IDENTIFICATION AND CHARACTERIZATION OF A 29-KILODALTON PROTEIN FROM MYCOBACTERIUM-TUBERCULOSIS CULTURE FILTRATE RECOGNIZED BY MOUSE MEMORY EFFECTOR-CELLS

Culture filtrate proteins from Mycobacterium tuberculosis induce prote ctive immunity in various animal models of tuberculosis. Two molecular mass regions (6 to 10 kDa and 24 to 36 kDa) of short-term culture fil trate are preferentially recognized by Th1 cells in animal models as w ell as by patients with minimal disease. In the present study, the 24- to 36-kDa region has been studied, and the T-cell reactivity has been mapped in detail. Monoclonal antibodies were generated, and one monoc lonal antibody, HYB 71-2, with reactivity against a 29-kDa antigen loc ated in the highly reactive region below the antigen 85 complex was se lected. The 29-kDa antigen (CFP29) was purified from M. tuberculosis s hort-term culture filtrate by thiophilic adsorption chromatography, an ion-exchange chromatography, and gel filtration, In its native form, C FP29 forms a polymer with a high molecular mass. CFP29 was mapped in t wo-dimensional electrophoresis gels as three distinct spots just below the antigen 85 complex component MPT59, CFP29 is present in both cult ure filtrate and the membrane fraction from M. tuberculosis, suggestin g that this antigen is released from the envelope to culture filtrate during growth, Determination of the N-terminal amino acid sequence all owed cloning and sequencing of the cfp29 gene. The nucleotide sequence showed 62% identity to the bacteriocin Linocin from Brevibacterium li nens, Purified recombinant histidine-tagged CFP29 and native CFP29 had similar T-cell stimulatory properties, and they both elicited the rel ease of high levels of gamma interferon from mouse memory effector cel ls isolated during the recall of protective immunity to tuberculosis. Interspecies analysis by immunoblotting and PCR demonstrated that CFP2 9 is widely distributed in mycobacterial species.