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The molecular basis for recognition of bacterial ligands at equine TLR2, TLR1 and TLR6.

Irvine KL, Hopkins LJ, Gangloff M, Bryant CE - Vet. Res. (2013)

Bottom Line: The EC50 of Pam2CSK4 was the same for equine and human TLR2/6, indicating amino acid differences between the two species' TLRs do not significantly affect ligand recognition.Molecular modelling indicates that the majority of non-conserved ligand-interacting residues are at the periphery of the TLR2 binding pocket and in the ligand peptide-interacting regions, which may cause subtle effects on ligand positioning.These results suggest that there are potentially important species differences in recognition of lipopeptides by TLR2/1, which may affect how the horse deals with bacterial infections.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB30ES, UK. ceb27@cam.ac.uk.

ABSTRACT
TLR2 recognises bacterial lipopeptides and lipoteichoic acid, and forms heterodimers with TLR1 or TLR6. TLR2 is relatively well characterised in mice and humans, with published crystal structures of human TLR2/1/Pam3CSK4 and murine TLR2/6/Pam2CSK4. Equine TLR4 is activated by a different panel of ligands to human and murine TLR4, but less is known about species differences at TLR2. We therefore cloned equine TLR2, TLR1 and TLR6, which showed over 80% sequence identity with these receptors from other mammals, and performed a structure-function analysis. TLR2/1 and TLR2/6 from both horses and humans dose-dependently responded to lipoteichoic acid from Staphylococcus aureus, with no significant species difference in EC50 at either receptor pair. The EC50 of Pam2CSK4 was the same for equine and human TLR2/6, indicating amino acid differences between the two species' TLRs do not significantly affect ligand recognition. Species differences were seen between the responses to Pam2CSK4 and Pam3CSK4 at TLR2/1. Human TLR2/1, as expected, responded to Pam3CSK4 with greater potency and efficacy than Pam2CSK4. At equine TLR2/1, however, Pam3CSK4 was less potent than Pam2CSK4, with both ligands having similar efficacies. Molecular modelling indicates that the majority of non-conserved ligand-interacting residues are at the periphery of the TLR2 binding pocket and in the ligand peptide-interacting regions, which may cause subtle effects on ligand positioning. These results suggest that there are potentially important species differences in recognition of lipopeptides by TLR2/1, which may affect how the horse deals with bacterial infections.

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Responses of human and equine TLR2/1 and TLR2/6 to LTA. SW620 cells were transiently transfected with human TLR2 (hTLR2) and human TLR1 (hTLR1), hTLR2 and human TLR6 (hTLR6), equine TLR2 (eqTLR2) and equine TLR1 (eqTLR1), or eqTLR2 and equine TLR6 (eqTLR6), together with the cognate species’ CD14 and reporter constructs NF-κB-luc and Renilla luciferase. Cells were stimulated 24 hours later with increasing doses of LTA from Staphylococcus aureus. Cells were stimulated for six hours, lysed, and analysed for luciferase activity. Data are from a representative experiment and expressed as triplicate mean ± standard error of the mean (SEM) for that experiment. (A) LTA dose-dependently stimulated human TLR2/1 and TLR2/6. Maximum stimulation of human TLR2/6 was higher than human TLR2/1. (B) LTA dose-dependently stimulated equine TLR2/1 and equine TLR2/6. Maximum stimulation of equine TLR2/6 was higher than TLR2/1. (C) Data for each curve were normalised for calculation of EC50, using medium alone as 0% and maxima for each curve (raw data) from (A) and (B) as 100%. EC50 values were not significantly different for human and equine receptor pairs, nor between equine receptor pairs. Human TLR2/1 and TLR2/6 had significantly different EC50 values.
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Figure 1: Responses of human and equine TLR2/1 and TLR2/6 to LTA. SW620 cells were transiently transfected with human TLR2 (hTLR2) and human TLR1 (hTLR1), hTLR2 and human TLR6 (hTLR6), equine TLR2 (eqTLR2) and equine TLR1 (eqTLR1), or eqTLR2 and equine TLR6 (eqTLR6), together with the cognate species’ CD14 and reporter constructs NF-κB-luc and Renilla luciferase. Cells were stimulated 24 hours later with increasing doses of LTA from Staphylococcus aureus. Cells were stimulated for six hours, lysed, and analysed for luciferase activity. Data are from a representative experiment and expressed as triplicate mean ± standard error of the mean (SEM) for that experiment. (A) LTA dose-dependently stimulated human TLR2/1 and TLR2/6. Maximum stimulation of human TLR2/6 was higher than human TLR2/1. (B) LTA dose-dependently stimulated equine TLR2/1 and equine TLR2/6. Maximum stimulation of equine TLR2/6 was higher than TLR2/1. (C) Data for each curve were normalised for calculation of EC50, using medium alone as 0% and maxima for each curve (raw data) from (A) and (B) as 100%. EC50 values were not significantly different for human and equine receptor pairs, nor between equine receptor pairs. Human TLR2/1 and TLR2/6 had significantly different EC50 values.

