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Infection and Immunity, September 2008, p. 4269-4281, Vol. 76, No. 9
0019-9567/08/$08.00+0     doi:10.1128/IAI.01735-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A Mycobacterium tuberculosis Rpf Double-Knockout Strain Exhibits Profound Defects in Reactivation from Chronic Tuberculosis and Innate Immunity Phenotypes

Departments of Medicine,¹ Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461²

Received 26 December 2007/ Returned for modification 14 February 2008/ Accepted 16 June 2008

Resuscitation-promoting factors (Rpfs), apparent peptidoglycan hydrolases, have been implicated in the reactivation of dormant bacteria. We previously demonstrated that deletion of rpfB impaired reactivation of Mycobacterium tuberculosis in a mouse model. Because M. tuberculosis encodes five Rpf paralogues, redundant functions among the family members might obscure rpf single-knockout phenotypes. A series of rpf double knockouts were therefore generated. One double mutant, rpfAB, exhibited several striking phenotypes. Consistent with the proposed cell wall-modifying function of Rpfs, rpfAB exhibited an altered colony morphology. Although rpfAB grew comparably to the parental strain in axenic culture, in vivo it exhibited deficiency in reactivation induced in C57BL/6 mice by the administration of nitric oxide synthase inhibitor (aminoguanidine) or by CD4⁺ T-cell depletion. Notably, the reactivation deficiency of rpfAB was more severe than that of rpfB in aminoguanidine-treated mice. A similar deficiency was observed in rpfAB reactivation from a drug-induced apparently sterile state in infected NOS2^–/– mice upon cessation of antimycobacterial therapy. Secondly, rpfAB showed a persistence defect not seen with the rpfB or rpfA single mutants. Interestingly, rpfAB exhibited impaired growth in primary mouse macrophages and induced higher levels of the proinflammatory cytokines tumor necrosis factor alpha and interleukin 6. Simultaneous reintroduction of rpfA and rpfB into the double-knockout strain complemented the colony morphology and macrophage cytokine secretion phenotypes. Phenotypes related to cell wall composition and macrophage responses suggest that M. tuberculosis Rpfs may influence the outcome of reactivation, in part, by modulating innate immune responses to the bacterium.

Published ahead of print on 30 June 2008.

Editor: F. C. Fang