In silico discovery of potential drug molecules to improve the treatment of isoniazid-resistant Mycobacterium tuberculosis

The emergence of multidrug-resistant Mycobacterium tuberculosis (M.tb) has become one of the major hurdles in the treatment of tuberculosis (TB). Drug-resistant M.tb has evolved with various strategies to avoid killing by the anti-tubercular drugs. Thus, there is a rising need to develop effective anti-TB drugs to improve the treatment of these strains. Traditional drug design approach has earned little success due to time and the cost involved in the process of development of anti-infective drugs. Numerous reports have demonstrated that several mutations in the drug target sites cause emergence of drug-resistant M.tb strains. In this study, we performed computational mutational analysis of M.tb inhA, fabD, and ahpC genes, which are the primary targets for first-line isoniazid (INH) drug. In silico virtual drug screening was performed to identify the potent drugs from a ChEMBL compound library to improve the treatment of INH-resistant M.tb. Further, these compounds were analyzed for their binding efficiency against active drug binding cavity of M.tb wild-type and mutant InhA, FabD and AhpC proteins. The drug efficacy of predicted lead compounds was verified by molecular docking using M.tb wild-type and mutant InhA, FabD and AhpC protein template models. Different in silico and pharmacophore analysis predicted three potent lead compounds with better drug-like properties against both M.tb wild-type and mutant InhA, FabD, and AhpC proteins as compared to INH drug, and thus may be considered as effective drugs for the treatment of INH-resistant M.tb strains. We hypothesize that this work may accelerate drug discovery process for the treatment of drug-resistant TB.

Communicated by Ramaswamy H. Sarma     

Commensal Pseudomonas protect Arabidopsis thaliana from a coexisting pathogen via multiple lineage-dependent mechanisms

Plants are protected from pathogens not only by their own immunity but often also by colonizing commensal microbes. In Arabidopsis thaliana, a group of cryptically pathogenic Pseudomonas strains often dominates local populations. This group coexists in nature with commensal Pseudomonas strains that can blunt the deleterious effects of the pathogens in the laboratory. We have investigated the interaction between one of the Pseudomonas pathogens and 99 naturally co-occurring commensals, finding plant protection to be common among non-pathogenic Pseudomonas. While protective ability is enriched in one specific lineage, there is also a substantial variation for this trait among isolates of this lineage. These functional differences do not align with core-genome phylogenies, suggesting repeated gene inactivation or loss as causal. Using genome-wide association, we discovered that different bacterial genes are linked to plant protection in each lineage. We validated a protective role of several lineage-specific genes by gene inactivation, highlighting iron acquisition and biofilm formation as prominent mechanisms of plant protection in this Pseudomonas lineage. Collectively, our work illustrates the importance of functional redundancy in plant protective traits across an important group of commensal bacteria.Subject terms: Microbial ecology, Plant ecology     

Efficient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Ealpha(52-68) yields an epitope that is similar to the one generated during constitutive presentation of I-Ealpha as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Ealpha is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.     

MHC class I-restricted presentation of maleylated protein binding to scavenger receptors

Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs.     

