Viable cells of Micrococcus luteus secrete a factor, which promotes the resuscitation and growth of dormant, nongrowing cells of the same organism. The resuscitationpromoting factor (Rpf) is a protein, which has been purified to homogeneity. In picomolar concentrations, it increases the viable cell count of dormant M. luteus cultures at least 100-fold and can also stimulate the growth of viable cells. Rpf also stimulates the growth of several other high G؉C Gram-positive organisms, including Mycobacterium avium, Mycobacterium bovis (BCG), Mycobacterium kansasii, Mycobacterium smegmatis, and Mycobacterium tuberculosis. Similar genes are widely distributed among high G؉C Gram-positive bacteria; genome sequencing has uncovered examples in Mycobacterium leprae and Mb. tuberculosis and others have been detected by hybridization in Mb. smegmatis, Corynebacterium glutamicum, and Streptomyces spp. The mycobacterial gene products may provide different targets for the detection and control of these important pathogens. This report is thus a description of a proteinaceous autocrine or paracrine bacterial growth factor or cytokine.The essential role of cytokines in controlling the activation, growth, and proliferation of eukaryotic cells is now widely recognized (1). These proteinaceous cell-signaling molecules include many growth factors that are widely distributed among vertebrates, and probably also invertebrates (2). Similar growth factors have also recently been found in unicellular organisms such as ciliates (3-6). In prokaryotic organisms, intercellular signaling usually involves small metabolites (e.g., N-acyl homoserine lactones) or peptides (7-16). Although specific interactions have been documented between vertebrate cytokines and prokaryotes (17-20), there are no known examples of autocrine or paracrine growth factors produced by prokaryotic microorganisms.The number of observable microbial cells in a natural sample often exceeds the number that can be cultured therefrom by orders of magnitude (21-23). It is not known in general whether such nonculturable (and often noncultured) cells are dead, are killed by our media, or are in a dormant state from which we could, in principle, resuscitate them with appropriate growth factors (24). After growth to stationary phase and starvation in spent growth medium, cells of the nonsporulating, Gram-positive bacterium Micrococcus luteus can enter a dormant state in which they can persist for at least 7 months. Whereas exponentially growing cultures have a viability of Ϸ100%, as estimated by comparing colony forming units (cfu) on agar plates with the total cell count determined microscopically, such dormant cultures can exhibit a viability of less than 10 Ϫ4 (25). The viable count of this type of culture as measured by the Most Probable Number (MPN) method also corresponds to the number of cfu. However, in the presence of sterile (filtered) culture supernatant from the late logarithmic phase of batch growth, resuscitation occurs and the viable count by MPN increases ...

SummaryMycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus . Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross-species activity against M. luteus , Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinitypurified antibodies inhibit bacterial growth in vitro , suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo . The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.

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...