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 ⌬rpfB, ⌬rpfD, and ⌬rpfE strains). In contrast, two multiple strains, KDT8 (⌬rpfA-mutation ⌬rpfC ⌬rpfB) and KDT9 (⌬rpfA ⌬rpfC ⌬rpfD), 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.The majority of individuals infected with Mycobacterium tuberculosis harbor a clinically latent infection in which the organism is able to persist or remain dormant within an otherwise healthy individual for prolonged periods of time (15). In a relatively small proportion of these individuals, the infection may reactivate to cause active disease. Since it is estimated that one-third of the world's population is latently infected with M. tuberculosis, the implications of reactivation tuberculosis for the global burden of disease are alarming (5). The molecular mechanisms by which M. tuberculosis persists or remains dormant and reactivates are poorly understood (12, 15) and may be attributed to the host immune response and/or the physiology of the organism itself.M. tuberculosis encodes a family of five proteins that bear significant similarity to the resuscitation promoting factor (Rpf) of Micrococcus luteus, which is a secreted protein essential for growth of this organism (9, 10). Studies on the growth stimulation and reactivation of cultures of M. luteus, Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis by M. luteus Rpf and other Rpf-like proteins strongly suggest that one or more of the M. tuberculosis Rpf-like proteins may play similar roles in mycobacterial growth and/or reactivation of latent infection (8, 10, 13). However, it was...

Resuscitation-promoting factors are important determinants of the pathophysiology inMycobacterium tuberculosisinfection

“…This contrasts with an intraperitoneal model of infection where DrpfAC was more attenuated for growth than DrpfAB although differences in host, strain or experimental conditions may account for this (Biketov et al, 2007). With triple rpf deletion mutants, the loss of rpfB from a DrpfAC mutant was more attenuating for growth in the mouse lung than rpfD loss (Downing et al, 2005).…”

New insights into the function of granulomas in human tuberculosis

“…Tufariello et al showed that rpf-deficient M. tuberculosis strains remain viable and that rpf genes are expressed during latent stages of M. tuberculosis in mouse lungs [57]. Knockout of three of the five rpf genes in M. tuberculosis apparently impaired mycobacterial growth and inhibited resuscitation [58]. Although more recent publications revealed the structure and properties of rpf gene products [59][60][61], the question remains as to whether this mechanism plays a decisive role in transformation of latent infection into active disease in human tuberculous lung lesions.…”

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).…”