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Infect. Immun. doi:10.1128/IAI.01735-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A double Rpf knockout Mycobacterium tuberculosis strain exhibits profound defects in reactivation from chronic tuberculosis and innate immunity phenotypes

Departments of Medicine, and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461

^* To whom correspondence should be addressed. Email: tufariel{at}aecom.yu.edu.

	Abstract

Resuscitation-promoting factors (Rpfs), apparent peptidoglycan (PG) hydrolases, have been implicated in reactivation of dormant bacteria. We previously demonstrated that deletion of rpfB impaired reactivation of Mycobacterium tuberculosis (Mtb) in a mouse model. Because Mtb encodes five Rpf paralogues, redundant function among the family members might obscure single rpf knock-out phenotypes. A series of double rpf 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 apparent sterile state in infected NOS2-/- mice upon cessation of anti-mycobacterial 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 TNF- and IL-6. Simultaneous reintroduction of rpfA and rpfB into the double knock-out strain complemented the colony morphology and macrophage cytokine secretion phenotypes. Phenotypes related to cell wall composition and macrophage responses suggest that Mtb Rpfs may influence the outcome of reactivation, in part, by modulating innate immune responses to the bacterium.