Structure-based design was used to develop a focused library of A-ring-modified diphenyl ether InhA inhibitors. From this library of analogs, two high-affinity alkyl-substituted diphenyl ethers, 6PP and 8PP, were selected for advanced study into their in vitro activity against Mycobacterium tuberculosis clinical isolates, their in vivo properties, and their signature response mode of action. 6PP and 8PP demonstrated enhanced activity against whole bacteria and showed activity in a rapid macrophage model of infection. In addition, transcriptional profiling revealed that the A-ring modifications of 6PP and 8PP increased the specificity of each analog for InhA. Both analogs had substantially longer half-lives in serum than did the parent compound, exhibited a fivefold reduction in cytotoxicity compared to the parent compound, and were well tolerated when administered orally at 300 mg/kg of body weight in animal models. Thus, the A-ring modifications increased the affinity and whole-cell specificity of the compounds for InhA and increased their bioavailability. The next step in optimization of the pharmacophore for preclinical evaluation is modification of the B ring to increase the bioavailability to that required for oral delivery.

CD4+ T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an Ag associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell Ag as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4+ T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate or inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, because recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by α-mannosidase, did not activate T cells. This study demonstrates that the universe of bacterial T cell Ags includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo.

Identification of Mycobacterium tuberculosis antigens inducing cellular immune responses is required to improve the diagnosis of and vaccine development against tuberculosis. To identify the antigens of M. tuberculosis that differentiated between tuberculosis (TB) patients and healthy contacts based on T cell reactivity, the culture filtrate of in vitro grown M. tuberculosis was fractionated by two-dimensional liquid phase electrophoresis and tested for the ability to stimulate T cells in a whole blood assay. This approach separated the culture filtrate into 350 fractions with sufficient protein quantity (at least 200 g of protein) for mass spectrometry and immunological analyses. High levels of interferon-␥ (IFN-␥) secretion were induced by 105 fractions in healthy contacts compared with TB patients (p < 0.05). Most interesting was the identification of 10 fractions that specifically induced strong IFN-␥ production in the healthy contact population but not in TB patients. Other immunological measurements showed 42 fractions that induced significant lymphocyte proliferative responses in the healthy contact group compared with the TB patients. The tumor necrosis factor-␣ response for most of the fractions did not significantly differ in the tested groups, and the interleukin-4 response was below the detectable range for all fractions and both study groups. Proteomic characterization of the 105 fractions that induced a significant IFN-␥ response in the healthy contacts compared with the TB patients led to the identification of 59 proteins of which 24 represented potentially novel T cell antigens. Likewise, the protein identification in the 10 healthy "contact-specific fractions" revealed 16 proteins that are key candidates as vaccine or diagnostic targets. Molecular & Cellular Proteomics 9:538 -549, 2010. Tuberculosis (TB)1 is a major health problem throughout the world. A recent World Health Organization report shows that TB has been increasing at a rate of 1% per year, and an estimated 9.2 million new cases arise each year (1). Although TB is preventable, there has been an increase in its incidence in recent years. Re-emergence of TB is mainly due to its association with human immunodeficiency virus infection (2) and also due to the occurrence of multidrugresistant strains of the causative agent, Mycobacterium tuberculosis (3).Vaccination of general population is cost effective and represents one of the best biological measures for disease control. The current vaccine against tuberculosis, Bacille Calmette-Gué rin (BCG), has been administered to more people than any other vaccine. The side effects of BCG are tolerable, and it prevents miliary and meningeal tuberculosis in young children. In striking contrast, it affords limited and highly variable protection (0 -80%) against pulmonary TB (4). Thus, BCG does not seem to be a satisfactory vaccine (5, 6) and necessitates exploration of newer strategies to improve BCG or to develop a more effective vaccine.One of the potential strategies for the development of an im...

