Mycobacterium tuberculosis must import iron from its host for survival, and its siderophore-dependent iron acquisition pathways are well established. Here we demonstrate a newly characterized pathway, whereby M. tuberculosis can use free heme and heme from hemoglobin as an iron source. Significantly, we identified the genomic region, Rv0202c – Rv0207c , responsible for the passage of heme iron across the mycobacterial membrane. Key players of this heme uptake system were characterized including a secreted protein and two transmembrane proteins, all three specific to mycobacteria. Furthermore, the crystal structure of the key heme carrier protein Rv0203 was found to have a unique fold. The discovery of a unique mycobacterial heme acquisition pathway opens new avenues of exploration into mycobacterial therapeutics.

To assess the role of glutamine synthetase (GS), an enzyme of central importance in nitrogen metabolism, in the pathogenicity of Mycobacterium tuberculosis, we constructed a glnA1 mutant via allelic exchange. The mutant had no detectable GS protein or GS activity and was auxotrophic for L-glutamine. In addition, the mutant was attenuated for intracellular growth in human THP-1 macrophages and avirulent in the highly susceptible guinea pig model of pulmonary tuberculosis. Based on growth rates of the mutant in the presence of various concentrations of L-glutamine, the effective concentration of L-glutamine in the M. tuberculosis phagosome of THP-1 cells was ϳ10% of the level assayed in the cytoplasm of these cells (4.5 mM), indicating that the M. tuberculosis phagosome is impermeable to even very small molecules in the macrophage cytoplasm. When complemented by the M. tuberculosis glnA1 gene, the mutant exhibited a wild-type phenotype in broth culture and in human macrophages, and it was virulent in guinea pigs. When complemented by the Salmonella enterica serovar Typhimurium glnA gene, the mutant had only 1% of the GS activity of the M. tuberculosis wild-type strain because of poor expression of the S. enterica serovar Typhimurium GS in the heterologous M. tuberculosis host. Nevertheless, the strain complemented with S. enterica serovar Typhimurium GS grew as well as the wild-type strain in broth culture and in human macrophages. This strain was virulent in guinea pigs, although somewhat less so than the wild-type. These studies demonstrate that glnA1 is essential for M. tuberculosis virulence.Glutamine and glutamate are central molecules in nitrogen metabolism. Glutamine is used as the nitrogen donor for many nitrogen-containing molecules in the cell and is synthesized from L-glutamate, ammonia, and ATP by the enzyme glutamine synthetase (GS) (33). The internal L-glutamine pool has been shown to be a sensor of external nitrogen limitation for Salmonella enterica serovar Typhimurium (21). GS is the only known biosynthetic pathway for the synthesis of glutamine and along with glutamate synthetase is responsible for ammonia assimilation under nitrogen-limiting growth conditions. In enteric bacteria, glutamate dehydrogenase can assimilate ammonia directly into glutamate at high concentrations of ammonia. However, for bacteria such as Mycobacterium tuberculosis which lack glutamate dehydrogenase, GS and glutamate synthetase are the sole means of ammonia assimilation. Due to its central role in nitrogen metabolism, GS is subject to varied and complex forms of transcriptional and posttranslational regulation as well as feedback inhibition by several products of glutamine metabolism (7,33).There are at least four major forms of GS (25). In enteric bacteria, a single glnA gene encodes a GS type I (GSI) enzyme, and glnA null mutants are glutamine auxotrophs. Other bacteria have been shown to possess two or three different types of GS. In the case of Sinorhizobium meliloti (formerly Rhizobium meliloti), all three GS genes...

Background: Mycobacterium tuberculosis has a probable nanocompartment (Mt-Enc). Results: Mt-Enc self-assembles into a 60-subunit cage that encapsulates enzymes via their C-terminal tails, which remain active within Mt-Enc. Conclusion: Cargo proteins are potentially involved in host oxidative stress response, suggesting that enzyme encapulation may be a mechanism to evade host immune assault. Significance: Mt-Enc may be utilized as a novel therapeutic delivery mechanism.

SummaryGlutamine synthetases (GS) are ubiquitous enzymes that play a central role in every cell's nitrogen metabolism. We have investigated the expression and activity of all four genomic Mycobacterium tuberculosis GS -GlnA1, GlnA2, GlnA3 and GlnA4 -and four enzymes regulating GS activity and/or nitrogen and glutamate metabolism -adenylyl transferase (GlnE), γ γ γ γ -glutamylcysteine synthase (GshA), UDP-N -acetylmuramoylalanine-D -glutamate ligase (MurD) and glutamate racemase (MurI). All eight genes are located in multigene operons except for gln A1, and all are transcribed in M. tuberculosis ; however, some are not translated or translated at such low levels that the enzymes escape detection. Of the four GS, only GlnA1 can be detected. Each of the eight genes, as well as the gln A1-gln E-gln A2 cluster, was expressed separately in Mycobacterium smegmatis , and its gene product was characterized and assayed for enzymatic activity by analysing the reaction products. In

Glutamine synthetase (GS) and superoxide dismutase (SOD), large multimeric enzymes that are thought to play important roles in the pathogenicity of Mycobacterium tuberculosis, are among the bacterium's major culture filtrate proteins in actively growing cultures. Although these proteins lack a leader peptide, their presence in the extracellular medium during early stages of growth suggested that they might be actively secreted. To understand their mechanism of export, we cloned the homologous genes (glnA1 and sodA) from the rapid-growing, nonpathogenic Mycobacterium smegmatis, generated glnA1 and sodA mutants of M. smegmatis by allelic exchange, and quantitated expression and export of both mycobacterial and nonmycobacterial GSs and SODs in these mutants. We also quantitated expression and export of homologous and heterologous SODs from M. tuberculosis. When each of the genes was expressed from a multicopy plasmid, M. smegmatis exported comparable proportions of both the M. tuberculosis and M. smegmatis GSs (in the glnA1 strain) or SODs (in the sodA strain), in contrast to previous observations in wild-type strains. Surprisingly, recombinant M. smegmatis and M. tuberculosis strains even exported nonmycobacterial SODs. To determine the extent to which export of these large, leaderless proteins is expression dependent, we constructed a recombinant M. tuberculosis strain expressing green fluorescent protein (GFP) at high levels and a recombinant M. smegmatis strain coexpressing the M. smegmatis GS, M. smegmatis SOD, and M. tuberculosis BfrB (bacterioferritin) at high levels. The recombinant M. tuberculosis strain exported GFP even in early stages of growth and at proportions very similar to those of the endogenous M. tuberculosis GS and SOD. Similarly, the recombinant M. smegmatis strain exported bacterioferritin, a large (ϳ500-kDa), leaderless, multimeric protein, in proportions comparable to GS and SOD. In contrast, high-level expression of the large, leaderless, multimeric protein malate dehydrogenase did not lead to extracellular accumulation because the protein was highly unstable extracellularly. These findings indicate that, contrary to expectations, export of M. tuberculosis GS and SOD in actively growing cultures is not due to a protein-specific export mechanism, but rather to bacterial leakage or autolysis, and that the extracellular abundance of these enzymes is simply due to their high level of expression and extracellular stability. The same determinants likely explain the presence of other leaderless proteins in the extracellular medium of actively growing M. tuberculosis cultures.Mycobacterium tuberculosis, the primary etiologic agent of tuberculosis, is one of the world's leading causes of death, killing 2 million persons annually worldwide (23). New modalities to combat M. tuberculosis and a greater understanding of the biology and immunology of this pathogen are high priorities of tuberculosis research.The extracellular proteins of M. tuberculosis have been the focus of many studies investigating ...