Rpf proteins are the factors of reactivation of the dormant forms of actinobacteria

As the response to unfavorable growth conditions, nonsporulating mycobacteria transform into the dormant state with the concomitant formation of the specialized dormant forms characterized by low metabolic activity and resistance to antibiotics. Such dormant cells can be reactivated under the influence of several factors including proteins of Rpf (Resuscitation promoting factor) family, which possess peptidoglycan hydrolase activity and were considered to belong to the group of the autocrine growth factors of the bacteria. Remarkable interest toward Rpf family is determined by its par-ticipation in resuscitation of the dormant forms of Mycobacterium tuberculosis, what in turn is the key element in resuscitation of the latent tuberculosis – an infectious disease that affects one third of the World’s population. Experiments with Rpf mutant forms and with strains deleted in these proteins revealed a relationship between the enzymatic activity of this protein and its ability to resuscitate mycobacteria both in vitro and in vivo. This review discusses possible mechanisms of Rpf action including those related to possible participation of the products of mycobacterial Rpf-mediated cell wall hydrolysis (muropeptides) as signaling molecules. The unique ability of Rpf proteins to resuscitate the dormant forms of mycobacteria and to stimulate their proliferation would allow these proteins to occupy their niche in medicine–in diagnostics and in creation of antituberculosis subunit vaccines.     

A family of autocrine growth factors in Mycobacterium tuberculosis

Mycobacterium 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. Affinity-purified 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.     

Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.     

Culturability of Mycobacterium tuberculosis cells isolated from murine macrophages: a bacterial growth factor promotes recovery

Very little is known about the culturability and viability of mycobacteria following their phagocytosis by macrophages. We therefore studied populations of the avirulent 'Academia' strain of Mycobacterium tuberculosis isolated from murine peritoneal macrophage lysates several days post-infection in vivo. The resulting bacterial suspensions contained a range of morphological types including rods, ovoid forms and coccoid forms. Bacterial viability measured using the MPN method (dilution to extinction in liquid medium) was often much higher than that measured by CFU (plating on solid medium). Viability in the MPN assay was further enhanced when the Micrococcus luteus protein, Rpf, was incorporated into the liquid culture medium at picomolar concentrations. Rpf is an example of a family of autocrine growth factors found throughout the high G+C cohort of Gram-positive bacteria including M. tuberculosis. M. tuberculosis cells obtained from macrophages had altered surface properties, as compared with bacteria grown in vitro. This was indicated by loss of the ability to adsorb bacteriophage DS6A, a reduced tendency to form clumps, acquisition of ethidium bromide stainability following heat treatment, and loss of Rpf-mediated resuscitation following freezing and thawing. These results indicate that a proportion of 'unculturable' M. tuberculosis cells obtained from macrophages is either injured or dormant and that these cells may be recovered or resuscitated using Rpf in liquid medium.     

Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation

The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.     

The rpf gene of Micrococcus luteus encodes an essential secreted growth factor

Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non-productively at the cell surface, as a truncated form of the protein, encompassing only the 'Rpf domain' is fully active. The C-terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti-Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid-located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf-like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease.     

Cell-cell interactions during formation and reactivation of "nonculturable" mycobacteria

To date, the possible existence of "nonculturable" (NC) but potentially viable forms has been shown for some bacteria. NC mycobacteria have attracted particular interest due to the assumption that the latent form of tuberculosis is associated with the conversion of its causative agent, Mycobacterium tuberculosis, into the NC state. A number of approaches have been developed to obtain NC forms of mycobacteria, but the mechanisms of transition into or from this state have been insufficiently studied. This review considers cell-cell communications involved in the formation and reactivation of NC forms of the bacteria M. smegmatis and M. tuberculosis. Special attention has been paid to the secreted Rpf family proteins, which belong to peptidoglycan hydrolases and participate in the resuscitation of NC mycobacteria.     

