Cloning, expression, and immunological evaluation of two putative secretedserine protease antigens of Mycobacterium tuberculosis

Culture filtrate proteins (CFP) of Mycobacterium tuberculosis have been sho wn to contain immunogenic components that elicit at least partial protectiv e immunity against Mycobacterium infection. To clone genes encoding some of the immunogenic proteins, we made a high-titer rabbit anti-CFP serum and u sed it to screen an BL tuberculosis genomic expression library in Escherich ia coil, In this paper, we describe the molecular cloning of two new protei n components of CFP and identified them as members of the serine protease g ene family. Their open reading frames contain N-terminal hydrophobic secret ory signals consistent with their detection in CFP. The predicted molecular masses of the mature, fully processed forms of both antigens are similar t o 32 kDa, in agreement with their observed sizes on immunoblots of CFP prob ed with polyclonal rabbit antisera made to the recombinant proteins, Thus, these proteins have been designated MTB32A and MTB32B. Interestingly, and d espite 66% amino acid sequence homology between the two proteins, polyclona l rabbit antisera made to each of the recombinant proteins were found to be specific for the respective immunizing antigens. The recombinant proteins were also evaluated in in vitro assays with donor peripheral blood mononucl ear cells (PBMC) from healthy purified protein derivative (PPD)-positive in dividuals of diverse ethnic backgrounds. MTB32A but not MTB32B stimulated P BMC from healthy PPD-positive donors but not from PPD-negative donors to pr oliferate and secrete gamma interferon, MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tube rculosis complex and the BCG strain of Mycobacterium bovis but absent in th e environmental mycobacterial species tested. In addition, nucleotide seque nce comparison of mtb32a of the avirulent H37Ra strain and the virulent Erd man strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical isolate CSU93, revealed 100% id entity. MTB32A, therefore, represents a candidate for inclusion in subunit vaccine development. Finally, the possible role of MTB32 serine proteases a s a virulence factor(s) during Mycobacterium spp. infection is discussed.