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Infection and Immunity, May 2005, p. 3038-3043, Vol. 73, No. 5
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.5.3038-3043.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Mutants of Mycobacterium tuberculosis Lacking Three of the Five rpf-Like Genes Are Defective for Growth In Vivo and for Resuscitation In Vitro

Katrina J. Downing,^1, Vladimir V. Mischenko,² Margarita O. Shleeva,³ Danielle I. Young,⁴ Michael Young,⁴ Arseny S. Kaprelyants,³ Alexander S. Apt,² and Valerie Mizrahi^1*

MRC/NHLS/WITS Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, School of Pathology of the University of the Witwatersrand and the National Health Laboratory Service, Johannesburg, South Africa,¹ Central Institute for Tuberculosis, Russian Academy of Medical Sciences,² Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia,³ Institute of Biological Sciences, University of Wales, Aberystwyth, Wales, United Kingdom⁴

Received 1 October 2004/ Returned for modification 18 November 2004/ Accepted 15 December 2004

Mycobacterium tuberculosis contains five genes, rpfA through rpfE, that bear significant homology to the resuscitation-promoting factor (rpf) gene of Micrococcus luteus, whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf-like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf-like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the three strains tested (the rpfB, rpfD, and rpfE strains). In contrast, two multiple strains, KDT8 (rpfA-mutation rpfC rpfB) and KDT9 (rpfA rpfC rpfD), which lack three of the five rpf-like genes, were significantly yet differentially attenuated in a mouse infection model. These mutants were also unable to resuscitate spontaneously in vitro, demonstrating the importance of the Rpf-like proteins of M. tuberculosis in resuscitation from the nonculturable state. These results strongly suggest that the biological functions of the five rpf-like genes of M. tuberculosis are not wholly redundant and underscore the potential utility of these proteins as targets for therapeutic intervention.

Editor: J. N. Weiser

Present address: Department of Molecular and Cell Biology and Institute of Infectious Disease and Molecular Medicine, P/Bag Rondebosch, University of Cape Town, Cape Town, South Africa.

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