Formation of nonculturable Mycobacterium tuberculosis and their regeneration

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long post-stationary phase. These cells were small (0.6-0.8 micron) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosis culture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

The Corynebacterium glutamicum gene pmt encoding a glycosyltransferase related to eukaryotic protein-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted proteins

Two-dimensional gel electrophoresis and immunoassays revealed several proteins of the secretory subproteome of Corynebacterium glutamicum to be glycosylated. By genome-wide searches for genes involved in glycosylation, the C. glutamicum gene cg1014 was found to exhibit significant similarity to eukaryotic protein-O-mannosyltransferases (PMTs) and to a recently identified orthologue of Mycobacterium tuberculosis, Rv1002c, which is responsible for protein-O-mannosylation. The putative membrane protein Cg1014 showed the same predicted transmembrane topology as Saccharomyces cerevisiae PMT1 and M. tuberculosis Rv1002c along with conserved amino acid residues responsible for catalytic activity. Deletion of the C. glutamicum pmt gene (cg1014) caused a complete loss of glycosylation of secreted proteins including the resuscitation promoting factor 2 (Rpf2), which is involved in intercellular communication and growth stimulation of C. glutamicum. Because the gene pmt as well as rpf genes are present in the genomes of all actinobacteria sequenced so far, this work provides new insights into bacterial protein glycosylation and new opportunities to elucidate the molecular mechanisms of Rpf activity in pathogenic growth and infection.     

Structural Changes and Cellular Localization of Resuscitation-Promoting Factor in Environmental Isolates of Micrococcus luteus

Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in “spent culture medium.” Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.     

Peptidoglycan fragments stimulate resuscitation of “non-culturable” mycobacteria

Resuscitation promoting factors (Rpfs), belonging to a family of secreted actinobacterial proteins with predicted peptidoglycan (PG) hydrolytic activities, participate in the reactivation of dormant cells. In the present study we demonstrate that a recombinant truncated form of Micrococcus luteus Rpf hydrolyzes isolated PG of Mycobacterium smegmatis and Mycobacterium tuberculosis liberating PG fragments of different size. These fragments possess stimulatory activity toward “non-culturable” dormant M. smegmatis and M. tuberculosis cells, similar to the activity of recombinant Rpf. Relatively large PG fragments (0.1–0.5 μm) obtained either by Rpf digestion or by PG ultrasonication revealed resuscitation activities when added in concentrations 0.1–0.2 μg/ml to the resuscitation medium. It is suggested that PG fragments could either directly activate the resuscitation pathway of dormant mycobacteria or serve as a substrate for endogenous Rpf, resulting in low molecular weight products with resuscitation activity. Whilst both suggestions are plausible, it was observed that PG-dependent resuscitation activity was suppressed by means of a specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate), which provides additional support for the second of these possibilities.     

Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling

Resuscitation-promoting factor (Rpf) is a muralytic enzyme that increases the culturability of dormant bacteria. Recently, considerable progress has been made in understanding the structure, function and physiological role of Rpfs in different organisms, most notably the major human pathogen, Mycobacterium tuberculosis , which encodes multiple rpf -like genes. A key unresolved question, however, concerns the relationship between the predicted biochemical activity of Rpfs – cleavage of the β-1,4 glycosidic bond in the glycan backbone of peptidoglycan – and their effect on culturability. In M. tuberculosis , the interaction between RpfB and the d,l -endopeptidase, Rpf interacting protein A (RipA), enables these proteins to synergistically degrade peptidoglycan to facilitate growth. Furthermore, the combined action of Rpfs with RipA and other peptidoglycan hydrolases might produce muropeptides that could exert diverse biological effects through host and/or bacterial signaling, the latter involving serine/threonine protein kinases. Here, we explore these possibilities in the context of the structure and composition of mycobacterial peptidoglycan. Clearly, a deeper understanding of the role of Rpfs and associated peptidoglycan remodeling enzymes in bacterial growth and culturability is necessary to establish the significance of dormancy and resuscitation in diseases such as tuberculosis, which are associated with long-term persistence of viable bacterial populations recalcitrant to antibiotic and immune clearance.     

