Functional divergence of TLR5 paralogs reveals distinct roles for TLR5M and TLR5S in salmonid immunity

Sensing of pathogen-associated molecular patterns (PAMPs) is a critical step in mounting effective immune responses against invading pathogens. Toll-like receptor 5 (TLR5) is central to this process through its recognition of bacterial flagellin. While the function of the canonical membrane-bound form (TLR5M) appears conserved across vertebrate species, gene duplication events have given rise to distinct TLR5 variants in some taxa. Notably, a soluble form (TLR5S), lacking transmembrane and TIR domains, has been identified. In this study, CRISPR/Cas9-mediated gene editing was employed in both cell lines and brown trout to generate TLR5M and TLR5S knockout models. Functional characterisation of knockout fish demonstrates that TLR5M serves as the primary receptor for bacterial flagellin in both a rainbow trout gonad cell line (RTG-2) and brown trout and is capable of mediating flagellin-induced immune responses independently of TLR5S. In contrast, disruption of TLR5S did not diminish flagellin-induced responses; rather, enhanced expression of certain immune genes was observed in TLR5S-deficient trout. Additionally, disruption of TLR5S, alone or in combination with TLR5M knockout, led to dysregulation of cytosolic inflammasome components in brown trout, suggesting a potential link between TLR5S and inflammasome signalling pathways. Collectively, these findings challenge previous assumptions of a synergistic role for TLR5S in flagellin recognition and instead support an alternative model in which TLR5S may negatively modulate immune responses under certain conditions.