Abstract
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.
Highlights
Tuberculosis (TB) is a major global health problem with 1/3rd 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 natural expression of pe35, ppe68, esxA, esxB and esxV in M. tuberculosis, BCG, M. avium, and M. vaccae was studied at mRNA level by reverse transcription-polymerase chain reaction (RT-PCR)
The results showed that mRNA corresponding to pe35, ppe68, esxA and esxB were expressed in M. tuberculosis (Figure 1a), only esxV was expressed in BCG (Figure 1b) and mRNA for none of these genes were expressed in M. avium and M. vaccae (Figures 1c and 1d, respectively)
Summary
Tuberculosis (TB) is a major global health problem with 1/3rd of the world population infected with Mycobacterium tuberculosis, about 9.3 million people developing active disease leading to 1.8 million deaths annually [1]. 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]
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