Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is the world's leading cause of death in humans from a single infectious agent. A safe and effective vaccine against this scourge is urgently needed. This study demonstrates that immunization with the 30-kDa major secretory protein, alone or in combination with other abundant extracellular proteins of M. tuberculosis, induces strong cell-mediated immune responses and substantial protective immunity against aerosol challenge with virulent M. tuberculosis bacilli in the highly susceptible guinea pig model of pulmonary tuberculosis. Protection is manifested by decreased clinical illness including decreased weight loss, reduced mortality, and decreased growth of M. tuberculosis in the lungs and spleens of immunized animals compared with sham-immunized controls. This study demonstrates that purified major extracellular proteins of M. tuberculosis are can-

Tuberculosis (TB) continues to ravage humanity, causing 2 million deaths per year. A vaccine against TB more potent than the current live vaccine, bacillus Calmette-Gué rin (BCG), is desperately needed. Using two commercially available strains of BCG as host strains, BCG Connaught and Tice, we have constructed two recombinant BCG vaccines stably expressing and secreting the 30-kDa major secretory protein of Mycobacterium tuberculosis (M. tb.), the primary causative agent of TB. We have tested the efficacy of the two strains in the highly susceptible guinea pig model of pulmonary TB, a model noteworthy for its close resemblance to human TB. Animals immunized with the recombinant BCG vaccines and challenged by aerosol with a highly virulent strain of M. tb. had 0.5 logs fewer M. tb. bacilli in their lungs and 1 log fewer bacilli in their spleens on average than animals immunized with their parental conventional BCG vaccine counterparts. Statistically, these differences were highly significant. Paralleling these results, at necropsy, animals immunized with the recombinant BCG vaccines had fewer and smaller lesions in the lung, spleen, and liver and significantly less lung pathology than animals immunized with the parental BCG vaccines. The recombinant vaccines are the first vaccines against TB more potent than the current commercially available BCG vaccines, which were developed nearly a century ago.

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...

We have investigated the activity and extra- (9), Legionella pneumophila Philadelphia 1 (10), Bacillus cereus (ATCC 14579), and Bacillus subtilis (ATCC 6051) were grown as described. For comparative glutamine synthetase assays, all bacteria were grown in 7H9 medium at pH 6.7 and 7.5 or in Sauton's medium (11).Purification of Glutamine Synthetase. Supernatant from 18 liters of M. tuberculosis Erdman strain cultures was filtered through Tuifryn 0.45-and 0.22-,um filters (Gelman) and concentrated by tangential flow through a polyethersulfone membrane (Filtron Technology, Northborough, MA). Proteins in this concentrate were precipitated with ammonium sulfate at 100% saturation, pelleted by centrifugation, and dialyzed against sorbitol buffer (10%6 sorbitol/10 mM potassium phosphate, pH 7.0/5 mM 2-mercaptoethanol/0.2 mM EDTA). The proteins were applied to DEAE-Sepharose CL-6B (Pharmacia) and glutamine synthetase was eluted at 0.5-1 M NaCl. The enzyme was further chromatographed on thiopropyl-Sepharose 6B (Pharmacia), eluted at 150-250 mM 2-mercaptoethanol, concentrated to 2.5 ml in a Diaflo unit (Amicon), and finally size fractionated on Sepharose 6B (Pharmacia). Enzymatically active fractions were pooled and stored at 40C. Protein concentrations were determined by the bicinchoninic acid reagent (Pierce). Proteins in the active fractions were analyzed by SDS/10% PAGE and stained with Coomassie brilliant blue R or silver nitrate. The N-terminal sequence of glutamine synthetase was determined on poly-(vinylidene difluoride) membranes at the University of California, Los Angeles, protein microsequencing facility with a Porton 2090 E amino acid sequencer.Assays of Glutamine Synthetase Activity. The enzyme was assayed both in the biosynthetic (forward) assay (glutamate + ATP + ammonia --glutamine + ADP + Pj) and in the transfer assay (glutamine + hydroxylamine y-glutamylhydroxamate + ammonia) as described (12). One unit of glutamine synthetase was defined as the amount of enzyme producing 1 pmol of Pi per min in the biosynthetic reaction or 1 pnmol of -glutamylhydroxamate per min in the transfer reaction.The pH optima of glutamine synthetase were determined for both assay systems for the range pH 6.0-9.0. The enzyme's cation requirements were also examined for both reactions. Cobalt(II) chloride, magnesium chloride, manganese chloride, or zinc(II) chloride was added at 50 mM for the biosynthetic reaction and at 3 mM for the transfer reaction.*To whom reprint requests should be addressed. 9342The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate vaccine, Mycobacterium bovis BCG, is urgently needed. We describe a recombinant BCG vaccine (rBCG30) expressing and secreting the 30-kDa major secretory protein of Mycobacterium 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 of M. tuberculosis survived 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 in Escherichia coli but 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.Tuberculosis continues as a major global health problem, especially in the developing world where 1 in 6 adults between the ages of 15 and 59 dies from this disease (10, 22). The AIDS epidemic, whose victims are severalfold more susceptible to tuberculosis than the general population, and the worldwide emergence of drug-resistant strains of Mycobacterium tuberculosis, the primary causative agent of tuberculosis, compound the problem (8,14). A better vaccine against tuberculosis is urgently needed (11, 23). The current vaccine, Mycobacterium bovis BCG, a live attenuated vaccine derived from the bovine tuberculosis bacillus in the early 1900s by Calmette and Guérin, while protective against disseminated forms of tuberculosis such as meningitis and miliary tuberculosis, is of inconsistent efficacy against pulmonary tuberculosis, the dominant form (9, 12).In a previous study (20), two recombinant BCG vaccines (rBCG30) overexpressing the major secretory protein of M. tuberculosis, a 30-kDa mycolyl transferase (2, 27), were described. This protein is not only the major secretory protein of M. tuberculosis in broth culture (19) but it is also among the major proteins of all M. tuberculosis proteins expressed in human macrophages (15, 21). Derived from the commercially available Connaught (Conn) and Tice strains of BCG, the recombinant vaccines were tested in the demanding guinea pig model of pulmonary tuberculosis. In contrast to other small animals used for models of tuberculosis, guinea pigs are much more susceptible to tuberculosis than humans, yet they develop disease that closely mimics human disease clinically, immunologically, and pathologically. Guinea pigs immunized with the recombinant vaccines and then challenged by aerosol...

