The role of neutrophils in tuberculosis (TB) resistance and pathology is poorly understood. Neutrophil reactions are meant to target the offending pathogen but may lead to destruction of the host lung tissue, making the defending cells an enemy. Here, we show that mice of the I/St strain which are genetically susceptible to TB show an unusually high and prolonged neutrophil accumulation in their lungs after intratracheal infection. Compared to neutrophils from more resistant A/Sn mice, I/St neutrophils display an increased mobility and tissue influx, prolonged lifespan, low expression of the CD95 (Fas) apoptotic receptor, relative resistance to apoptosis, and an increased phagocytic capacity for mycobacteria. Segregation genetic analysis in (I/St × A/Sn)F₂hybrids indicates that the alleles of I/St origin at the chromosome 3 and 17 quantitative trait loci which are involved in the control of TB severity also determine a high level of neutrophil influx. These features, along with the poor ability of neutrophils to restrict mycobacterial growth compared to that of lung macrophages, indicate that the prevalence of neutrophils in TB inflammation contributes to the development of pathology, rather than protection of the host, and that neutrophils may play the role of a “Trojan horse” for mycobacteria.

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 Δ rpfB , ΔrpfD , and Δ rpfE strains). In contrast, two multiple strains, KDT8 (Δ rpfA -mutation ΔrpfC ΔrpfB ) and KDT9 (Δ rpfA ΔrpfC ΔrpfD ), 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 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 α-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.

Bacteriocins (Bcn) are natural peptides that are secreted by several taxonomically distant bacteria and exert bactericidal activity against other bacterial species. Their capacity to inhibit growth of virulent Mycobacterium tuberculosis H37Rv was evaluated in this study. Five different Bcn were isolated and purified from bacterial culture supernatants, their amino acid sequence was determined, and activity against mycobacteria assessed in three different models: in vitro mycobacterial cultures, in vitro infection of mouse macrophages and in vivo high-dose infection of inbred mice. In the in vitro model, four out of five Bcn exhibited stronger antimycobacterial activity than equal concentrations of a widely used anti-TB antibiotic, rifampicin. These Bcn were non-toxic for mouse macrophages at a concentration of 0.1 mg/L (>MIC₉₀ of these compounds). Pure Bcn did not inhibit mycobacterial growth within murine macrophages when added at 0.01–0.1 mg/L, suggesting that at physiologically tolerable concentrations these molecules do not penetrate through the membrane of eukaryotic cells. However, when administered as a complex with phosphatidylcholine–cardiolipin liposomes, Bcn5 (selected as a model compound due to its cytotoxicity and antimycobacterial activity regular titration curves) demonstrated capacity both to inhibit intracellular growth of M. tuberculosis and to prolong survival of mice in an acute TB model. 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.