Duplicated TLR5 of zebrafish functions as a heterodimeric receptor

Abstract

Toll-like receptor 5 (TLR5) of mammals, birds, and reptiles detects bacterial flagellin and signals as a homodimeric complex. Structural studies using truncated TLR5b of zebrafish confirm the homodimeric TLR5-flagellin interaction. Here we provide evidence that zebrafish (Danio rerio) TLR5 unexpectedly signals as a heterodimer composed of the duplicated gene products drTLR5b and drTLR5a. Flagellin-induced signaling by the zebrafish TLR5 heterodimer increased in the presence of the TLR trafficking chaperone UNC93B1. Targeted exchange of drTLR5b and drTLR5a regions revealed that TLR5 activation needs a heterodimeric configuration of the receptor ectodomain and cytoplasmic domain, consistent with ligand-induced changes in receptor conformation. Structure-guided substitution of the presumed principal flagellin-binding site in human TLR5 with corresponding zebrafish TLR5 residues abrogated human TLR5 activation, indicating a species-specific TLR5-flagellin interaction. Our findings indicate that the duplicated TLR5 of zebrafish underwent subfunctionalization through concerted coevolution to form a unique heterodimeric flagellin receptor that operates fundamentally differently from TLR5 of other species.

Keywords: TLR5; flagellin; heterodimer; subfunctionalization; zebrafish.

Fig. 1.

Induction of NF-κB by zebrafish TLR5b (drTLR5b) and drTLR5a upon stimulation with purified flagellins and bacterial lysates. (A and C–H) HeLa-57A cells were transfected with drTLR5b, drTLR5a, hTLR5 or drTLR5b and drTLR5a combined as indicated. Control cells were transfected with empty vector. Cells were stimulated (5 h) with vehicle (–) or 1 µg mL⁻¹ of purified recombinant FliC^SE or FliC^PA flagellin (A, C, E, and G) or 2 µg mL⁻¹ of total protein from lysates of S. Enteritidis, P. aeruginosa,or A. hydrophila (D, F, and H). Note that the NF-κB response of the drTLR5 heterodimer is higher when stimulated with bacterial lysate compared with purified recombinant flagellin. (B) Fluorescence microscopy of empty vector or FLAG-tagged drTLR5b transfected HeLa-57A cells stained with M2 α-FLAG and DAPI for nuclear visualization. (White scale bars: 10 µm.) NF-κB activity is represented by luciferase activity in RLUs. Values are the mean ± SEM of three independent experiments (A, C, E, and G) or a representative of three independent experiments (D, F, and H) all performed in duplicate.

Fig. 2.

Effect of zebrafish UNC93B1 (drUNC93B1) on localization of drTLR5a and drTLR5b. Confocal microscopy on HeLa-57A cells transfected with (A) drTLR5-HA and drTLR5b-FLAG or (B) drTLR5a-HA and drTLR5b-FLAG and untagged drUNC93B1. Merge images show nuclei stained with DAPI (blue). White boxes in merge images indicate the magnified area shown for each channel on the right of merge images. Images were selected from three independent experiments, and three representative images are shown for each transfected group. (Scale bars in merge images: 10 µm.)

Fig. 3.

Effect of drUNC93B1 on drTLR5a- and drTLR5b-mediated NF-κB activation. HeLa-57A cells expressing drUNC93B1, drTLR5a, and/or drTLR5b in the indicated combinations were stimulated (5 h) with vehicle (–) or 1 µg mL⁻¹ FliC^SE. Data shows NF-κB activity represented by luciferase activity in RLUs. Values are the mean ± SEM of three independent experiments performed in duplicate.

Fig. 4.

Functionality of the N-terminal part of the ectodomain of drTLR5b and drTLR5a. (A) Schematic representation of WT and chimeric TLR5 constructs. N-ECD indicates the N-terminal part of the ectodomain ranging from the NTLRR to LRR14; C-ECD indicates the C-terminal part of the ectodomain ranging from LRR15 to the CTLRR. (B and C) HeLa-57A cells transfected with drUNC93B1 and the indicated receptor combinations were stimulated (5 h) with vehicle (–) or 1 µg mL⁻¹ FliC^SE. Data show NF-κB activity represented by luciferase activity in RLUs. Values are the mean ± SEM of three independent experiments performed in duplicate.

Fig. 5.

Structural modeling and functionality of the flagellin-binding hotspot in hTLR5, drTLR5a, and drTLR5b. (A) Superposition of unbound drTLR5b (PDB ID: 3v44; purple) and a model of drTLR5a based on 3v44 (drTLR5a-3v44; orange). The red arrow indicates the flagellin-binding hotspot that forms a loop between LRR9 and LRR10. (B) Alignment of the putative flagellin-binding hotspot of hTLR5 and drTLR5a with drTLR5b. Purple-colored residues in drTLR5b are involved in flagellin binding (see ref. 19). (C) Superposition of unbound drTLR5b (PDB ID: 3v44; purple) and a model of hTLR5 based on 3v44 (hTLR5-3v44; green). (D) HeLa-57A cells transfected with WT hTLR5 (hTLR5 WT), hTLR5 containing the hotspot of drTLR5a (hTLR5-hs5a), or the hotspot of drTLR5b (hTLR5-hs5b) were stimulated (5 h) with vehicle (–) or 1 µg mL⁻¹ FliC^SE. Data show NF-κB activity represented by luciferase activity in RLUs. Values are the mean ± SEM of three independent experiments performed in duplicate.

Fig. 6.

Heterodimerization of the ectodomain of drTLR5a and drTLR5b. (A) Schematic representation of WT and chimeric TLR5 constructs. N-ECD indicates the N-terminal part of the ectodomain ranging from NTLRR to LRR14. C-ECD indicates the C-terminal part of the ectodomain ranging from LRR15 to the CTLRR. (B–D) HeLa-57A cells transfected with drUNC93B1 and the indicated receptor combinations were stimulated (5 h) with vehicle (–) or 1 µg mL⁻¹ FliC^SE. Data show NF-κB activity represented by luciferase activity in RLUs. Values are the mean ± SEM of three independent experiments performed in duplicate.

Fig. 7.

Heterodimerization of the ICD of drTLR5a and drTLR5b. (A) Schematic representation of WT and chimeric TLR5 constructs. (B–D) HeLa-57A cells transfected with drUNC93B1 and the indicated receptor combinations were stimulated (5 h) with vehicle (–) or 1 µg mL⁻¹ FliC^SE. Data show NF-κB activity represented by luciferase activity in RLUs. Values are the mean ± SEM of three independent experiments performed in duplicate.