Identification and characteristics of temperature-sensitive sites of Pseudomonas plecoglossicida based on aptamers

Abstract

Pseudomonas plecoglossicida can infect Pseudosciaena crocea and cause visceral white spot disease, resulting in substantial economic losses to the P. crocea industry. Pseudomonas plecoglossicida is a temperate-sensitive bacterium and only infects P. crocea below 22 ℃. Aptamers are oligonucleotide molecules selected by the systematic evolution of ligands by exponential enrichment (SELEX) and can identify all kinds of targets. In the present study, five aptamers (M17, M19, M48, M56 and M81) for P. plecoglossicida cultured at 18 ℃ were selected by SELEX and based on comparative analysis of the high frequency sequences. The five aptamers had significantly higher affinities for the pathogenic P. plecoglossicida at 18 ℃ than for the nonpathogenic P. plecoglossicida at 28 ℃ and the other six bacteria (Escherichia coli, Edwardsiella tarda, Aeromonas hydrophila, Vibiro harveryi, Vibiro alginolyticus, Pseudomonas aeruginosa) (P < 0.01). The affinity constants (Kd) and maximum affinity (Am) of these aptamers for P. plecoglossicida were determined, with Kd values of M17 (23.35 ± 6.40), M19 (66.37 ± 14.73), M48 (105.91 ± 21.41), M56 (31.16 ± 5.03) and M81 (52.63 ± 13.82) nmol/L and with Am values of M17 (1041.26 ± 57.08), M19 (2291.76 ± 171.96), M48 (1258.51 ± 21.41), M56 (1307.70 ± 54.01) and M81 (543.27 ± 44.16) nmol/L, respectively. The secondary structures of the aptamers were analyzed, and the aptamer binding proteins of the pathogenic P. plecoglossicida were isolated by magnetic separation and purified by polyacrylamide gel electrophoresis. Mass spectrometry revealed ribosomal proteins to be the main aptamer binding proteins. The secondary and tertiary structures and subcellular localization of these aptamer binding proteins were also studied and analyzed. These findings provide a foundation for the temperature-sensitive pathogenic mechanism of P. plecoglossicida in P. crocea. This is the first report on aptamers that recognize temperature-sensitive sites in P. plecoglossicida.