# Molecular Cloning and Expression Responses to Streptococcus agalactiae and Aeromonas veronii of TLR19, TLR20, and TLR21 in Schizothorax prenanti

## TL;DR

This study identifies and characterizes three TLRs in Schizothorax prenanti, showing their role in immune responses to bacterial infections.

## Contribution

The study reports the molecular cloning and expression analysis of TLR19, TLR20, and TLR21 in a cold-water fish species.

## Key findings

- The three TLRs (TLR19, TLR20, TLR21) in S. prenanti have conserved domain structures typical of TLRs.
- Expression of these TLRs increased significantly in response to bacterial challenges in immune-related tissues.
- Phylogenetic analysis shows spTLR19 and spTLR20 are closely related to Cyprinus carpio homologues.

## Abstract

As essential pattern recognition receptors of the innate immune system, Toll-like receptors (TLRs) are critical for pathogen recognition in teleosts. The transitional cold-water fish Schizothorax prenanti, distributed from low- to high-altitude regions, serves as an important species for elucidating immune mechanisms in cold-water fish and adaptations to environments with altitude variation. Three TLRs (TLR19, TLR20, TLR21) were identified from S. prenanti, each possessing the conserved domain architecture typical of TLRs and involved in antibacterial immunity. These findings enhance understanding of TLR functions in teleost innate immunity and provide a foundation for elucidating disease resistance and immune regulation in S. prenanti.

Toll-like receptors (TLRs) are essential pattern recognition receptors of the innate immune system and play critical roles in pathogen invasion in teleosts. In this study, we identified and characterized full-length open reading frames of three TLRs belonging to the TLR11 subfamily from Schizothorax prenanti, termed spTLR19 (2868 bp), spTLR20 (2835 bp), and spTLR21 (2946 bp), encoding 955, 944, and 981 amino acids, respectively. All three proteins exhibited the conserved domain architecture typical of TLRs, comprising a leucine-rich repeat (LRR) domain, a transmembrane region, and a Toll/IL-1 receptor (TIR) domain. Phylogenetic and homology analyses revealed that spTLR19 and spTLR20 clustered most closely with their homologues from Cyprinus carpio, while spTLR21 showed the highest similarity to Onychostoma macrolepis TLR21. Expression profiling showed that these TLRs were ubiquitously expressed across examined tissues, with relatively higher expression in immune-related tissues such as spleen and gills. Furthermore, challenge with Streptococcus agalactiae and Aeromonas veronii significantly up-regulated the expression of spTLR19, spTLR20, and spTLR21 in spleen, liver, and gills, suggesting their involvement in antibacterial immune responses. These findings enhance the functional understanding of the teleost TLR11 subfamily and provide a foundation for elucidating disease resistance and immune regulation in S. prenanti.