Mycobacterium tuberculosis and other pathogenic mycobacteria export abundant quantities of proteins into their extracellular milieu when growing either axenically or within phagosomes of host cells. One major extracellular protein, the enzyme glutamine synthetase, is of particular interest because of its link to pathogenicity. Pathogenic mycobacteria, but not nonpathogenic mycobacteria, export large amounts of this protein. Interestingly, export of the enzyme is associated with the presence of a poly-l-glutamate/glutamine structure in the mycobacterial cell wall. In this study, we investigated the influence of glutamine synthetase inhibitors on the growth of pathogenic and nonpathogenic mycobacteria and on the poly-l-glutamate/glutamine cell wall structure.The inhibitor l-methionine-S-sulfoximine rapidly inactivated purified M. tuberculosis glutamine synthetase, which was 100-fold more sensitive to this inhibitor than a representative mammalian glutamine synthetase. Added to cultures of pathogenic mycobacteria, l-methionine- S-sulfoximine rapidly inhibited extracellular glutamine synthetase in a concentration-dependent manner but had only a minimal effect on cellular glutamine synthetase, a finding consistent with failure of the drug to cross the mycobacterial cell wall. Remarkably, the inhibitor selectively blocked the growth of pathogenic mycobacteria, all of which release glutamine synthetase extracellularly, but had no effect on nonpathogenic mycobacteria or nonmycobacterial microorganisms, none of which release glutamine synthetase extracellularly. The inhibitor was also bacteriostatic for M. tuberculosis in human mononuclear phagocytes (THP-1 cells), the pathogen's primary host cells. Paralleling and perhaps underlying its bacteriostatic effect, the inhibitor markedly reduced the amount of poly-l-glutamate/glutamine cell wall structure in M. tuberculosis.Although it is possible that glutamine synthetase inhibitors interact with additional extracellular proteins or structures, our findings support the concept that extracellular proteins of M. tuberculosis and other pathogenic mycobacteria are worthy targets for new antibiotics. Such proteins constitute readily accessible targets of these relatively impermeable organisms, which are rapidly developing resistance to conventional antibiotics.

No abstract

Mycobacterium tuberculosis remains one of the world's most important pathogens. Responsible for millions of new cases of tuberculosis annually (1), it is the leading cause of death from a single infectious agent. The emergence of multidrug-resistant M. tuberculosis underscores the need for new approaches to combat this pathogen (1).We recently have identified glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] as an important determinant of M. tuberculosis pathogenesis (2-4). An enzyme that plays a central role in nitrogen metabolism in every cell, glutamine synthetase is 1 of 10 proteins released in large quantity into the bacterium's extracellular milieu, whether the bacterium is growing axenically or intraphagosomally in human mononuclear phagocytes, the primary host cells (2). Of great interest, extracellular release of glutamine synthetase is unique to pathogenic mycobacteria and correlated with the presence of a poly-L-glutamate͞ glutamine (P-L-glx) component in the cell wall of these organisms, suggesting that not only is the enzyme involved in the synthesis of this heteropolymer but also that its presence is significant to virulence (4). Treatment of bacteria with L-methionine-Ssulfoximine, an irreversible inhibitor of glutamine synthetase, results in a decrease in extracellular glutamine synthetase activity, a marked reduction in the amount of cell wall P-L-glx, and inhibition of bacterial growth both in broth culture and in human macrophages (4).Antisense oligodeoxyribonucleotides (ODNs), which can base pair with a gene's transcript, constitute a new technology for the control of gene expression in prokaryotes and eukaryotes, including mammalian cells (5, 6). In addition, this technology shows promise as a means for developing new chemotherapeutic agents against human diseases, including antibiotics against such pathogens as Plasmodium falciparum, Toxoplasma gondii, and HIV (7-10). An antisense PS-ODN for treatment of cytomegalovirus retinitis in AIDS patients became the first antisense ODN-based drug approved for human use in the U.S. (11).In this report, we have utilized modified antisense ODNs, in which all internucleoside linkages are phosphorothioates (PSODNs), to study the expression and function of M. tuberculosis glutamine synthetase and the feasibility of using antisense PSODNs as antimicrobial agents against this pathogen. We find that glutamine synthetase-specific antisense PS-ODNs substantially inhibit the expression and activity of M. tuberculosis glutamine synthetase, the amount of the P-L-glx structure in the mycobacterial cell wall, and bacterial replication. These observations are consistent with entry of the antisense PS-ODNs into the bacterial cytoplasm. Materials and Methods PS-ODN Selection and Preparation.Three target sites for the binding of the antisense PS-ODNs were chosen (Table 1). One site, located near the 5Ј end of the glutamine synthetase mRNA, corresponds to codons 4-9 of the glutamine synthetase mRNA translation product, starting from the N-termi...