AMycobacterium tuberculosisRpf Double-Knockout Strain Exhibits Profound Defects in Reactivation from Chronic Tuberculosis and Innate Immunity Phenotypes

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Abstract
Resuscitation-promoting factors (Rpfs), apparent peptidoglycan hydrolases, have been implicated in the reactivation of dormant bacteria. We previously demonstrated that deletion ofrpfBimpaired reactivation ofMycobacterium tuberculosisin a mouse model. BecauseM. tuberculosisencodes five Rpf paralogues, redundant functions among the family members might obscurerpfsingle-knockout phenotypes. A series ofrpfdouble knockouts were therefore generated. One double mutant, ΔrpfAB, exhibited several striking phenotypes. Consistent with the proposed cell wall-modifying function of Rpfs, ΔrpfABexhibited an altered colony morphology. Although ΔrpfABgrew 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 ΔrpfABwas more severe than that of ΔrpfBin aminoguanidine-treated mice. A similar deficiency was observed in ΔrpfABreactivation from a drug-induced apparently sterile state in infected NOS2−/−mice upon cessation of antimycobacterial therapy. Secondly, ΔrpfABshowed a persistence defect not seen with the ΔrpfBor ΔrpfAsingle mutants. Interestingly, ΔrpfABexhibited impaired growth in primary mouse macrophages and induced higher levels of the proinflammatory cytokines tumor necrosis factor alpha and interleukin 6. Simultaneous reintroduction ofrpfAandrpfBinto the double-knockout strain complemented the colony morphology and macrophage cytokine secretion phenotypes. Phenotypes related to cell wall composition and macrophage responses suggest thatM. tuberculosisRpfs may influence the outcome of reactivation, in part, by modulating innate immune responses to the bacterium.

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