The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membraneperforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly + rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly + rBCG (∆ureC hly + rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. ∆ureC hly + rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of ∆ureC hly + rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.
Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis, expression of one gene from this pathway, lipB, encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked activesite complex with decanoic acid. We provide evidence that the transferase functions as a cysteine͞lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid͞base catalysts. Analysis by MS reveals that the LipB catalytic reaction proceeds by means of an internal thioesteracyl intermediate. Structural comparison of LipB with lipoate protein ligase A indicates that, despite conserved structural and sequence active-site features in the two enzymes, 4-phosphopantetheine-bound octanoic acid recognition is a specific property of LipB.catalytic dyad ͉ lipoic acid ͉ x-ray structure ͉ thioester formation ͉ mass spectrometry S everal multicomponent enzyme complexes that catalyze key metabolic reactions in the citric acid cycle and single-carbon metabolism are posttranslationally modified by attachment to lipoic acid (1). These systems share a domain that covalently binds lipoic acid by means of an amide bond to the -amino group of a conserved exposed lysine residue. In many organisms, lipoylation is catalyzed by two separate enzymes, lipoyl protein ligase A (LplA) or octanoyl-[acyl carrier protein]-protein transferase (LipB; see Fig. 5, which is published as supporting information on the PNAS web site). Although LplA uses exogenous lipoic acid, LipB transfers endogenous octanoic acid, which is attached by means of a thioester bond to the 4Ј-phosphopantetheine cofactor of acyl carrier protein (ACP) onto lipoyl domains (2-4). These octanoylated domains are converted into lipoylated derivatives by the S-adenosyl-L-methioninedependent enzyme, lipoyl synthase (LipA), which catalyzes the insertion of sulfur atoms into the six-and eight-carbon positions of the corresponding fatty acid (5-7). This process bypasses the requirement for an exogenous supply of lipoic acid.In bacteria, enzymes involved in lipoylation have gained increasing attention because of their implication in pathogenicity. For instance, a Listeria monocytogenes mutant strain lacking LplA has been found to be defective in its ability to grow in the host cytosol and is less virulent in animals because of its dependence on host-derived lipoic acid (8). Mice Lias Ϫ/Ϫ (LipA) null variants die during embryogenesis, indicating that the mammalian pathway is related to the bacterial LipB͞LipA pathway and is essenti...
Sterol 14␣-demethylase (CYP51), a major checkpoint in membrane sterol biosynthesis, is a key target for fungal antibiotic therapy. We sought small organic molecules for lead candidate CYP51 inhibitors. The changes in CYP51 spectral properties following ligand binding make CYP51 a convenient target for highthroughput screening technologies. These changes are characteristic of either substrate binding (type I) or inhibitor binding (type II) in the active site. We screened a library of 20,000 organic molecules against Mycobacterium tuberculosis CYP51 (CYP51 Mt ), examined the top type I and type II binding hits for their inhibitory effects on M. tuberculosis in broth culture, and analyzed them spectrally for their ability to discriminate between CYP51 Mt and two reference M. tuberculosis CYP proteins, CYP130 and CYP125. We determined the binding mode for one of the top type II hits, ␣-ethyl-N-4-pyridinyl-benzeneacetamide (EPBA), by solving the X-ray structure of the CYP51 Mt -EPBA complex to a resolution of 1.53 Å. EPBA binds coordinately to the heme iron in the CYP51 Mt active site through a lone pair of nitrogen electrons and also through hydrogen bonds with residues H259 and Y76, which are invariable in the CYP51 family, and hydrophobic interactions in a phylumand/or substrate-specific cavity of CYP51. We also identified a second compound with structural and binding properties similar to those of EPBA, 2-(benzo[d]-2,1,3-thiadiazole-4-sulfonyl)-2-amino-2-phenyl-N-(pyridinyl-4)-acetamide (BSPPA). The congruence between the geometries of EPBA and BSPPA and the CYP51 binding site singles out EPBA and BSPPA as lead candidate CYP51 inhibitors with optimization potential for efficient discrimination between host and pathogen enzymes.In eukaryotic organisms, cytochrome P450 enzymes play important roles in many systems, including the biosynthesis of cholesterol, steroid hormones, and vitamins; the control of cardiovascular physiology and systemic blood pressure; drug metabolism; and chemical toxicology and carcinogenesis (23). With respect to their potential for pharmacological development, eukaryotic P450 enzymes may be divided into two groups, drug targets and drug-metabolizing enzymes. Wellestablished P450 drug targets include (i) the aromatase CYP19, required for the conversion of androgens into estrogens (30) and a key target in the treatment of breast cancer; (ii) sterol 14␣-demethylases (CYP51), required for the biosynthesis of membrane sterols, including cholesterol in animals, ergosterol in fungi, and a variety of C-24-modified sterols in plants and protozoa (2), and a key target in the treatment of diseases caused by infectious microbes; and (iii) other biosynthetic sterol hydroxylases (20). The isoform-specific inhibition of P450 enzymes offers promise for the development of therapeutic, insecticidal, and herbicidal agents (7). High-throughput screening (HTS) libraries of small organic molecules and potential drugs against P450 enzymes may thus be key to selecting high-quality compounds in the lead identificat...
