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...

The disaccharide trehalose is the major free sugar in the cytoplasm of mycobacteria; it is a constituent of cell wall glycolipids, and it plays a role in mycolic acid transport during cell wall biogenesis. The pleiotropic role of trehalose in the biology of Mycobacterium tuberculosis and its absence from mammalian cells suggests that its biosynthesis may provide a useful target for novel drugs. However, there are three potential pathways for trehalose biosynthesis in M. tuberculosis, and the aim of the present study was to introduce mutations into each of the pathways to determine whether or not they are functionally redundant. The results show that the OtsAB pathway, which generates trehalose from glucose and glucose-6-phosphate, is the dominant pathway required for M. tuberculosis growth in laboratory culture and for virulence in a mouse model. Of the two otsB homologues annotated in the genome sequence of M. tuberculosis, only OtsB2 (Rv3372) has a functional role in the pathway. OtsB2, trehalose-6-phosphate phosphatase, is strictly essential for growth and provides a tractable target for high throughput screening. Inactivation of the TreYZ pathway, which can generate trehalose from ␣-1,4-linked glucose polymers, had no effect on the growth of M. tuberculosis in vitro or in mice. Deletion of the treS gene altered the late stages of pathogenesis of M. tuberculosis in mice, significantly increasing the time to death in a chronic infection model. Because the TreS enzyme catalyzes the interconversion of trehalose and maltose, the mouse phenotype could reflect either a requirement for synthesis of additional trehalose or, conversely, a requirement for breakdown of stored trehalose to liberate free glucose.

SUMMARYOne approach to study the role of distinct cellular mechanisms in susceptibility/resistance to tuberculosis (TB) is to compare parameters of response to infection in the lungs of mouse strains exhibiting genetically determined differences in TB susceptibility/severity. Interstrain differences in antimycobacterial macrophage reactions, T cell responses & inflammation in the lungs of TB-susceptible I/St, TBresistant A/Sn and (I/St ¥ A/Sn)F1 mice were analysed following intratracheal inoculation of 10 3 CFUs of M. tuberculosis H37Rv. The antimycobacterial responses in the lungs of susceptible I/St mice were characterized by: (i) increased inflammatory infiltration by all major immune cell subsets; (ii) decreased type 1 cytokine production; (iii) impaired antimycobacterial activity of lung macrophages; (iv) unusually high proliferation of lung T lymphocytes. Differences in several parameters of anti-TB immunity between susceptible and resistant mice corresponded well to the polygenic pattern of TB control previously established in this mouse model. Importantly, lung macrophages isolated from noninfected mice were unable to respond to IFN-g by increasing their mycobactericidal function, but between weeks 3 and 5 of the infection this capacity developed in all mice. However, by this time point susceptible but not resistant mice demonstrated a pronounced decrease in IFN-g production by lung cells. This chain of events may explain the inability of I/St mice to control both early and chronic TB infection.

Mice of the I/St and A/Sn inbred strains display a severe and moderate course, respectively, of disease caused by Mycobacterium tuberculosis. Earlier, we showed that the response to mycobacterial antigens in I/St mice compared to that in A/Sn mice is shifted toward Th2-like reactivity and a higher proliferative activity and turnover of T cells. However, the physiologic basis for different expressions of tuberculosis severity in these mice remains largely unknown. Here, we extend our previous observations with evidence that I/St interstitial lung macrophages are defective in the ability to inhibit mycobacterial growth and to survive following in vitro infection with M. tuberculosis H37Rv. A unique feature of this phenotype is its exclusive expression in freshly isolated lung macrophages. The defect is not displayed in ex vivo macrophages obtained from the peritoneal cavity nor in macrophages developed in vitro from progenitors extracted from various organs, including the lung itself. In addition, we show that, in sharp contrast to peritoneal macrophages, the mycobactericidal capacity of lung macrophages is not elevated in the presence of exogenous gamma interferon. Our data suggest that the in vivo differentiation in a particular anatomical microenvironment determines the pattern of macrophage-mycobacterium interaction. Thus, caution should be exercised when conclusions based upon the results obtained in a particular in vitro system are generalized to the functions of all phagocytes during M. tuberculosis infection.The identification of genes and their alleles that confer resistance versus susceptibility to tuberculosis (TB) provides deep insight into basic mechanisms of immunity and pathology. Of utmost importance is establishing genotyping-based approaches for identifying the 10% of individuals who are at high risk of progression to clinical TB after primary infection (8). Although there is substantial evidence for the role of genetic factors in human susceptibility to TB (13,42,57), the identification of human TB susceptibility genes has been complicated by polygenic control of the trait (7,30,45,53) and inability to clearly delineate clinical phenotypes for stratified genetic analysis (5, 55). Unlike rare mutant alleles of the genes IFN␥R and IL-12R that determine extremely high susceptibility to mycobacterial infections in humans (2, 16, 31, 43)-resembling that in mice bearing disrupted genes for several key cytokines (14, 15, 25, 26)-genes with modest effects rarely produce clear phenotypes. Due to a limited power to detect such genes and to choose "major" genes among numerous candidates, little is known concerning the human genetics of TB control. Variations in NRAMP1 (1, 6) and/or NRAMP1-linked loci on the human chromosome 2q35 segment (28), as well as the class II HLA genes (10,27,40), have been shown to be linked to or associated with susceptibility to mycobacterial infections in humans. However, the overall effect of NRAMP1 and HLA genes on TB infection is acknowledged to be weak, and other genes are c...

The generation of effector, IFN-γ producing T lymphocytes and their accumulation at sites of infection are critical for host protection against various infectious diseases. The activation and differentiation of naive T lymphocytes into effector memory cells starts in lymphoid tissues, but it is not clear whether the Ag-experienced cells that leave lymph nodes (LN) are mature or if they undergo further changes in the periphery. We have previously shown that CD44highCD62Llow effector CD4 T lymphocytes generated during the course of mycobacterial infection can be segregated into two subsets on the basis of CD27 receptor expression. Only the CD27low subset exhibited a high capacity for IFN-γ secretion, indicating that low CD27 expression is characteristic of fully differentiated effector CD4 T lymphocytes. We demonstrate now that CD27low IFN-γ-producing CD4 T lymphocytes accumulate in the lungs but are rare in LNs. Several factors contribute to their preferential accumulation. First, CD27low CD4 T lymphocytes present in the LN are highly susceptible to apoptosis. Second, circulating CD27low CD4 T cells do not enter the LN but efficiently migrate to the lungs. Third, CD27high effector CD4 T cells that enter the lungs down-regulate CD27 expression in situ. In genetically heterogeneous mice that exhibit varying susceptibility to tuberculosis, the accumulation of mature CD27low CD4 T cells in the lungs correlates with the degree of protection against infection. Thus, we propose that terminal maturation of effector CD4 T lymphocytes in the periphery provides the host with efficient local defense and avoids potentially harmful actions of inflammatory cytokines in lymphoid organs.

Given that the mechanism of Bcn bactericidal activity differs from that of all commonly used antibiotics, their possible involvement in complex TB therapies deserves further study.

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Mycobacterial spread to lymphoid organs preceded spread to the initially uninfected contralateral lung. Genetic differences in susceptibility to tuberculosis are associated with differences in dynamics of the immune response, rather than differences in mycobacterial trafficking.