[Show abstract][Hide abstract] ABSTRACT: We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center 'bridge-helix cap,' comprising the 'bridge-helix N-terminal hinge,' 'F-loop,' and 'link region.' We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.
[Show abstract][Hide abstract] ABSTRACT: Identification of unique leads represents a significant challenge in drug discovery. This hurdle is magnified in neglected diseases such as tuberculosis. We have leveraged public high-throughput screening (HTS) data to experimentally validate a virtual screening approach employing Bayesian models built with bioactivity information (single-event model) as well as bioactivity and cytotoxicity information (dual-event model). We virtually screened a commercial library and experimentally confirmed actives with hit rates exceeding typical HTS results by one to two orders of magnitude. This initial dual-event Bayesian model identified compounds with antitubercular whole-cell activity and low mammalian cell cytotoxicity from a published set of antimalarials. The most potent hit exhibits the in vitro activity and in vitro/in vivo safety profile of a drug lead. These Bayesian models offer significant economies in time and cost to drug discovery.
[Show abstract][Hide abstract] ABSTRACT: A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery.
Current opinion in microbiology 08/2011; 14(5):532-43. · 7.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The competition for L-arginine between the inducible nitric oxide synthase and arginase contributes to the outcome of several parasitic and bacterial infections. The acquisition of L-arginine, however, is important not only for the host cells but also for the intracellular pathogen. In this study we observe that strain AS-1, the Mycobacterium bovis BCG strain lacking the Rv0522 gene, which encodes an arginine permease, perturbs l-arginine metabolism in J774.1 murine macrophages. Infection with AS-1, but not with wild-type BCG, induced l-arginine uptake in J774.1 cells. This increase in L-arginine uptake was independent of activation with gamma interferon plus lipopolysaccharide and correlated with increased expression of the MCAT1 and MCAT2 cationic amino acid transport genes. AS-1 infection also enhanced arginase activity in resting J774.1 cells. Survival studies revealed that AS-1 survived better than BCG within resting J774.1 cells. Intracellular growth of AS-1 was further enhanced by inhibiting arginase and ornithine decarboxylase activities in J774.1 cells using L-norvaline and difluoromethylornithine treatment, respectively. These results suggest that the arginine-related activities of J774.1 macrophages are affected by the arginine transport capacity of the infecting BCG strain. The loss of Rv0522 gene-encoded arginine transport may have induced other cationic amino acid transport systems during intracellular growth of AS-1, allowing better survival within resting macrophages.
Journal of Bacteriology 08/2006; 188(13):4830-40. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glutathione is a tripeptide and antioxidant, synthesized at high levels by cells during the production of reactive oxygen and nitrogen intermediates. Glutathione also serves as a carrier molecule for nitric oxide in the form of S-nitrosoglutathione. Previous studies from this laboratory have shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. Glutathione is not transported into the cells as a tripeptide. Extracellular glutathione is converted to a dipeptide due to the action of transpeptidase, and the dipeptide is then transported into the bacterial cells. The processing of glutathione and S-nitrosoglutathione is brought about by the action of the enzyme gamma-glutamyl transpeptidase. The function of gamma-glutamyl transpeptidase is to cleave glutathione and S-nitrosoglutathione to the dipeptide (Cys-Gly), which is then transported into the bacterium by the multicomponent ABC transporter dipeptide permease. We have created a mutant strain of Mycobacterium tuberculosis lacking this metabolic enzyme. We investigated the sensitivity of this strain to glutathione and S-nitrosoglutathione compared to that of the wild-type bacteria. In addition, we examined the role of glutathione and/or S-nitrosoglutathione in controlling the growth of intracellular M. tuberculosis inside mouse macrophages.
Journal of Bacteriology 03/2006; 188(4):1364-72. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We demonstrate that Mycobacterium tuberculosis grown in vitro is sensitive to glutathione and its derivative S-nitrosoglutathione. Furthermore, our infection studies with J774.1 macrophages indicate that glutathione is essential for the control of the intracellular growth of M. tuberculosis. This study indicates the important role of glutathione in the control of macrophages by M. tuberculosis.
Infection and Immunity 04/2005; 73(3):1886-9. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen and nitrogen intermediates are important antimicrobial defense mechanisms of macrophages and other phagocytic cells. While reactive nitrogen intermediates have been shown to play an important role in tuberculosis control in the murine system, their role in human disease is not clearly established. Glutathione, a tripeptide and antioxidant, is synthesized at high levels by cells during reactive oxygen intermediate and nitrogen intermediate production. Glutathione has been recently shown to play an important role in apoptosis and to regulate antigen-presenting-cell functions. Glutathione also serves as a carrier molecule for nitric oxide, in the form of S-nitrosoglutathione. Previous work from this laboratory has shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. A mutant strain of Mycobacterium bovis BCG, defective in the transport of small peptides such as glutathione, is resistant to the toxic effect of glutathione and S-nitrosoglutathione. Using the peptide transport mutant as a tool, we investigated the role of glutathione and S-nitrosoglutathione in animal and human macrophages in controlling intracellular mycobacterial growth.
Infection and Immunity 05/2003; 71(4):1864-71. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using a Mycobacterium bovis BCG mutant (AS1) lacking a Bacillus subtilis L-arginine transporter homolog, we demonstrate here that the interaction between intracellular mycobacteria and the macrophage with respect to L-arginine transport and metabolism is quite complex. Intracellular AS1 stimulates macrophage L-arginine transport and accumulates 2.5-fold more (3)H label derived from L-arginine than does the wild type. These studies suggest that the accumulation of (3)H label reflects the acquisition of metabolites of L-arginine produced by the macrophage.
Infection and Immunity 03/2003; 71(2):1011-5. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: L-arginine uptake systems in macrophages play a role in regulating nitric oxide synthesis via the inducible L-arginine nitric oxide pathway. This paper describes the association of L-arginine transport with nitric oxide production in human peripheral blood monocyte-derived macrophages and in peritoneal macrophages from control and inducible nitric oxide synthase knock out C57BL6 mice. Experiments performed with human macrophages suggested that little or no nitric oxide was produced in human macrophages in vitro and that human macrophages exhibit a different arginine transport-specific response to stimuli compared with rodent macrophages. We conclude that increased L-arginine transport in both human and murine macrophages is dependent on the requirement for intracellular nitric oxide.