Resuscitation Promoting Factors: a Family of Microbial Proteins in Survival and Resuscitation of Dormant Mycobacteria

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an extraordinarily successful pathogen of humankind. It has been estimated that up to one-third of the world’s population is infected with M. tuberculosis, and this population is an important reservoir for disease reactivation. Resuscitation promoting factor (Rpf) is a secretory protein, which was first reported in Micrococcus luteus. There are five functionally redundant Rpf-like proteins found in M. tuberculosis. Rpf promotes the resuscitation of dormant bacilli to yield normal, viable colony forming bacteria. All Rpfs share a conserved domain of about 70 amino acids and possess a lysozyme-like activity. The structural studies of the conserved domain suggest that Rpfs could be considered as a c-type lysozyme and lytic transglycosylases. Recently a novel class of nitrophenylthiocyanates (NPT) inhibitors of the muralytic activity of Rpf were reported which opens a new approach in the study of cell-wall hydrolyzing enzymes. This review describes molecular and structural studies conducted on Rpf proteins, their role in the resuscitation of dormant bacteria, in the reactivation of latent infection and identification of low molecular weight inhibitors of resuscitation promoting factors.     

Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

Background

Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs.

Methodology/Principal Findings

Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC₅₀ 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC₅₀. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations.

Conclusions/Significance

NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.     

A partner for the resuscitation-promoting factors of Mycobacterium tuberculosis

Many cases of active tuberculosis are thought to result from the reactivation of dormant Mycobacterium tuberculosis from a prior infection, yet remarkably little is known about the mechanism by which these non-sporulating bacteria reactivate. A family of extracellular bacterial proteins, known as resuscitation-promoting factors (Rpfs), has previously been shown to stimulate growth of dormant mycobacteria. While Rpf proteins are clearly peptidoglycan glycosidases, the mechanism and role of Rpf in mediating reactivation remains unclear. Here we use a yeast two-hybrid screen to identify potential binding partners of RpfB and report the interaction between RpfB and a putative mycobacterial endopeptidase, which we named Rpf-interacting protein A (RipA). This interaction was confirmed by in vitro and in vivo co-precipitation assays. The interacting domains map to the C-termini of both proteins, near predicted enzymatic domains. We show that RipA is a secreted, cell-associated protein, found in the same cellular compartment as RpfB. Both RipA and RpfB localize to the septa of actively growing bacteria by fluorescence microscopy. Finally, we demonstrate that RipA is capable of digesting cell wall material and is indeed a peptidoglycan hydrolase. The interaction between these two peptidoglycan hydrolases at the septum suggests a role for the complex in cell division, possibly during reactivation.     

Evaluation of Rpf protein of Micrococcus luteus for cultivation of soil actinobacteria

Rpf protein, a kind of resuscitation promoting factor, was first found in the culture supernatant of Micrococcus luteus. It can resuscitate the growth of M. luteus in “viable but non-culture, VBNC” state and promote the growth of Gram-positive bacteria with high G + C content. This paper investigates the resuscitating activity of M. luteus ACCC 41016^T Rpf protein, which was heterologously expressed in E. coli, to cells of M. luteus ACCC 41016^T and Rhodococcus marinonascens HBUM200062 in VBNC state, and examines the effect on the cultivation of actinobacteria in soil. The results showed that the recombinant Rpf protein had resuscitation effect on M. luteus ACCC 41016^T and R. marinonascens HBUM200062 in VBNC state. 83 strains of actinobacteria, which were distributed in 9 families and 12 genera, were isolated from the experimental group with recombinant Rpf protein in the culture medium. A total of 41 strains of bacteria, which were distributed in 8 families and 9 genera, were isolated from the control group without Rpf protein. The experimental group showed richer species diversity than the control group. Two rare actinobacteria, namely HBUM206391^T and HBUM206404^T, were obtained in the experimental group supplemented with Rpf protein. Both may be potential new species of Actinomadura and Actinokineospora, indicating that the recombinant expression of M. luteus ACCC 41016^T Rpf protein can effectively promote the isolation and culture of actinobacteria in soil.     

