siRNA targeting Atp5a1 gene encoding ATPase α, the ligand of Peg fimbriae, reduced Salmonella Enteritidis adhesion.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a zoonotic pathogen that can infect both humans and animals. Among the 13 types of fimbrial operons in S. Enteritidis, the highly conserved Peg fimbriae play a crucial role in the adhesion and invasion of S. Enteritidis into host cells but are not well studied. In this study, we identified the ATP synthase subunit alpha (ATPase α) as a ligand of Peg fimbriae using ligand blotting and mass spectrometry techniques. We confirmed the in vitro binding of ATPase α to the purified adhesion protein (PegD). Furthermore, we used siRNA to suppress the expression of ATPase α gene Atp5a1 in Leghorn male hepatoma (LMH) cells, which resulted in a significant reduction in the adhesion rate of S. Enteritidis to the cells (P < 0.05). The findings in this study provide insight into the mechanism of S. Enteritidis infection through Peg fimbriae and highlight the importance of ATPase α in the adhesion process.RESEARCH HIGHLIGHTS Ligand blotting was performed to screen the ligand of S. Enteritidis Peg fimbriae.Binding assay confirmed that ATPase α is the ligand of the Peg fimbriae.siRNA targeting ATPase α gene (Atp5a1) significantly reduced S. Enteritidis adhesion.

Detection of Fibronectin-Binding Proteins of Streptococcus pyogenes Using Ligand Blot Analysis.

Streptococcus pyogenes utilizes extracellular cellular matrix (ECM) proteins to adhere to human tissues and internalize into host cells. Fibronectin (Fn) is one of the most abundant ECM proteins and targeted by a wide variety of secreted Fn-binding proteins (Fbps) of S. pyogenes. However, prior to detailed kinetic analysis of that binding process, evaluations of the ability of S. pyogenes strains to bind to Fn as well as interactions of target molecules with Fn are required. In this chapter, we present routine procedures for ligand blot analysis with labeled human Fn, using bacterial cell wall extracts prepared by either enzymatic digestion of cells or extraction with a denaturing agent.

Synthetic fusion-protein containing domains of Bt Cry1Ac and Allium sativum lectin (ASAL) conferred enhanced insecticidal activity against major lepidopteran pests.

Different transgenic crop plants, developed with δ-endotoxins of Bacillus thuringiensis (Bt) and mannose-specific plant lectins, exhibited significant protection against chewing and sucking insects. In the present study, a synthetic gene (cry-asal) encoding the fusion-protein having 488 amino acids, comprising DI and DII domains from Bt Cry1Ac and Allium sativum agglutinin (ASAL), was cloned and expressed in Escherichia coli. Ligand blot analysis disclosed that the fusion-protein could bind to more number of receptors of brush border membrane vesicle (BBMV) proteins of Helicoverpa armigera. Artificial diet bioassays revealed that 0.025 µg/g and 0.50 µg/g of fusion-protein were sufficient to cause 100% mortality in Pectinophora gossypiella and H. armigera insects, respectively. As compared to Cry1Ac, the fusion-protein showed enhanced (8-fold and 30-fold) insecticidal activity against two major lepidopteran pests. Binding of fusion-protein to the additional receptors in the midgut cells of insects is attributable to its enhanced entomotoxic effect. The synthetic gene, first of its kind, appears promising and might serve as a potential candidate for engineering crop plants against major insect pests.