Apoptosis and activation of macrophages play an important role in the host response to mycobacterial infection involving TNF-α as a critical autocrine mediator. The underlying mechanisms are still ill-defined. Here, we demonstrate elevated levels of methylglyoxal (MG), a small and reactive molecule that is usually a physiological product of various metabolic pathways, and advanced glycation end products (AGE) during mycobacterial infection of macrophages, leading to apoptosis and activation of macrophages. Moreover, we demonstrate abundant AGE in pulmonary lesions of tuberculosis (TB) patients. Global gene expression profiling of MG-treated macrophages revealed a diverse spectrum of functions induced by MG, including apoptosis and immune response. Our results not only provide first evidence for the involvement of MG and AGE in TB, but also form a basis for novel intervention strategies against infectious diseases in which MG and AGE play critical roles.
A universal step in the biosynthesis of membrane sterols and steroid hormones is the oxidative removal of the 14␣-methyl group from sterol precursors by sterol 14␣-demethylase (CYP51). This enzyme is a primary target in treatment of fungal infections in organisms ranging from humans to plants, and development of more potent and selective CYP51 inhibitors is an important biological objective. Our continuing interest in structural aspects of substrate and inhibitor recognition in CYP51 led us to determine (to a resolution of 1.95 Å ) the structure of CYP51 from Mycobacterium tuberculosis (CYP51 Mt ) cocrystallized with 4,4-dihydroxybenzophenone (DHBP), a small organic molecule previously identified among top type I binding hits in a library screened against CYP51 Mt . The newly determined CYP51 Mt -DHBP structure is the most complete to date and is an improved template for three-dimensional modeling of CYP51 enzymes from fungal and prokaryotic pathogens. The structure demonstrates the induction of conformational fit of the flexible protein regions and the interactions of conserved Phe-89 essential for both fungal drug resistance and catalytic function, which were obscure in the previously characterized CYP51 Mt -estriol complex. DHBP represents a benzophenone scaffold binding in the CYP51 active site via a type I mechanism, suggesting (i) a possible new class of CYP51 inhibitors targeting flexible regions, (ii) an alternative catalytic function for bacterial CYP51 enzymes, and (iii) a potential for hydroxybenzophenones, widely distributed in the environment, to interfere with sterol biosynthesis. Finally, we show the inhibition of M. tuberculosis growth by DHBP in a mouse macrophage model.2 is a cytochrome P450 (P450, CYP) heme thiolate containing enzyme involved in biosynthesis of membrane sterols, including cholesterol in animals, ergosterol in fungi, and a variety of C24-modified sterols in plants and protozoa in most organisms in biological kingdoms from bacteria to animals (1). CYP51 has been a therapeutic target for several generations of azole antifungal agents including fluconazole, voriconazole, itraconazole, ravuconazole, and posaconazole (2). These drugs inhibit microbial growth by disrupting biosynthesis of ergosterol, a major component of fungal membrane. Protozoa share with fungi the requirement of ergosterol and ergosterol-related sterols for cell viability and proliferation (3). Inhibition of sterol biosynthesis has been proven to be effective in trypanosomatids (3-5) and Leishmania spp (6), which cause such tropical diseases as African sleeping sickness, Chagas disease, and leishmaniasis.Although mammalian CYP51 enzymes perform the same catalytic reaction (7) as their fungal and protozoan orthologs (1), they share relatively modest overall sequence identity (within 30%) with them. This accounts for the reduced sensitivity of mammalian CYP51 to azole and triazole drugs. Despite the lack of the full sterol biosynthetic pathway in Mycobacterium tuberculosis (8), and hence, de novo sterol biosynthesis...
Tuberculosis (TB) is a major global health threat caused by Mycobacterium tuberculosis (Mtb). It is further fueled by the HIV pandemic and by increasing incidences of multidrug resistant Mtb-strains. Rv2827c, a hypothetical protein from Mtb, has been implicated in the survival of Mtb in the macrophages of the host. The threedimensional structure of Rv2827c has been determined by the three-wavelength anomalous diffraction technique using bromide-derivatized crystals and refined to a resolution of 1.93 Å . The asymmetric unit of the orthorhombic crystals contains two independent protein molecules related by a non-crystallographic translation. The tertiary structure of Rv2827c comprises two domains: an N-terminal domain displaying a winged helix topology and a C-terminal domain, which appears to constitute a new and unique fold. Based on structural homology considerations and additional biochemical evidence, it could be established that Rv2827c is a DNA-binding protein. Once the understanding of the structure-function relationship of Rv2827c extends to the function of Rv2827c in vivo, new clues for the rational design of novel intervention strategies may be obtained.
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