immunoglobulin G) and addition of substrate for color development, as described previously [30]. The color intensity was measured by determining the optical density (OD) at 405 nm. Antigen uncoated wells, and antigen coated wells but without adding primary antibody were used as negative controls. The results were expressed as E/C, which is defined as: E/C = OD in antigen coated wells with primary and secondary antibodies/ OD in antigen uncoated wells with primary and secondary antibodies. The values of E/C> 2 were considered positive.

Western immunoblotting
Western immunoblotting was performed with rabbit sera and the purified recombinant proteins; sonicates, cell walls and culture filtrate of M. tuberculosis; and sonicates of M. bovis BCG, M. avium and M. vaccae using standard procedures [31]. In brief, the PE35, PPE68, ESXA, ESXB and ESXV recombinant proteins, mycobacterial sonicates, MT-CF and MT-CW were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes according to standard procedures [32,33].The membranes were blocked, incubated with rabbit sera (diluted 1:100), followed by incubation with alkaline phosphatase conjugated anti-rabbit IgG and substrate, as described previously [28].  (Figures 1c and  1d, respectively).

Characterization of the reactivity of antigen-specific antibodies raised in rabbits
The GST-free purified recombinant PE35, PPE68, ESXA, ESXB and ESXV proteins were used to raise antigen-specific antibodies in rabbits. Sera were obtained from all the rabbits before immunization and tested in Western immunoblots to confirm the absence of preexisting antibodies to the immunizing proteins (data not shown). Following immunization, sera were collected and tested for reactivity in Western immunoblots with pure proteins. The results showed that antibody reactivity was observed in the sera of all animals after the second booster, but only with the specific proteins that were used for immunization (Figure 2a, b, c, d, e), thus showing antigen-specificity of the antibodies. To determine the heterogeneity of response and the number of epitopes of recognized by rabbit antibodies, overlapping synthetic peptides corresponding to each protein were also tested with the sera using ELISA. The results showed that the antibodies, present in the sera of immunized rabbits, recognized several epitopes from different regions of each protein (Figure 3, results shown for PE35 and PPE68). These results suggest that each of the tested protein had several antibody-inducing epitopes scattered throughout the sequence.

Expression of PE35, PPE68, ESXA, ESXB, ESXV proteins in M. tuberculosis and other mycobacteria
The rabbit anti-sera containing antigen-specific polyclonal antibodies were used as probes in Western immunoblots to determine the natural expression of each protein in M. tuberculosis H 37 Rv, BCG, M. avium, and M. vaccae using sonicates of these mycobacteria. The results showed that proteins reactive with anti-PE35, -PPE68, -ESXA, -ESXB and -ESXV antibodies were present in M. tuberculosis H 37 Rv sonicate (Figure 4a, b, c, d, e); whereas, the BCG sonicate showed reactivity only with sera containing anti-EXSV antibodies (Figure 4e) and none of the sera showed antibody reactivity with sonicates of M. avium and M. vaccae ( Figure.4a, b, c, d, e). The rabbit anti-sera against

