φYeO3-12 phage tail fiber Gp17 as a promising high specific tool for recognition of Yersinia enterocolitica pathogenic serotype O:3

Abstract

Yersiniosis is an infectious zoonotic disease caused by two enteropathogenic species of Gram-negative genus Yersinia: Yersinia enterocolitica and Yersinia pseudotuberculosis. Pigs and other wild and domestic animals are reservoirs for these bacteria. Infection is usually spread to humans by ingestion of contaminated food. Yersiniosis is considered a rare disease, but recent studies indicate that it is overlooked in the diagnostic process therefore the infections with this bacterium are not often identified. Reliable diagnosis of Yersiniosis by culturing is difficult due to the slow growth of the bacteria easily overgrown by other more rapidly growing microbes unless selec-tive growth media is used. Phage adhesins recognizing bacteria in a specific manner can be an excellent diagnostic tool, es-pecially in the diagnosis of pathogens difficult for culturing. In this study, it was shown that Gp17, the tail fiber protein (TFP) of phage φYeO3-12, specifically recognizes only the pathogenic Yersinia enterocolitica serotype O:3 (YeO:3) bacteria. The ELISA test used in this work confirmed the specific interaction of this protein with YeO:3 and demonstrated a promising tool for developing the pathogen recognition method based on phage adhesins.

Keywords: Diagnostic; ELISA; Phage; Phage adhesins; Tail fiber protein; Yersinia enterocolitica; Yersiniosis.

Fig. 1

SDS-PAGE of TFP-Gp17. A Samples from all purification steps by nickel-affinity chromatography. Lanes: 1, supernatant from bacterial lysate; 2, flow through 1 (FT1) containing the unbound proteins; 3, elution fraction 1 (elution with buffer A supplemented with 250 mM imidazole); 4, elution fraction after desalting with ammonium sulfate; 5,7, FT2, containing the unbound proteins after cleavage with the TEV protease; 6, elution fraction 2. B TFP-Gp17 before SEC chromatography is shown in the lane 6. C Protein fractions after SEC chromatography; TFP-Gp17 in 4 lane is indicated by the red arrow. D The H/MTFP-Gp17 protein after SEC chromatography is indicated in lane by the red arrow

Fig. 2

H/MTFP-Gp17—based ELISA for specific detection of YeO:3 (10⁵ CFU/ml). The bound H/MTFP-Gp17 was detected by HRP-conjugated IgG anti-HisTag monoclonal antibody. The color development was measured at 450 nm on a microplate reader. SD was calculated from 3 replicates

Fig. 3

The sensitivity the H/MTFP-Gp17—based ELISA for specific detection of YeO:3 bacteria. The wells were coated with YeO:3 bacteria at indicated concentrations. For the detection H/MTFP-Gp17 at 8.5 µg/ml was used, and the bound H/MTFP-Gp17 was detected as described in Fig. 4 legend. As the negative control R1 strain was used. SD was calculated from 3 replicates. T-test was performed between tested strains and control strain R1 (*- P ≤ 0.05; **- P ≤ 0.01; ***- P ≤ 0.001; ****- P ≤ 0.0001)

Fig. 4

Sandwich-type preparation method based on immunogold labelling to visualize by TEM the interaction of H/MTFP-Gp17 with bacterial cell surface

Fig. 5

Specific interaction of H/MTFP-Gp17 with YeO:3 bacteria visualized by TEM using immunogold labelling. A R1 mutant, the negative reaction with Protein A-Gold, B YeO:3 wild type strain 6471/76-c, positive reaction with Protein A-Gold