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

Abstract

SummaryMycobacterium 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.

Rapid and spontaneous loss of phthiocerol dimycocerosate (PDIM) from Mycobacterium tuberculosis grown in vitro: implications for virulence studies

“…The availability of sequence information has also contributed to the situation where the vast majority of recombinant M. tuberculosis clones, including those used for in vivo pathogenesis and vaccination experiments, have now been generated in the H37Rv background. Unfortunately, our data presented herein add to a mounting body of recent evidence (Andreu & Gibert, 2008;Kana et al, 2008;Manjunatha et al, 2006) indicating that a substantial proportion of H37Rv stock cultures held around the world contain a high percentage of cells lacking PDIM -a cell-wall-associated wax that is essential for full virulence in vivo.…”

“…Mayer Goren and colleagues first noted that H37Rv had a propensity for losing the ability to synthesize PDIM during in vitro culture back in 1974 (Goren et al, 1974). That this phenomenon occurs relatively frequently is evident from three recent publications that document the loss of PDIM from H37Rv (Andreu & Gibert, 2008;Kana et al, 2008;Manjunatha et al, 2006).…”

Novel regulatory roles of cAMP receptor proteins in fast-growing environmental mycobacteria

“…A CRP-binding site is also present in the promoter regions of both whiB4 and whiB6 genes in M. smegmatis, suggesting that Crp2 could directly regulate these whiB genes. M. tuberculosis possesses five rpf genes, rpfA-E (Downing et al, 2004(Downing et al, , 2005Kana et al, 2008). In M. tuberculosis, a CRP-binding site was identified in the promoter region of rpfA, and rpfA is directly regulated by CRP (Rickman et al, 2005).…”

“…S5(d). Microarray analysis showed that Crp2 regulates genes with diverse biological functions, including genes encoding Sdh1, the WhiB4 and WhiB6 of WhiB family proteins, which have been implicated in many cellular processes (Larsson et al, 2012), and RpfE, one of five resuscitation-promoting factor proteins (Rpfs) that are required for the resuscitation of dormant cells (Kana et al, 2008) (Table S3). The M. tuberculosis genome encodes seven WhiB proteins (WhiB1-7) and their homologues are present in M. smegmatis.…”

Mycobacterium tuberculosis RpfE crystal structure reveals a positively charged catalytic cleft

“…13,14 The combined deletions of at least three rpf genes produce cell-growth defects in vitro, uncovering apparent functional specialization of Rpfs in Mtb. 13 Consistent with this idea, the Mtb rpfB deletion mutant fails to resuscitate in mice, 3 while rpfE is individually essential for switching mycobacterial cultures from slow to fast growth in a chemostat. 15 These results provide functional data suggesting that RpfB and RpfE are the most important Rpf family members.…”