Culture filtrate
proteins (CFP) of Mycobacterium tuberculosis have been shown to contain immunogenic components that elicit at least partial protective immunity against
Mycobacterium infection. To clone genes encoding some of the immunogenic
proteins, we made a high-titer rabbit anti-CFP serum and used it to screen an M.
tuberculosis genomic expression library in Escherichia coli. In this paper, we describe the molecular cloning of two new
protein components of CFP and identified them as members of the
serine protease gene family. Their open reading frames contain N-terminal hydrophobic secretory signals consistent with their detection in CFP. The predicted molecular masses of the mature, fully processed forms of both
antigens are approximately 32 kDa, in agreement with their observed sizes on immunoblots of CFP probed with polyclonal rabbit
antisera made to the
recombinant proteins. Thus, these
proteins have been designated MTB32A and MTB32B. Interestingly, and despite 66% amino acid sequence homology between the two
proteins, polyclonal 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 mononuclear cells (PBMC) from healthy purified
protein derivative (
PPD)-positive individuals of diverse ethnic backgrounds. MTB32A but not MTB32B stimulated PBMC from healthy
PPD-positive donors but not from
PPD-negative donors to proliferate and secrete
gamma interferon. MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M.
tuberculosis complex and the BCG strain of Mycobacterium bovis but absent in the environmental mycobacterial species tested. In addition, nucleotide sequence comparison of mtb32a of the avirulent H37Ra strain and the virulent Erdman strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical isolate CSU93, revealed 100% identity. MTB32A, therefore, represents a candidate for inclusion in
subunit vaccine development. Finally, the possible role of MTB32
serine proteases as a
virulence factor(s) during Mycobacterium spp.
infection is discussed.