Discovery And Exploration Of Riemerella Anatipestifer Hemophore, As An Antibacterial Target

The iron and heme utilization has been one of the hotspots in current research. In this study, we focused on the heme acquisition system of RA, and discovered and characterized an extracellular heme-binding protein hemophore. Subsequently, we found a potential hemophore gene family by bioinformatics analysis, and then we identified the function of one hemophore after cloning, prokaryotic expression and purification. Eventually, to achieve the purpose of anti-RA, according to the feature of hemophore targeted transport heme, we found SDNS (C32H22N6Na2O6S2), a heme-analogue drug, and preliminary explore its effect in anti-RA. The main results as follows:1. Discovery and characterization an extracellular heme-binding protein, hemophore, secreted by RA.Owing to the expression of bacterial heme uptake proteins is regulated by the iron ions concentration or heme concentration in the environment, we tried to use the iron ions restrictive medium or heme contained medium to culture RA. Then we extracted the secreted proteins and identified them whether can binding heme. As a result, we found a small heme-binding protein from LB medium containing heme but not from iron ions restrictive TSB medium. It indicated the heme-binding protein expression is regulated by the heme concentration, not by iron ions concentration in the environment. Further, we used heme-agarose affinity chromatography to pull down the secreted heme-binding proteins. The results also confirmed the existance of this protein. Above experiments had shown that RA encoded and secreted an extracellular heme-binding proteins, and we named it RA hemophore.2. R. anatipestifer encodes potential hemophoreBecause RA could secrete hemophore, we tried to search the gene by bioinformatics analysis. Finally we found a gene, annotated "extracellular heme-binding protein" in RA-GD genome, may be the potential hemophore. In the four complete genomes of RA, we found that the gene and its homologous genes should blong to a gene family including 3 genes with sequence similarity above 70% by sequence alignment, and we named them hemophore1, hemophore2, hemophore3 respectively. Except for RA-CH-1 only encoded hemophore1 and hemophore2 with double copy, the others encoded hemophore1, hemophore2, hemophore3 with single copy. Signal peptide prediction shows that this family proteins contain signal peptide, and protein subcellular localization prediction shows that the porteins mainly locates extracellular. To further verifying the function of potential hemophore, we cloned, prokaryotic expressed and purified hemophorel. Eventually, we identified the function of hemophore1 and the result shown that hemophorel could bind heme.3. R. anatipestifer hemophore may be a potential antibacterial drug targetIn vitro, isolation and culture of RA required blood contained medium. However, in vivo, the blood is an important organization of RA infection. So we speculate that the heme in blood plays an important role in its growth, and we also proved it by genome analysis (Wang X. et al,2014). If we block the demand for heme, we can inhibit the growth of RA. According to the principle of competitive inhibition, we found a heme-analogue SDNS, and verified its bacteriostasis in vitro. The result shown that SDNS could significantly inhibit the growth of RA. Because of the secretion of hemophore is the first step for bacteria utilizing heme, we speculate that hemophore is an important target protein for SDNS. The antibacterial effects and safety evaluation of SDNS remain to further study in vivo.