Discussion
The antigens encoded by M. tuberculosis-specific RDs may be useful in the specific diagnosis of TB and development of new vaccines [7,8]. However, it is required that antigens selected for these applications should only be expressed in M. tuberculosis and not in other mycobacteria, particularly in BCG vaccine and environmental  [23]. The expression of PPE68 and ESXA has also been shown by other workers in M™. tuberculosis but not in BCG [34,35]. However, to our knowledge, this is the first study to demonstrate the expression of all these genes at mRNA level in M. tuberculosis, only ESXV in M. bovis BCG and none of them in the environmental mycobacterial species of M. avium and M. vaccae.
The expression of pe35, ppE68, esxA, esxB and esxV genes was studied at the protein level by using polyclonal antibodies raised in rabbits against all of the five pure recombinant proteins. The specificity of the antibodies was confirmed by Western immunoblot analysis, which demonstrated that sera from pre-immunized rabbits did not have antibodies to any of these proteins, and the sera from immunized rabbits had antibodies reactive with the immunizing proteins only. These results suggest that the rabbits used were not exposed to M. tuberculosis and the epitopes of a given protein recognized by antibodies were not crossreactive with other proteins. When sera containing these antigen-specific antibodies were tested for expression of the respective proteins in M. tuberculosis and other mycobacteria (BCG, M. vaccae and M. avium), it was found that the sera containing antibodies against all of the proteins reacted with the sonicates of M. tuberculosis H 37 Rv, only sera containing anti-ESXV antibodies reacted with BCG sonicate and none of the sera reacted with M. avium and M. vaccae sonicates. By using synthetic peptides in ELISA, we found that the antibodies in rabbit sera recognized several epitopes in these proteins. The lack of reactivity of these sera with BCG (in case of PE35, PPE68, ESXA and ESXB) and M. avium and M. vaccae suggest that, in addition to lacking the respective full-length genes as demonstrated by RT-PCR, these mycobacteria do not express proteins with crossreactive epitopes.
The results of RT-CR and Western immunoblots confirm the expression of ESXV mRNA and proteins in M. tuberculosis and BCG, while PE35, PPE68, ESXA and ESXB mRNA and proteins are expressed in M. tuberculosis only. The expression of PE35, PPE68, ESXA and ESXB in M. tuberculosis but not in BCG and other mycobacteria was expected because the respective genes are present in RD1 genomic region, which is deleted in BCG and many other mycobacteria [9,36]. However, the expression of ESXV in M. tuberculosis as well as in BCG was not expected because this gene is present in RD9 segment of M. tuberculosis, which is also deleted in BCG [9,36]. To resolve this discrepancy, a protein BLAST search was performed using the sequence of ESXV by accessing Tuberculist world-wide web server (http://genolist.pasteur.fr/TubercuList/ ) to determine the presence of a homolog in M. tuberculosis which may be identical/similar to ESXV. The search showed that ESXV is 100% identical with another protein of ESX-family, i.e. ESXI [16]. The gene predicted to encode ESXI is present in the genome region of M. tuberculosis which is shared with BCG. Thus, two identical copies of the same gene occur in M. tuberculosis, one copy in RD9 as ESXV, which is deleted in BCG, and the other copy outside RD9 as ESXI, which is conserved in BCG.
Further experiments were performed to determine the sub-cellular localization of the proteins included in this study by testing MT-CF and MT-CW in Western immunoblots using rabbit sera containing antigen-specific antibodies. The results of these experiments show that antigen-specific antibodies present in rabbit sera reacted with proteins present in the both MT-CF and MT-CW preparations of M. tuberculosis. These results suggest that all of these proteins were either actively secreted or released from the growing organisms in 14 days culture period, but they were also entrapped within the cell wall structure during in vitro growth of M. tuberculosis. Consistent with our study, association of PPE68 with mycobacterial cells and cell walls has been shown by demonstrating that this protein was mainly detected in the lysate and cell wall fractions and smaller amounts were present in the membrane fraction [34,35]. Furthermore the presence of ESXA and ESXB has been reported in culture filtrates, cell walls, cytosol and cell membrane of M. tuberculosis [36][37][38].
The presence of all of these proteins in the cell walls would suggest that live mycobacteria may not be must to study the in vivo immunogenicity of RD1 and RD9 proteins included in the study. In line with this suggestion, we have recently shown that immunization of guinea-pigs with killed M. tuberculosis induced delayed type hypersensitivity responses to all these proteins [16]. On the other hand, live mycobacteria would have been essential for immunogenicity studies if these proteins were only present in culture filtrate but not in cytoplasmic or cell wall preparations.
In conclusion, our study demonstrates that the five major antigenic proteins encoded by RD1, i.e. PE35, PPE68, ESXA, ESXB, and RD9, i.e. ESXV, are expressed in M. tuberculosis at both mRNA and protein levels, whereas only ESXV is expressed in M. bovis BCG and none of these proteins are expressed in the environmental mycobacterial species of M. avium and M. vaccae. As these proteins are major antigens encoded by RD1 and RD9 of M. tuberculosis [39][40][41][42][43], they could be useful in tuberculosis control both in relation to specific diagnosis and development of new vaccines.