The glycosylated cell surface protein Rpf2, containing a resuscitation-promoting factor motif,is involved in intercellular communication of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The genome of Corynebacterium glutamicum ATCC 13032 contains two genes, rpf1 and rpf2, encoding proteins with similarities to the essential resuscitation-promoting factor (Rpf) of Micrococcus luteus. Both the Rpf1 (20.4 kDa) and Rpf2 (40.3 kDa) proteins share the so-called Rpf motif, a highly conserved protein domain of approximately 70 amino acids, which is also present in Rpf-like proteins of other gram-positive bacteria with a high G+C content of the chromosomal DNA. Purification of the C. glutamicum Rpf2 protein from concentrated supernatants, SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified modified Rpf2 variants with increased or reduced mobility when compared with the calculated size of Rpf2. A Western blot-based enzyme immunoassay demonstrated glycosylation of the Rpf2 variants with higher molecular masses. Galactose and mannose were identified as two components of the oligosaccharide portion of the Rpf2 glycoprotein by capillary gas chromatography coupled to mass spectrometry. The Rpf2 protein was localized on the surface of C. glutamicum with the use of immuno-fluorescence microscopy. C. glutamicum strains with defined deletions in the rpf1 or rpf2 gene or simultaneous deletions in both rpf genes were constructed, indicating that the rpf genes are neither individually nor collectively essential for C. glutamicum. The C. glutamicum rpf double mutant displayed slower growth and a prolonged lag phase after transfer of long-stored cells into fresh medium. The addition of supernatant from exponentially growing cultures of the rpf double mutant, the wild type or C. glutamicum strains with increased expression of the rpf1 or rpf2 gene significantly reduced the lag phase of long-stored wild-type and rpf single mutant strains, but addition of purified His-tagged Rpf1 or Rpf2 did not. In contrast, the lag phase of the C. glutamicum rpf double mutant was not affected upon addition of these culture supernatants.     

Cell-cell interactions during the formation and reactivation of “nonculturable” mycobacteria

To date, the possible existence of “nonculturable” (NC) but potentially viable forms has been shown for some bacteria. NC mycobacteria have attracted particular interest due to the assumption that the latent form of tuberculosis is associated with the conversion of its causative agent, Mycobacterium tuberculosis, into the NC state. A number of approaches have been developed to obtain NC forms of mycobacteria, but the mechanisms of transition into or from this state have been insufficiently studied. This review considers cell-cell communications involved in the formation and reactivation of NC forms of the bacteria M. smegmatis and M. tuberculosis. Special attention has been paid to the secreted Rpf family proteins, which belong to peptidoglycan hydrolases and participate in the resuscitation of NC mycobacteria.     

微生物VBNC状态形成及复苏机制

99%以上的微生物因处于活的但非可培养(viable but non-culturable,VBNC)状态而无法分离培养。复苏促进因子(resuscitation-promoting factors,Rpfs)是培养获取VBNC菌的最重要突破。结合课题组近十余年从环境功能视角利用Rpf复苏培养VBNC菌的研究,本文在阐述微生物VBNC状态的形成及复苏进展的基础上,从VBNC菌形成及复苏过程出发,探究"探索因子"与群体感应的内在关系。并总结了课题组利用Rpf所复苏培养的具有潜在环境功能的VBNC菌种。本论文将为揭示微生物VBNC状态的形成及复苏机制提供新的思路,并为认识和重新评价Rpf法复苏培养VBNC菌在污染环境微生物修复中的作用提供理论依据。     

Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis

The RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo, each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP-1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP-10 and ESAT-6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild-type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.