Low dose insemination in synchronized gilts

Conventional insemination techniques in pigs require 2 to 3 x 10(9) sperm/dose. When using the latest high-speed sperm-sorting technology, one can still sort only about 5 to 6 million sperm of each sex per hour. The objective of the present study was to find the minimal sperm concentration at a low-insemination volume in pigs without diminishing fertilization rate and litter size using surgical deep intra-uterine insemination (IUI). Semen from 3 boars was collected and diluted with Androhep to 5 x 10(8), 1 x 10(8), 1 x 10(7), 5 x 10(6) or 1 x 10(6) sperm/0.5 ml. In trial 1, 109 prepuberal gilts were synchronized and surgically inseminated into the tip of each uterine horn 32 h or 38 h after hCG treatment or at the time of ovulation, respectively. Pregnant gilts were allowed to go to term. Pregnancy and farrowing rates did not differ significantly except at the lowest sperm concentration if inseminated 32 h or 38 h after hCG treatment (p < 0.05). No differences were found among insemination groups for the total number of piglets, number of piglets born alive, stillborn piglets, and mummified fetuses. In trial 2, 34 gilts were inseminated as described above 32 h after hCG. Additionally, 9 gilts were inseminated once nonsurgically with 1 x 10(9) sperm as controls. Gilts were slaughtered 48 h after insemination, and embryos were recovered. Embryos were cultured in NCSU 23 (120 h), evaluated morphologically and stained with fluorescent dye (Hoechst 33342) to visualize nuclei. Recovery rates varied between 71.4% and 84.4%. Fertilization rate of the lowest sperm concentration (1 x 10(6) sperm/horn) differed significantly (p < 0.05) from all other groups. Cleavage rates at specific developmental stages did not differ. After 5 days of in vitro culture, embryos developed to morulae and blastocysts. No differences were found for these stages. In conclusion, no major differences were found between insemination groups as long as the sperm dosage was at least 10 million sperm per gilt. The low volume was sufficient for successful deep intra-uterine insemination. Embryo development was comparable to the controls.     

Fatty Acids Induce Chloride Permeation in Rat Liver Mitochondria by Activation of the Inner Membrane Anion Channel (IMAC)

The inner membrane of freshly isolated mammalian mitochondria is poorly permeable to Cl(-). Low, nonlytic concentrations (< or =30 microM) of long-chain fatty acids or their branched-chain derivatives increase permeation of Cl(-) as indicated from rapid large-scale swelling of mitochondria suspended in slightly alkaline KCl medium (supplemented with valinomycin). Myristic, palmitic, or 5-doxylstearic acid are powerful inducers of Cl(-) permeation, whereas lauric, phytanic, stearic, or 16-doxylstearic acid stimulate Cl(-) permeation in a lesser extent. Fatty acid-induced Cl(-) permeation across the inner membrane correlates well with the property of nonesterified fatty acids to release endogenous Mg(2+) from mitochondria. Myristic acid stimulates anion permeation in a selective manner, similar as was described for A23187, an activator of the inner membrane anion channel (IMAC). Myristic acid-induced Cl(-) permeation is blocked by low concentrations of tributyltin chloride (IC(50) approximately 1.5 nmol/mg protein). Moreover, myristic acid activates a transmembrane ion current in patch-clamped mitoplasts (mitochondria with the outer membrane removed) exposed to alkaline KCl medium. This current is best ascribed to the opening of an ion channel with a single-channel conductance of 108 pS. We propose that long-chain fatty acids can activate IMAC by withdrawal of Mg(2+) from intrinsic binding sites.     

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.     

CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis

The epithelial cell adhesion molecule CEACAM1 (carcinoembryonic antigen cell adhesion molecule-1) is down-regulated in colon, prostate, breast, and liver cancer. Here we show that CEACAM1-4S, a splice form with four Ig-like ectodomains and a short cytoplasmic domain (14 amino acids), directly associates with annexin II, a lipid raft-associated molecule, which is also down-regulated in many cancers. Annexin II was identified using a glutathione S-transferase pull-down assay in which the cytoplasmic domain of CEACAM-4S was fused to glutathione S-transferase, the fusion protein was incubated with cell lysates, and isolated proteins were sequenced by mass spectrometry. The interaction was confirmed first by reciprocal immunoprecipitations using anti-CEACAM1 and anti-annexin II antibodies and second by confocal laser microscopy showing co-localization of CEACAM1 with annexin II in mammary epithelial cells grown in Matrigel. In addition, CEACAM1 co-localized with p11, a component of the tetrameric AIIt complex at the plasma membrane, and with annexin II in secretory vesicles. Immobilized, oriented peptides from the cytoplasmic domain of CEACAM1-4S were shown to directly associate with bovine AIIt, which is 98% homologous to human AIIt, with average KD values of about 30 nM using surface plasmon resonance, demonstrating direct binding of functionally relevant AIIt to the cytoplasmic domain of CEACAM1-4S.     

Specificity determinants of human cathepsin s revealed by crystal structures of complexes

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.