Proteins secreted by Mycobacterium tuberculosis are usually targets of immune responses in the infected host. Here we describe a search for secreted proteins that combined the use of bioinformatics and phoA' fusion technology. The 3,924 proteins deduced from the M. tuberculosis genome were analyzed with several computer programs. We identified 52 proteins carrying an NH(2)-terminal secretory signal peptide but lacking additional membrane-anchoring moieties. Of these 52 proteins-the TM1 subgroup-only 7 had been previously reported to be secreted proteins. Our predictions were confirmed in 9 of 10 TM1 genes that were fused to Escherichia coli phoA', a marker of subcellular localization. These findings demonstrate that the systematic computer search described in this work identified secreted proteins of M. tuberculosis with high efficiency and 90% accuracy.
MPT53 is a secreted protein of Mycobacterium tuberculosis. Southern transfer and hybridization showed mpt53 to be conserved in the M. tuberculosis complex and to have homology with DNA from Mycobacterium avium and other nontuberculous mycobacteria. However, anti-MPT53 polyclonal antibodies detected no antigen in the culture filtrates of M. avium and other nontuberculous mycobacteria. MPT53 of M. tuberculosis induced strong, tuberculosis-specific antibody responses in guinea pigs but induced no delayed-type hypersensitivity. Involvement in immune responses during human tuberculosis was very modest.Proteins secreted into the extracellular environment by Mycobacterium tuberculosis are usually targets of immune responses in the infected host. Thus, the filtrate of M. tuberculosis cultures has constituted an important source of antigens that induce protective immunity and immune responses having diagnostic value (reviewed in references 1, 6, and 20). In the 1980s and early 1990s S. Nagai and his collaborators purified several proteins from culture filtrates of M. tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG). These proteins were termed MPT (for M. tuberculosis) or MPB (for M. bovis BCG) followed by a number indicating the relative mobility during nondenaturing polyacrylamide gel electrophoresis (13). Of the MPT and MPB proteins known to be secreted (18), many elicit immune responses that are specific for the M. tuberculosis complex and therefore of diagnostic value (for example, MPT64 [2], MPB70 [7], and MPT63 [12]). Others confer protective immunity (MPT44, MPT59, and MPT45, i.e., the members of the Ag85 complex [8,17]). While all other genes encoding MPT and MPB proteins were identified and sequenced in the pregenome era, the mpt53 gene was identified only recently (19) from the analysis of the NH 2 -terminal sequence of the purified protein (13) and from the genome sequence of M. tuberculosis (mpt53 is Rv2878c) (5). MPT53 is a 15-kDa protein (13) that induces antibody responses in tuberculous cattle (19). In the present work, we characterized the specificity of MPT53 for the M. tuberculosis complex and the involvement of MPT53 in immune responses to tuberculosis (TB) in guinea pigs and in humans.The distribution of mpt53 among tuberculous and nontuberculous mycobacteria was determined by Southern transfer and hybridization. The gene was present in the DNA of members of the M. tuberculosis complex (Fig. 1, lanes 1, 4, and 8, and data not shown). Analysis of 50 clinical isolates of M. tuberculosis identified one isolate bearing a chromosomal deletion encompassing mpt53 (data not shown), indicating a relative gene instability. We next investigated the distribution of mpt53 among nontuberculous mycobacteria. Analysis of the partial M. avium genome sequence (http://www.tigr.org/tdb/) by BLAST homology programs indicated that M. avium contains a homolog of mpt53 (Ϸ80% identity with nucleotide and amino acid sequences corresponding to the extracellular MPT53 protein of M. tuberculosis) (data not sho...
Proteins secreted by Mycobacterium tuberculosis are targets of host immune responses and as such are investigated for vaccine and immunodiagnostics development. Computer-driven searches of the M. tuberculosis H 37 Rv genome had previously identified 45 novel secreted proteins. Here, we report the characterization of these antigens in terms of specificity for the M. tuberculosis complex and the ability to induce human immune responses. BLAST homology searches and Southern hybridization identified 10 genes that were either specific for the M. tuberculosis complex or found in only two nontuberculous mycobacterial species of minor medical significance. Selected recombinant proteins were purified from Escherichia coli cells and tested for the ability to elicit antibody responses in tuberculosis patients. Reactivity of the serum panel was ' 36% with at least one of five novel proteins (Rv0203, Rv0603, Rv1271c, Rv1804c and Rv2253), 56% with the 38 kDa lipoprotein, a M. tuberculosis antigen known to be highly seroreactive, and 68% with a combination of Rv0203, Rv1271c and the 38 kDa antigen. Thus, at least five novel secreted proteins induce antibody responses during active disease; some of these proteins may increase the sensitivity of serological assays based on the 38 kDa antigen.
In [1], Abstract, line 10, the molecular sizes of some of the fusion proteins were published incorrectly. The correct version of line 10 is ' . . . antibodies at the expected molecular mass of 70, 61, 68, 71, 66 and 72 kDa . . . '.In addition, an incorrect version of Figure 2 was published on page 386. Lanes C and F (panel A) should have been labelled lanes F and C, respectively.The correct version of Figure 2 is shown below.Reference 1 Ahmad S, El-Shazly S, Mustafa AS, Al-Attiyah R. Mammalian cell-entry proteins encoded by the mce3 operon of Mycobacterium tuberculosis are expressed during natural infection in humans. Scand
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