Toll-like Receptor Expression in Pelodiscus sinensis Reveals Differential Responses after Aeromonas hydrophila Infection

<b>Background</b>: Toll-like receptor (TLR), as an important pattern recognition receptor, is a bridge between non-specific immunity and specific immunity, and plays a vital role in the disease resistance of aquatic animals. However, the function of TLR in <i>Pelodiscus sinensis</i> is still unclear. <b>Methods and Results</b>: The sequence characteristics and homology of three TLRs <i>(PsTLR2</i>, <i>PsTLR3</i> and <i>PsTLR5</i>) were determined in this investigation. Their annotation and orthologies were supported by phylogenetic analysis, functional domain prediction, and sequence similarity analysis. qPCR showed that the identified TLRs were expressed in all tissues, among the high expression of <i>PsTLR5</i> in the brain and liver and the high expression of <i>PsTLR2</i> and <i>PsTLR3</i> in the liver. <i>PsTLR2</i> mRNA expression increased 6.7-fold in the liver 12 h after <i>Aeromonas hydrophila</i> infection, while the mRNA expression of <i>PsTLR3</i> was down-regulated by 0.29 times in liver and 0.31 times in spleen. The mRNA expression of <i>PsTLR5</i> was significantly up-regulated in four immune tissues, and it was up-regulated by 122.8 times in the spleen after 72 h infection. Finally, the recombinant proteins of extracellular LRR domains of these three TLRs were obtained by prokaryotic expression technology, and the binding tests were performed to discover their ability of binding pathogenic microorganisms. Microbial binding test showed that rPsTLR2, rPsTLR3 and rPsTLR5 can combine <i>A. hydrophila</i>, <i>Edwardsiella tarda</i>, <i>Vibrio parahaemolyticus</i>, <i>Staphylococcus aureus</i>, <i>Streptococcus agalactiae</i> and <i>Candida albicans</i>, while rPsTLR3 can bind <i>A. hydrophila</i>, <i>E. tarda</i>, <i>V. parahaemolyticus</i> and <i>C. albicans</i>. <b>Conclusions</b>: Our findings suggested that TLRs may be crucial to turtles' innate immune response against microbes.