Mutants of Mycobacterium tuberculosis lacking three of the five rpf-like genes are defective for growth in vivo and for resuscitation in vitro

Abstract

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 DeltarpfB, DeltarpfD, and DeltarpfE strains). In contrast, two multiple strains, KDT8 (DeltarpfA-mutation DeltarpfC DeltarpfB) and KDT9 (DeltarpfA DeltarpfC DeltarpfD), 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.

FIG. 1.

Resuscitation of nonculturable M. tuberculosis strains in the MPN assay. Serially diluted samples of nonculturable cells prepared as described in Materials and Methods were employed to inoculate multiple tubes for the MPN assay. Representative tubes for successive dilutions are shown for strains H37Rv and KDT8.

FIG. 2.

Growth of M. tuberculosis H37Rv and rpf-like mutant strains in the organs of C57BL/6JCit (B6) mice. (A) Single rpf-like mutants. Diamond, wild type; triangle, ΔrpfA; X, ΔrpfB; asterisk, ΔrpfC; circle, ΔrpfD; square, ΔrpfE. (B) Triple rpf-like mutants. Open diamond, wild type; open circle, KDT8 (ΔrpfA ΔrpfC ΔrpfB); filled triangle, KDT9 (ΔrpfA ΔrpfC ΔrpfD). Mice were infected with 10⁴ bacilli of M. tuberculosis by the intravenous route, and bacillary loads in the lungs and spleen were determined by CFU assessment over a 16-week infection period as described in Materials and Methods. The top graph in each panel represents the bacillary growth in the lungs, whereas the bottom graph represents growth in the spleens of the infected animals. Each point represents the mean for four mice per group, and the error bars denote the standard deviations. The lung bacillary loads that differ significantly from those of the wild type are denoted by an asterisk above the relevant data points (P < 0.05 to 0.01). The P values of the spleen bacillary loads that differ significantly from those of the wild type are provided in the text.