Prediction of the binding interface between monoclonal antibody m102.4 and Nipah attachment glycoprotein using structure-guided alanine scanning and computational docking.

Nipah Virus (NiV) has been designated as a priority disease with an urgent need for therapeutic development by World Health Organization. The monoclonal antibody m102.4 binds to the immunodominant NiV receptor-binding glycoprotein (GP), and potently neutralizes NiV, indicating its potential as a therapeutic agent. Although the co-crystal structure of m102.3, an m102.4 derivative, in complex with the GP of the related Hendra Virus (HeV) has been solved, the structural interaction between m102.4 and NiV is uncharacterized. Herein, we used structure-guided alanine-scanning mutagenesis to map the functional epitope and paratope residues that govern the antigen-antibody interaction. Our results revealed that the binding of m102.4 is mediated predominantly by two residues in the HCDR3 region, which is unusually small for an antibody-antigen interaction. We performed computational docking to generate a structural model of m102.4-NiV interaction. Our model indicates that m102.4 targets the common hydrophobic central cavity and a hydrophilic rim on the GP, as observed for the m102.3-HeV co-crystal, albeit with Fv orientation differences. In summary, our study provides insight into the m102.4-NiV interaction, demonstrating that structure-guided alanine-scanning and computational modeling can serve as the starting point for additional antibody reengineering (e.g. affinity maturation) to generate potential therapeutic candidates.

Copper chloride induces antioxidant gene expression but reduces ability to mediate H2O2 toxicity in Xanthomonas campestris.

Copper (Cu)-based biocides are currently used as control measures for both fungal and bacterial diseases in agricultural fields. In this communication, we show that exposure of the bacterial plant pathogen Xanthomonas campestris to nonlethal concentrations of Cu(2+) ions (75 µM) enhanced expression of genes in OxyR, OhrR and IscR regulons. High levels of catalase, Ohr peroxidase and superoxide dismutase diminished Cu(2+)-induced gene expression, suggesting that the production of hydrogen peroxide (H2O2) and organic hydroperoxides is responsible for Cu(2+)-induced gene expression. Despite high expression of antioxidant genes, the CuCl2-treated cells were more susceptible to H2O2 killing treatment than the uninduced cells. This phenotype arose from lowered catalase activity in the CuCl2-pretreated cells. Thus, exposure to a nonlethal dose of Cu(2+) renders X. campestris vulnerable to H2O2, even when various genes for peroxide-metabolizing enzymes are highly expressed. Moreover, CuCl2-pretreated cells are sensitive to treatment with the redox cycling drug, menadione. No physiological cross-protection response was observed in CuCl2-treated cells in a subsequent challenge with killing concentrations of an organic hydroperoxide. As H2O2 production is an important initial plant immune response, defects in H2O2 protection are likely to reduce bacterial survival in plant hosts and enhance the usefulness of copper biocides in controlling bacterial pathogens.

Gene cloning and characterization of a novel highly organic solvent tolerant lipase from Proteus sp. SW1 and its application for biodiesel production.

Proteus sp. SW1 was found to produce an extracellular solvent tolerant lipase. The gene, lipA, encoding a bacterial lipase, was cloned from total Proteus sp. SW1 DNA. lipA was predicted to encode a 287 amino acid protein of 31.2 kDa belonging to the Group I proteobacterial lipases. Purified His-tagged LipA exhibited optimal activity at pH 10.0 and 55°C. It was highly stable in organic solvents retaining 112% of its activity in 100% isopropanol after 24 h, and exhibited more than 200% of its initial activity upon exposure to 60% acetone, ethanol, and hexane for 18 h. Biodiesel synthesis reactions, using a single step addition of 13% an acyl acceptor ethanol, showed that LipA was highly effective at converting palm oil into biodiesel.

The hdhA gene encodes a haloacid dehalogenase that is regulated by the LysR-type regulator, HdhR, in Sinorhizobium meliloti.

The plasmid pSymA, in the nitrogen-fixing soil bacterium, Sinorhizobium meliloti, carries a 750-bp ORF (SMa1978) designated, hdhA, which encodes a novel dehalogenase that can detoxify haloacid compounds, showing a preference for haloacetic acids. Purified His-tagged HdhA demonstrated the apparent ability to dehalogenate chloroacetic acid and trifluoroacetic acid. In addition, upstream of hdhA, a gene encoding a lysR-type transcription regulator denoted, hdhR (SMa1979), has been identified to be a transcriptional repressor of hdhA expression. In an hdhR knockout mutant, hdhA promoter activity was markedly increased. Purified 32-kDa His-tagged HdhR repressed expression of hdhA by specifically binding to the promoter region of hdhA, as demonstrated by gel mobility shift assay and DNase I foot printing experiments. Moreover, the pesticide, pentachlorophenol, was also found to induce hdhA expression via HdhR. Site-directed mutants, in which the Cys residues at positions 160 and 192 in HdhR were changed to Ser, were constructed. C160S and C192S single mutants showed diminished HdhR-mediated repression of hdhA expression, while a C160S:C192S double mutant could no longer repress expression of hdhA.