Immune responses and protective efficacy of outer membrane protein ExbB of Pseudomonas fluorescens against Aeromonas hydrophila and Pseudomonas fluorescens affecting Carassius auratus

Abstract

The outer membrane protein (ExbB) of Pseudomonas fluorescens present in the outermost layer may possess immune functions and potential applications in subunit vaccines. ExbB bioinformatic analysis elucidated that the relationship among Pseudomonas species was closer than others, and anti-ExbB serum might provide cross-protective ability to resist Pseudomonas bacterial infection in animals. ExbB was obtained via molecular cloning, prokaryotic expression, and purification. The optimal expression conditions were a strain OD 600 value of 1.0 and IPTG concentration of 0.3 mmol/L, inducing time of 3 h, and inducing temperature of 32℃. Mice immunized to ExbB could significantly increase (p < 0.05) the spleen index and leukocyte phagocytosis parameters such as phagocytic percentage (PP) and phagocytic index (PI). The specific antiserum titer (1:12,800) was obtained, which had an in vitro immune recognition effect on both P. fluorescens and Aeromonas hydrophila. Passive immunization results for anti-ExbB mouse serum to Carassius auratus showed that ExbB serum could significantly enhance PP (p < 0.05) and PI (p < 0.05). Moreover, the passive protective rate of anti-ExbB serum against P. fluorescens infection was 54% (p < 0.05), and the passive cross-protective rate against A. hydrophila was 38.4% (p < 0.05). Furthermore, the expression of immune-related genes such as IL-6, IL-8, IL-1β, and TNF-α was significantly decreased (p < 0.05) in kidneys and gills after challenging with P. fluorescens, while the downward trend was not obvious after challenging with A. hydrophila. The trend changes of antioxidant-related factors such as malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were not obvious after bacterial challenge. Interestingly, there was a significant reduction in the histopathological lesions in the kidney, spleen, and intestine of C. auratus challenged by P. fluorescens and A. hydrophila. Altogether, the results suggest that ExbB has an immune function in passive protection against P. fluorescens and passive cross-protection against A. hydrophila and can boost resistance to infection against the most dangerous bacterial pathogens infecting freshwater fish.