New chemotherapeutics are urgently required to control the tuberculosis pandemic. We describe a new pathway from trehalose to α-glucan in Mycobacterium tuberculosis comprising four enzymatic steps mediated by TreS, Pep2, GlgE (which has been identified as a maltosyltransferase that uses maltose 1-phosphate) and GlgB. Using traditional and chemical reverse genetics, we show that GlgE inactivation causes rapid death of M. tuberculosis in vitro and in mice through a self-poisoning accumulation of maltose 1-phosphate. Poisoning elicits pleiotropic phosphosugar-induced stress responses promoted by a self-amplifying feedback loop where trehalose-forming enzymes are upregulated. Moreover, the pathway from trehalose to α-glucan exhibited a synthetic lethal interaction with the glucosyltransferase Rv3032, which is involved in biosynthesis of polymethylated α-glucans, because key enzymes in each pathway could not be simultaneously inactivated. The unique combination of maltose 1-phosphate toxicity and gene essentiality within a synthetic lethal pathway validates GlgE as a distinct potential drug target that exploits new synergistic mechanisms to induce death in M. tuberculosis.

Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (

This unit includes protocols for the genetic manipulation of Mycobacterium tuberculosis, including nucleic acid extraction (plasmid DNA, genomic DNA, and mRNA), and methods for electroporation (transformation), transduction (including allelic exchange), and transposon mutagenesis. Considerations for working with M. tuberculosis at Biosafety Level 3 containment are also discussed. Curr. Protoc. Microbiol. 6:10A.2.1‐10A.2.21. © 2007 by John Wiley & Sons, Inc.

Tuberculosis (TB) remains a global health burden for which safe vaccines are needed. BCG has limitations as a TB vaccine so we have focused on live attenuated Mycobacterium tuberculosis mutants as vaccine candidates. Prior to human studies, however, it is necessary to demonstrate safety in non-human primates (NHP). In this study, we evaluate the safety and efficacy of two live attenuated M. tuberculosis double deletion vaccine strains mc26020 (ΔlysA ΔpanCD) and mc26030 (ΔRD1 ΔpanCD) in cynomolgus macaques. In murine models, mc26020 is rapidly cleared while mc26030 persists. Both mc26020 and mc26030 were safe and well tolerated in cynomolgus macaques. Following a high-dose intrabronchial challenge with virulent M. tuberculosis, mc26020-vaccinates were afforded a level of protection intermediate between that elicited by BCG vaccination and no vaccination. BCG vaccinates had reduced tuberculosis-associated pathology and improved clinical scores as compared to saline and mc26030 vaccinates, but survival did not differ among the groups.