Delivery of antituberculosis drugs by nanoparticles offers potential advantages over free drug, including the potential to target specifically the tissues and cells that are infected by Mycobacterium tuberculosis , thereby simultaneously increasing therapeutic efficacy and decreasing systemic toxicity, and the capacity for prolonged release of drug, thereby allowing less-frequent dosing. We have employed mesoporous silica nanoparticle (MSNP) drug delivery systems either equipped with a polyethyleneimine (PEI) coating to release rifampin or equipped with cyclodextrin-based pH-operated valves that open only at acidic pH to release isoniazid (INH) into M. tuberculosis -infected macrophages. The MSNP are internalized efficiently by human macrophages, traffic to acidified endosomes, and release high concentrations of antituberculosis drugs intracellularly. PEI-coated MSNP show much greater loading of rifampin than uncoated MSNP and much greater efficacy against M. tuberculosis- infected macrophages. MSNP were devoid of cytotoxicity at the particle doses employed for drug delivery. Similarly, we have demonstrated that the isoniazid delivered by MSNP equipped with pH-operated nanovalves kill M. tuberculosis within macrophages significantly more effectively than an equivalent amount of free drug. These data demonstrate that MSNP provide a versatile platform that can be functionalized to optimize the loading and intracellular release of specific drugs for the treatment of tuberculosis.

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate vaccine,MycobacteriumbovisBCG, is urgently needed. We describe a recombinant BCG vaccine (rBCG30) expressing and secreting the 30-kDa major secretory protein ofMycobacterium tuberculosis, the primary causative agent of TB, that affords greater survival after challenge than parental BCG in the highly demanding guinea pig model of pulmonary TB. Animals immunized with rBCG30 and then challenged by aerosol with a highly virulent strain ofM. tuberculosissurvived significantly longer than animals immunized with conventional BCG. The parental and recombinant vaccine strains are comparably avirulent in guinea pigs, as they display a similar pattern of growth and clearance in the lung, spleen, and regional lymph nodes. The pMTB30 plasmid encoding the 30-kDa protein is neither self-transmissible nor mobilizable to other bacteria, including mycobacteria. The pMTB30 plasmid can be stably maintained inEscherichia colibut is expressed only in mycobacteria. The recombinant and parental strains are sensitive to the same antimycobacterial antibiotics. rBCG30, the first vaccine against TB more potent than nearly century-old BCG, is being readied for human clinical trials.

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 ∼10% 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.

Summary: Glutamine 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‐glutamylcysteine synthase (GshA), UDP‐N‐acetylmuramoylalanine‐d‐glutamate ligase (MurD) and glutamate racemase (MurI). All eight genes are located in multigene operons except for glnA1, 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 glnA1–glnE–glnA2 cluster, was expressed separately in Mycobacterium smegmatis, and its gene product was characterized and assayed for enzymatic activity by analysing the reaction products. In M. smegmatis, all four recombinant‐overexpressed GS are multimeric enzymes exhibiting GS activity. Whereas GlnA1, GlnA3 and GlnA4 catalyse the synthesis of l‐glutamine, GlnA2 catalyses the synthesis of d‐glutamine and d‐isoglutamine. The generation of mutants in M. tuberculosis of the four glnA genes, murD and murI demonstrated that all of these genes except glnA1 are nonessential for in vitro growth. l‐methionine‐S,R‐sulphoximine (MSO), previously demonstrated to inhibit M. tuberculosis growth in vitro and in vivo, strongly inhibited all four GS enzymes; hence, the design of MSO analogues with an improved therapeutic to toxic ratio remains a promising strategy for the development of novel anti‐M. tuberculosis drugs.