Molecular characterization and expression analysis of four toll-like receptors (TLR) genes: TLR2, TLR5S, TLR14 and TLR22 in Mastacembelus armatus under Aeromonas veronii infection

Abstract

Toll-like receptors (TLRs) are important pattern recognition receptors that can recognize pathogen-associated molecular patterns (PAMPs), which in turn activate immune cells in vivo to perform immune functions. In this study, two TLR1 subfamily members (TLR2 and TLR14), one TLR5 subfamily member (TLR5S) and one TLR11 subfamily member (TLR22) genes were first identified in Mastacembelus armatus. In MaTLR2, MaTLR14 and MaTLR22, three main domains were identified, including leucine-rich repeat (LRR) domain, a transmembrane domain (TM) and an intracellular Toll/IL-1 receptor (TIR) domain as in most TLRs with no TM domain and TIR domain in MaTLR5S. The tissue expression of MaTLR2, MaTLR5S, MaTLR14 and MaTLR22 showed that they were ubiquitously expressed in all tested tissues. After infection with Aeromonas veronii, expression of MaTLR2, MaTLR14 and MaTLR22 was all downregulated in spleen and liver, but upregulated in kidney. By contrast, MaTLR5S showed upregulation in all three organs after bacterial infection. The amino acid sequence identity of MaTLR2 with other teleosts varied from 64.7% to 76.8%, while MaTLR5S exhibited a range of 70.9%–75.9%. MaTLR14 demonstrated a higher degree of similarity, with sequence identities ranging from 74% to 83.3%. In contrast, MaTLR22 showed the most variability, with sequence identities spanning from 42.5% to 74.6%. Phylogenetic analysis of TLR genes of M. aramatus clustered with Perciformes fishes, which aligned with traditional taxonomic classifications. The site model of PAML was used to detect the robust positively selected sites in extant teleost fishes. In total, 20, 12, 19 and 6 sites in the subsets of teleost TLR2, TLR5S, TLR14 and TLR22 were separately identified, indicating that the teleost TLR2, TLR5S, TLR14 and TLR22 had been subject to positive selection pressures. Our results will lay a good function for better understanding the potential function of TLRs in antibacterial process and their co-evolution with pathogens.