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Biotechnology and Applied Biochemistry (2001) 33, (71–74) (Printed in Great Britain)

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Anthrax-toxin-mediated delivery of a 19 kDa antigen of Mycobacterium tuberculosis into the cytosol of mammalian cells

Varsha Mehra*†, Hemant Khanna*, Ramesh Chandra† and Yogendra Singh*¹

*Centre for Biochemical Technology, Mall Road, Delhi-110007, India, and †Ambedkar Centre for Biomedical Research, University of Delhi, Delhi-110007, India

Abbreviations used: PA, protective antigen; PA63, 63 kDa proteolytically activated fragment of PA; lethal factor; LFn, N-terminal 254 amino acids of LF; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMEM, Dulbecco's modified Eagle's medium; CTL, cytotoxic T-lymphocyte.

¹ To whom correspondence should be addressed (e-mail ysingh@cbt.res.in).

PA63, the proteolytically activated 63 kDa fragment of protective antigen (PA, 83 kDa), mediates translocation of lethal factor (LF) and oedema factor into the cytosol. The N-terminal 254 amino acids of LF (LFn) are required for binding to PA63 and mediating translocation of active ligands fused to either the N- or C-terminus. Here we report translocation of a 19 kDa antigen of Mycobacterium tuberculosis into the cytosol of mammalian cells when fused to the C-terminus of LFn (LFn-19kDa). The fusion protein was non-toxic to J774A.1 macrophage cells in combination with PA and retained the ability to bind to PA63 when incubated with Chinese hamster ovary K1 cells. The data show the efficacy of anthrax toxin to mediate translocation of M. tuberculosis antigens into the cytosol of mammalian cells and may prove useful in delivering proteins and peptides carrying immunodominant mycobacterial antigens into the cytosol.

Received 29 September 2000/4 December 2000; accepted 7 December 2000

Portland Press Ltd © 2001