The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro

Abstract

Mycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

Fig. 1

Stepwise deletion of rpfA-E genes in M. tuberculosis H37Rv. The arrows represent in-frame deletions introduced by allelic exchange mutagenesis to produce the strains whose names are underlined. For the sake of simplicity, the mutant strains are referred to throughout the text according to their abbreviated genotypes, which are given beneath the strain names and are named according to the order in which the rpf-like genes were deleted.

Fig. 2. Delayed colony formation and SDS hypersensitivity of Rpf-deficient mutants of M. tuberculosis.

A. Log-phase cultures of selected strains were serially diluted and plated onto Middlebrook 7H11 agar. Plates were incubated for 18 days before scoring for growth. Deletions and/or complementation of specific rpf-like genes are shown by single or double-headed arrows. B. Serial dilutions of log-phase cultures of selected strains were spotted on 7H11 agar with or without SDS at a concentration of 0.01%. Plates were incubated for 10 days before scoring for growth. The relatively poor growth of the ΔACBE and ΔACBED strains on 7H11 agar is due to their delayed colony formation on this medium (A).

Fig. 3. Growth and survival of multiple rpf-like mutants in human monocytes and in B6D2/F₁ mice.

A. Human monocytes were infected with H37Rv (♦), ΔACBD (▪) or ΔACBE (▴) and growth was monitored over 4 days. Experiments were conducted with cells isolated from two healthy donors. B. Growth and survival of quadruple mutants in mouse lungs. B6D2/F₁ mice were infected with H37Rv (♦), ΔACBD (▪) or ΔACBE (▴) and the bacillary loads in the lungs of the infected animals were determined by cfu assessment over a period of 240 days. Each point represents the mean of three mice per group and the error bars denote the standard deviations. The lung bacillary loads that differ significantly from those of the wild-type control are denoted by an asterisk above the relevant data point (P < 0.0001 for all points except ΔACBD at 240 days, where P = 0.0024). C. Effect of complementation of ΔACBE with rpfC, rpfD and rpfE genes on growth in mouse lungs. B6D2/F₁ mice were infected with H37Rv (♦), ΔACBE (▴) or ΔACBE + CDE (□) and lung bacillary loads were assessed over 77 days. The individual bacillary loads or the mean of two or three infected mice per group are shown.