Characterization analysis of TLR5a and TLR5b immune response after different bacterial infection in grass carp (Ctenopharyngodon idella)

Abstract

Toll-like receptor (TLR) is an important pattern recognition receptor, which specifically recognizes microbial components, and TLR5 recognizes bacterial flagellin in vertebrates and invertebrates. In this study, two forms of TLR5 (TLR5a and TLR5b) were identified in grass carp (Ctenopharyngodon idella). Aeromonas hydrophila and Staphylococcus aureus were used to investigate the role of grass carp TLR5a and TLR5b against bacteria (flagellate and non-flagellate) in innate immunity, and the expression of TLR5a and TLR5b genes and proteins were detected in immune-related tissues. Quantitative real-time polymerase chain reaction results showed that TLR5a and TLR5b genes of grass carp were highly expressed in the liver, spleen, and head kidney, and their expression patterns were similar in tissues. Meanwhile, the TLR5b gene expression was higher than TLR5a in most tissues. Following exposure to A. hydrophila and S. aureus, the expression levels of TLR5a and TLR5b genes in the liver, spleen, and head kidney were up-regulated significantly. Moreover, the downstream gene, NF-κB, was up-regulated significantly. After A. hydrophila infection, the expression of TLR5a gene was up-regulated in the liver and spleen at 24 h, while TLR5b was up-regulated at 6 h. In the head kidney, TLR5a was up-regulated at 6 h, while TLR5b was up-regulated at 6 h and 12 h. After S. aureus infection, TLR5a and TLR5b were up-regulated at 6 h in the liver and 12 h in the spleen. However, in the head kidney, TLR5a was down-regulated, while TLR5b was up-regulated. Compared with TLR5a, TLR5b had a higher expression level and stronger response to pathogen stimulation. The immunofluorescence results showed that TLR5a and TLR5b proteins in the liver of grass carp infected with A. hydrophila and S. aureus were similar but different in the spleen and head kidney. The results indicated that TLR5a and TLR5b play a critical role in resisting bacterial infection, and TLR5a and TLR5b had obvious tissue and pathogen specificity. TLR5b may play a major role in immune tissues, while TLR5a may play an auxiliary regulatory role in early infection. In addition, TLR5a and TLR5b have an irreplaceable regulatory role in response to flagellate and non-flagellate bacteria. This lays a foundation to explore further the role of TLR5 in resisting flagellate and non-flagellate infections in fish and provides a reference for the innate immunity research of grass carp.

Keywords: Aeromonas hydrophila; Gene expression; Grass carp; Immunofluorescence; Innate immunity; Staphylococcus aureus.