Ali Nasser Eddine

Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin

The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membrane-perforating 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 (DeltaureC hly+ rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. DeltaureC 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 DeltaureC hly+ rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.

Small-Molecule Scaffolds for CYP51 Inhibitors Identified by High-Throughput Screening and Defined by X-Ray Crystallography

ABSTRACT Sterol 14α-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 high-throughput 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 (CYP51Mt), 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 CYP51Mt and two reference M. tuberculosis CYP proteins, CYP130 and CYP125. We determined the binding mode for one of the top type II hits, α-ethyl-N-4-pyridinyl-benzeneacetamide (EPBA), by solving the X-ray structure of the CYP51Mt-EPBA complex to a resolution of 1.53 Å. EPBA binds coordinately to the heme iron in the CYP51Mt 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 phylum- and/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.

Critical role of methylglyoxal and AGE in mycobacteria-induced macrophage apoptosis and activation.

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.

Antigen 85C inhibition restricts Mycobacterium tuberculosis growth through disruption of cord factor biosynthesis

ABSTRACT The antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital for Mycobacterium tuberculosis due to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential for M. tuberculosis virulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition of M. tuberculosis division in broth culture. I3-AG85 also inhibited M. tuberculosis survival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistant M. tuberculosis strains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of the M. tuberculosis envelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of the M. tuberculosis envelope. I3-AG85 inhibited the division of and reduced TDM synthesis in an M. tuberculosis strain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design.

X-ray structure of 4,4'-dihydroxybenzophenone mimicking sterol substrate in the active site of sterol 14alpha-demethylase (CYP51).

A universal step in the biosynthesis of membrane sterols and steroid hormones is the oxidative removal of the 14α-methyl group from sterol precursors by sterol 14α-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 1.95Å) the structure of CYP51 from Mycobacterium tuberculosis (CYP51Mt) co-crystallized with 4,4′-dihydroxybenzophenone (DHBP), a small organic molecule previously identified among top type I binding hits in a library screened against CYP51Mt. The newly determined CYP51Mt-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 CYP51Mt-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.

A Multicistronic DNA Vaccine Induces Significant Protection against Tuberculosis in Mice and Offers Flexibility in the Expressed Antigen Repertoire

ABSTRACT Concerns about the safety and efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) emphasize the need for alternative tuberculosis (TB) vaccines. DNA vaccines are interesting candidates but are limited by the restricted antigen repertoire that they express. Traditional polycistronic vectors are large and have imbalanced expression. Recent advances in molecular genetics and cellular immunology have paved the way toward the rational design of an efficacious vaccine. We exploited self-cleaving peptide 2A from the foot-and-mouth disease virus, because of its small size and high cleavage activity, to generate an efficient TB DNA vaccine (V-2A). V-2A expresses three mycobacterial antigens, Rv3407, Ag85A, and HspX, in a single open reading frame joined by the 2A sequences, which lead to the segmentation of the long translated polypeptide into individual proteins by posttranslational modification. Our in vitro measurements revealed no differences at the transcriptional or translational level between V-2A and the monocistronic expression of the individual antigens. Mice vaccinated with V-2A developed antigen-specific cellular and humoral responses against all three antigens, imparting protection against Mycobacterium tuberculosis aerosol challenge equivalent to that imparted by BCG. These results have important implications for the rational design and development of efficacious recombinant subunit vaccines.

Structural diversity in the six-fold redundant set of acyl-CoA carboxyltransferases in Mycobacterium tuberculosis

Mycobacterium tuberculosis contains multiple versions of the accA and accD genes that encode the α‐ and β‐subunits of at least three distinct multi‐functional acyl‐CoA carboxylase complexes. Because of its proposed involvement in pathogenic M. tuberculosis survival, the high‐resolution crystal structure of the β‐subunit gene accD5 product has been determined and reveals a hexameric 356 kDa complex. Analysis of the active site properties of AccD5 and homology models of the other five M. tuberculosis AccD homologues reveals unexpected differences in their surface composition, providing a molecular rational key for a sorting mechanism governing correct acyl‐CoA carboxylase holo complex assembly in M. tuberculosis.

Reductive methylation to improve crystallization of the putative oxidoreductase Rv0765c from Mycobacterium tuberculosis

Rv0765c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and crystallized. The native protein crystallized in a hexagonal crystal form which diffracted to 7 A resolution. In an attempt to improve the quality of the Rv0765c crystals, the protein was modified by reductive methylation using dimethylaminoborane and formaldehyde. The modified protein crystallized under different conditions in a tetragonal crystal form, from which diffraction data could be collected to a resolution of 3.2 A. In both crystal forms of Rv0765c, the asymmetric unit contained two copies of the protein molecule.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv2827c from Mycobacterium tuberculosis.

The hypothetical protein Rv2827c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and then crystallized. Preliminary X-ray diffraction data analysis suggests the presence of two translationally related molecules in the asymmetric unit of the orthorhombic crystals.