Francisella tularensis causes disease (tularemia) in a large number of mammals, including man. We previously demonstrated enhanced efficacy of conventional antibiotic therapy for tularemia by postexposure passive transfer of immune sera developed against a F. tularensis LVS membrane protein fraction (MPF). However, the protein composition of this immunogenic fraction was not defined. Proteomic approaches were applied to define the protein composition and identify the immunogens of MPF. MPF consisted of at least 299 proteins and 2-D Western blot analyses using sera from MPF-immunized and F. tularensis LVS-vaccinated mice coupled to liquid chromatography–tandem mass spectrometry identified 24 immunoreactive protein spots containing 45 proteins. A reverse vaccinology approach that applied labeling of F. tularensis LVS surface proteins and bioinformatics was used to reduce the complexity of potential target immunogens. Bioinformatics analyses of the immunoreactive proteins reduced the number of immunogen targets to 32. Direct surface labeling of F. tularensis LVS resulted in the identification of 31 surface proteins. However, only 13 of these were reactive with MPF and/or F. tularensis LVS immune sera. Collectively, this use of orthogonal proteomic approaches reduced the complexity of potential immunogens in MPF by 96% and allowed for prioritization of target immunogens for antibody-based immunotherapies against tularemia.

M ycobacterium leprae infections lead to human leprosy, characterized by disfiguring skin lesions, nerve damage, and eventually permanent disability (1). As part of the disease process, M. leprae is phagocytized by numerous cell types, including dendritic cells (DCs) and epidermal DCs known as Langerhans cells (LCs) (2-4). The cell envelopes of pathogenic Mycobacterium spp., including M. leprae, are rich in mannosylated macromolecules such as mannose-capped lipoarabinomannan (ManLAM), lipomannan (LM), phosphatidylinositol mannosides (PIM) and presumably glycoproteins (5, 6). These glycoconjugates contribute to the interaction of pathogenic mycobacteria with host phagocytes, and the C-type lectin receptors (CLRs) that decorate the surface of phagocytes have been shown to directly bind specific mannosylated glycoconjugates of Mycobacterium spp (7). For DCs, the constitutive CLRs are the mannose receptor (CD206) and the dendritic cell-specific intracellular adhesion moleculegrabbing nonintegrin (DC-SIGN; CD209). However, langerin (CD207) is a CLR specific to LCs. DC-SIGN and langerin recognize the sugar residues of macromolecules produced by pathogens and altered self-antigens in a calcium-dependent manner. These CLRs are comprised of a short cytoplasmic domain, a transmembrane domain, and, on the outer surface of the cell, an extracellular domain (ECD) that encompasses the highly conserved carbohydrate recognition domains (CRDs) and the hydrophobic neck domain. The CRDs determine sugar-binding specificity, and DC-SIGN and langerin possess an EPN (Glu-Pro-Asn) motif that is generally specific to oligosaccharides containing mannose (Man), fucose, glucose, or N-acetylglucosamine residues (8). The ␣-helix coiled-coil formation in the neck domains is responsible for the trimeric and tetrameric structures of langerin and DC-SIGN, respectively, and these multimeric conformations promote carbohydrate spatial specificity (9, 10). Some of mycobacterial ligands of DC-SIGN have been defined. Specifically, DC-SIGN recognizes the ␣(1¡2) linkages of ManLAM and PIM 6 (11,12). The 45-kDa and 19-kDa glycoproteins of Mycobacterium tuberculosis are also DC-SIGN ligands based on their inhibition of bacterial cell uptake via DC-SIGNtransfected cell lines (13). However, it should be noted that studies to directly compare the binding affinity of protein and lipoglycan ligands for C-type lectins or to define the relative contribution of each ligand have not been performed.We previously demonstrated that langerin-positive LCs present cell wall antigens of M. leprae to CD1a-restricted T cells, resulting in T cell proliferation and gamma interferon (IFN-␥) production (2). This study demonstrated the biological significance for the interaction of M. leprae ligands with the langerin of LCs; however, the M. leprae binding partners of this CLR were not elucidated. Thus, in the present work we sought to characterize the biochemical and biophysical features of the M. leprae glycoconjugates recognized by langerin.