Cloning, expression and significance of MPT53 for identification of secreted proteins of Mycobacterium tuberculosis

Based on our N-terminal amino acid sequence of MPT53 and a deduced DNA sequ ence, we searched for the corresponding gene in the Mycobacterium tuberculo sis genomic sequence at the Sanger centre, localizing mpt53 close to mpt70 and mpt83. The gene was cloned and expressed, followed by purification of M PT53 to homogeneity from recombinant M. smegmatis culture fluid. In MPT53 t here is 60 % identity with the active site of thioredoxin of M. tuberculosi s (MPT46) with two cysteins in a CXXC motif, but MPT53 could not serve as a n alternative substrate far thioredoxin reductase. Testing for IgM and IgG1 anti-MPT53 in cattle sera showed that MPT53 is immunogenic following natur al and experimental infection with M. bovis. Cloning of mpt53 represents cl oning of the last of the 10 proteins originally defined as 'secreted protei ns' of M. tuberculosis and M. bovis based on determination of their 'Locali zation index' (LI) (J Gen Microbiol 1991; 137: 875-84). The need for a prec ise definition of the term 'secreted protein' is discussed. So far we have observed full concordance between occurrence of an LI value indicating secr etion of a protein and occurrence of a signal sequence in the corresponding gene. Signal sequence independent protein secretion in mycobacteria may oc cur for a limited number of proteins and remains to be established. (C) 199 9 Academic Press.