Molecular and immunological characterization of Mycobacterium tuberculosis CFP-10, an immunodiagnostic antigen missing in Mycobacterium bovis BCG

Abstract

In order to identify antigens that may be used in the serodiagnosis of active tuberculosis (TB), we screened a Mycobacterium tuberculosis genomic expression library with a pool of sera from patients diagnosed with active pulmonary TB. The sera used lacked reactivity with a recombinant form of the M. tuberculosis 38-kDa antigen (r38kDa), and the goal was to identify antigens that might complement r38kDa in a serodiagnostic assay. Utilizing this strategy, we identified a gene, previously designated lhp, which encodes a 100-amino-acid protein referred to as culture filtrate protein 10 (CFP-10). The lhp gene is located directly upstream of esat-6, within a region missing in M. bovis BCG. Immunoblot analysis demonstrated that CFP-10 is present in M. tuberculosis CFP, indicating that it is likely a secreted or shed antigen. Purified recombinant CFP-10 (rCFP-10) was shown to be capable of detecting specific antibody in a percentage of TB patients that lack reactivity with r38kDa, most notably in smear-negative cases, where sensitivity was increased from 21% for r38kDa alone to 40% with the inclusion of rCFP-10. In smear-positive patient sera, sensitivity was increased from 49% for r38kDa alone to 58% with the inclusion of rCFP-10. In addition, rCFP-10 was shown to be a potent T-cell antigen, eliciting proliferative responses and gamma interferon production from peripheral blood mononuclear cells in 70% of purified protein derivative-positive individuals without evident disease. The responses to this antigen argue for the inclusion of rCFP-10 in a polyvalent serodiagnostic test for detection of active TB infection. rCFP-10 could also contribute to the development of a recombinant T-cell diagnostic test capable of detecting exposure to M. tuberculosis.

FIG. 1

Amino acid sequences of native CFP-10, rCFP-10, and rΔCFP-10. Residues that are identical in all three proteins are represented by dots.

FIG. 2

Southern blot analysis of the lhp gene. Genomic DNAs (2.5 μg) from mycobacterial strains were digested with PstI, separated by agarose gel electrophoresis, and blotted onto Nytran. The lhp gene was labeled with [³²P]dCTP by random oligonucleotide primers and used as a probe. Molecular size markers (M) in kilobases are shown.

FIG. 3

Characterization of native CFP-10. M. tuberculosis H37Rv lysate (2.5 μg; lane 1), 2.5 μg of CFP (lane 2), 2.5 μg of the cytoplasmic fraction (lane 3), 50 ng of rCFP-10 (lane 4), no sample (lane 5), and 2.5 μg of the membrane fraction (lane 6) were subjected to SDS-PAGE, transferred to nitrocellulose, and reacted with rabbit antiserum generated against rΔCFP-10. Molecular masses are shown in kilodaltons.

FIG. 4

Purification of rCFP-10. Expression and purification of rCFP-10 are shown with uninduced (lane 2) and induced (lane 3) E. coli lysates and 5 μg of purified protein (lane 4). Molecular masses are shown in kilodaltons (lane 1).