Evaluation of a novel Mycobacterium tuberculosis protein in the immunodiagnosis of TB

Source: Singh KK, Sharma N, Vargas D, Liu Z, Belisle JT, Potharaju V,

 Wanchu A, Behera D, Laal S. Peptides of a novel Mycobacterium tuberculosis

-specific cell wall protein for immunodiagnosis of tuberculosis. J Infect Dis.

2009 Aug15;200(4):571-81

 

Mycobacterium tuberculosis is an opportunistic pathogen that causes nearly 2-3 million deaths each year. One of the common methods of diagnosis is by microscopic identification of acid fast bacilli in sputum samples. However, the procedure is tedious, and requires multiple samples. Also, the success rate of correct diagnosis has been reported to be 30-60%. Thus, rapid, point-of-care diagnostic tests are required. Currently, there is no sero-diagnostic test that provides accurate diagnosis of M. tuberculosis infection. Many studies are going on to discover and identify new proteins of this pathogen, which can then be used to develop sero-diagnostic tests.

The authors of this paper previously identified a proline-threonine repetitive protein (PTRP; Rv0538) in aerosol infected rabbits. Earlier studies have shown that it is present in the cell wall. The authors of this paper also observed in their previous study that this protein does not have a homolog in M. avium and M. leprae.

Thus, the previous findings of immunogenicity of PRTP in rabbits, cell wall localization of this protein and its absence in the M. avium and M. leprae, made the authors to further characterize this protein and study its diagnostic potential.

The results obtained are discussed below:

1. ptrp is specific for M. tuberculosis (complex): The authors performed a protein BLAST analysis of PTRP. The results showed nearly 100% identity with a protein in M. tuberculosis CDC1551, M. tuberculosis F11, M. tuberculosis C, M. tuberculosis H37Ra, M. bovis AF2122/97 and M. bovis BCG strain. No such identity was observed in pathogenic non-tuberculous mycobacterial species. Nucleotide BLAST analysis of ptrp gene identified nearly 100% identical genes in Mycobacterium tuberculosis complex species and clinical isolates but not in the non-tuberculous mycobacterial species. The specificity of ptrp to the M. tuberculosis complex was further confirmed by Southern hybridization.
   
2. 
   Expression and purification of rPTRP: The PTRP protein was expressed and purified and its molecular weight was nearly 52kDa. Its identity was confirmed by sequencing.
   
3. PTRP is a cell wall protein of M. tuberculosis: To confirm the localization of this protein, sub-cellular protein fractions of M. tuberculosis were separated on a Western Blot and probed with pre-immune IgG, anti-PTRP IgG and anti-MS (malate synthase) IgG antibodies. Anti-PTRP antibodies identified a nearly 52KDa protein in total cell wall (TCW), SDS extracted cell wall (SDS-CW) of M. tuberculosis. Some weak reactivity was also observed in whole cell lysate (WCL), but no similar protein was observed in culture filtrates. It has been shown through earlier studies that malate synthase (MS) of M. tuberculosis is anchored to the cell wall. Thus, to estimate the relative abundance of PTRP in different cell preparations, the authors used anti-malate synthase (MS) IgG. With quantity of MS in each fraction considered to be 100%, WCLs were found to contain less PTRP than the TCW and SDS-CW. Finally, the presence of PTRP on cell wall was confirmed by immuno-electron microscopy.
   
4. Immunogenicity of PTRP in patients with TB: To determine the immunogenicity of PTRP in patients with TB, the reactivity of rPTRP was examined with serum specimens from patients with TB and healthy controls. The investigators observed that serum antibodies from 4 of the 6 HIV-negative TB positive and 5 of 6 HIV positive and TB positive patients displayed strong reactivity with rPTRP. None of the 6 HIV negative PPD positive, 6 HIV negative PPD negative or 6 HIV positive, TB negative patients showed any reactivity with rPDRP.
   
5. Identification of immunogenic epitopes of PTRP: The authors next checked the diagnostic potential of PTRP. Thus, they synthesized fifty-four overlapping peptides (20 aa in length with a 10-aa overlap; PT1–PT54) covering the entire PTRP sequence, each linked with a biotin residue at the N-terminal. Out of these 54, 53 peptides were tested for their reactivity with serum specimens from 13 PPD-negative and 23 PPD-positive healthy control subjects. No significant difference was observed in the reactivity of 49 peptides. No control serum tested positive with 22 of 53 peptides. The remaining peptides were recognized by the antibodies in only one or two serum specimens. When the 53 peptides were tested for their reactivity with serum specimens from HIV negative, TB positive patients, 16 of 53 peptides were recognized by antibodies from more than 40% of the patients with TB. Of these 16 peptides, 12 were reproducibly recognized by antibodies in specimens from more than 40% of patients with TB. Out of these 12 peptides, 4 were recognized by antibodies from specimens from more than 50% of patients with TB. These are PT9, PT13, PT40 and PT41.
   
6. Reactivity of immunodominant PTRP peptides with serum specimens from other classes of patients with TB: The reactivity of 4 immunodominant peptides was checked with serum specimens from different groups of patients and controls. 25-45% of smear negative, HIV negative, TB positive patients' serum specimens recognized these 4 immunodominant peptides. These peptides were also recognized by serum specimens from 60-76% of the HIV positive TB positive patients.
   

Concluding remarks:

In authors words: "These results demonstrate that immunodominant epitopes of carefully selected M. tuberculosis specific proteins can be used to devise a simple peptide based serodiagnostic test for TB."