Physicochemical and antigenic properties of the urea-extracted surface structures of <i>Yersinia pseudotuberculosis</i> O:1b

Abstract

Immuno-serological diagnostic tools particularly identifying pathogen antigens are the most important methods of pseudotuberculosis studies. The main immunodominant and species-specific antigens located in the surface structures of the bacterial cell are of practical interest. Thereby the aim of the work was to isolate and characterize biologically active surface structures of the pseudotuberculosis microbe. Here, the living cells of Y. pseudotuberculosis 3704 (O:1b) were lysed by using 9 M urea solution to extract antigens localized in the microbial surface structures. The subcellular fractions obtained such as outer membranes (OM), urea extract (UE) and isolated protein-lipopolysaccharide complex (PLPSC) are characterized by physical and chemical parameters. The protein content in the preparations ranged from 42% to 53%. The polypeptide band of the OM preparation, UE polypeptide and PLPSC for pseudotuberculosis microbe was presented by 14, 16 and 9 major polypeptides with molecular weight ranging from 13.9 kDa to 131.5 kDa, 13.5 kDa to 101.6 kDa, and 20.7 kDa to 66.6 kDa, respectively. Proteolytically active proteins and polypeptides were detected in isolated subcellular fractions (OM and UE) by using the radial enzyme diffusion test and substrate-gel electrophoresis found to be presented by 4 and 7 polypeptides with molecular weight ranging from 28.0 kDa to 118.0 kDa and 29.2 kDa to 97.7 kDa in the OM and UE preparation, respectively. The subcellular fractions obtained are capable to exhibit immunogenic activity after inoculation to experimental animals and antigenic activity while interacting with specific antibodies in the radial immunodiffusion (RID) assay and antibodies labeled with colloidal silver nanoparticles in dot immunoassay (DIA). OM and PLPSC preparations in DIA with immunoglobulins isolated from experimental antisera and labeled with colloidal silver nanoparticles were detected at a concentration of 0.12 g/ml (dry weight), cells of strain Y. pseudotuberculosis 3704 at a concentration of 3, 9 106 m.c./ml, which is similar to the results of DIA with immunoglobulins isolated from commercial pseudotuberculosis antiserum (St. Petersburg) and labeled with nanoparticles of colloidal silver. Thus, the subcellular fractions of pseudotuberculosis microbe isolated by using urea as a lysing and decontaminating agent retain their antigenic and immunogenic properties and enzymatic activity suggesting about their potential benefits for use to improve early diagnostics of pseudotuberculosis.