Mentions: SW620 cells were transiently transfected with reporter constructs and one of four receptor combinations: human TLR2+TLR1+CD14, human TLR2+TLR6+CD14, equine TLR2+TLR1+CD14 or equine TLR2+TLR6+CD14, and stimulated with increasing doses of ligand. EC50 and maximum stimulation values were determined at each heterodimer as measures of ligand potency and efficacy respectively. LTA efficacy was significantly higher at TLR2/6 than TLR2/1 for both species’ heterodimers (Figure 1A and 1B). The EC50 for LTA at human TLR2/1 (16.82 ng/mL (95% C.I. 9.82-26.49)) was significantly lower than that at human TLR2/6 (63.59 ng/mL (95% C.I. 51.47-78.56)), whereas potency at the equine TLR2 heterodimers was not significantly different (equine TLR2/1 EC50 = 27.11 ng/mL (95% C.I. 16.22-48.29); equine TLR2/6 EC50 = 39.32 ng/mL (95% C.I. 22.31-69.29); Figure 1C). The calculated EC50 values were also not significantly different between species for LTA at TLR2/1 or at TLR2/6. These findings show that S. aureus LTA activates both TLR2/1 and TLR2/6 analogously in the two species, with no clear species-specific behaviour.


The molecular basis for recognition of bacterial ligands at equine TLR2, TLR1 and TLR6.

Irvine KL, Hopkins LJ, Gangloff M, Bryant CE - Vet. Res. (2013)

Responses of human and equine TLR2/1 and TLR2/6 to LTA. SW620 cells were transiently transfected with human TLR2 (hTLR2) and human TLR1 (hTLR1), hTLR2 and human TLR6 (hTLR6), equine TLR2 (eqTLR2) and equine TLR1 (eqTLR1), or eqTLR2 and equine TLR6 (eqTLR6), together with the cognate species’ CD14 and reporter constructs NF-κB-luc and Renilla luciferase. Cells were stimulated 24 hours later with increasing doses of LTA from Staphylococcus aureus. Cells were stimulated for six hours, lysed, and analysed for luciferase activity. Data are from a representative experiment and expressed as triplicate mean ± standard error of the mean (SEM) for that experiment. (A) LTA dose-dependently stimulated human TLR2/1 and TLR2/6. Maximum stimulation of human TLR2/6 was higher than human TLR2/1. (B) LTA dose-dependently stimulated equine TLR2/1 and equine TLR2/6. Maximum stimulation of equine TLR2/6 was higher than TLR2/1. (C) Data for each curve were normalised for calculation of EC50, using medium alone as 0% and maxima for each curve (raw data) from (A) and (B) as 100%. EC50 values were not significantly different for human and equine receptor pairs, nor between equine receptor pairs. Human TLR2/1 and TLR2/6 had significantly different EC50 values.
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Figure 1: Responses of human and equine TLR2/1 and TLR2/6 to LTA. SW620 cells were transiently transfected with human TLR2 (hTLR2) and human TLR1 (hTLR1), hTLR2 and human TLR6 (hTLR6), equine TLR2 (eqTLR2) and equine TLR1 (eqTLR1), or eqTLR2 and equine TLR6 (eqTLR6), together with the cognate species’ CD14 and reporter constructs NF-κB-luc and Renilla luciferase. Cells were stimulated 24 hours later with increasing doses of LTA from Staphylococcus aureus. Cells were stimulated for six hours, lysed, and analysed for luciferase activity. Data are from a representative experiment and expressed as triplicate mean ± standard error of the mean (SEM) for that experiment. (A) LTA dose-dependently stimulated human TLR2/1 and TLR2/6. Maximum stimulation of human TLR2/6 was higher than human TLR2/1. (B) LTA dose-dependently stimulated equine TLR2/1 and equine TLR2/6. Maximum stimulation of equine TLR2/6 was higher than TLR2/1. (C) Data for each curve were normalised for calculation of EC50, using medium alone as 0% and maxima for each curve (raw data) from (A) and (B) as 100%. EC50 values were not significantly different for human and equine receptor pairs, nor between equine receptor pairs. Human TLR2/1 and TLR2/6 had significantly different EC50 values.
Mentions: SW620 cells were transiently transfected with reporter constructs and one of four receptor combinations: human TLR2+TLR1+CD14, human TLR2+TLR6+CD14, equine TLR2+TLR1+CD14 or equine TLR2+TLR6+CD14, and stimulated with increasing doses of ligand. EC50 and maximum stimulation values were determined at each heterodimer as measures of ligand potency and efficacy respectively. LTA efficacy was significantly higher at TLR2/6 than TLR2/1 for both species’ heterodimers (Figure 1A and 1B). The EC50 for LTA at human TLR2/1 (16.82 ng/mL (95% C.I. 9.82-26.49)) was significantly lower than that at human TLR2/6 (63.59 ng/mL (95% C.I. 51.47-78.56)), whereas potency at the equine TLR2 heterodimers was not significantly different (equine TLR2/1 EC50 = 27.11 ng/mL (95% C.I. 16.22-48.29); equine TLR2/6 EC50 = 39.32 ng/mL (95% C.I. 22.31-69.29); Figure 1C). The calculated EC50 values were also not significantly different between species for LTA at TLR2/1 or at TLR2/6. These findings show that S. aureus LTA activates both TLR2/1 and TLR2/6 analogously in the two species, with no clear species-specific behaviour.

Bottom Line: The EC50 of Pam2CSK4 was the same for equine and human TLR2/6, indicating amino acid differences between the two species' TLRs do not significantly affect ligand recognition.Molecular modelling indicates that the majority of non-conserved ligand-interacting residues are at the periphery of the TLR2 binding pocket and in the ligand peptide-interacting regions, which may cause subtle effects on ligand positioning.These results suggest that there are potentially important species differences in recognition of lipopeptides by TLR2/1, which may affect how the horse deals with bacterial infections.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB30ES, UK. ceb27@cam.ac.uk.

ABSTRACT
TLR2 recognises bacterial lipopeptides and lipoteichoic acid, and forms heterodimers with TLR1 or TLR6. TLR2 is relatively well characterised in mice and humans, with published crystal structures of human TLR2/1/Pam3CSK4 and murine TLR2/6/Pam2CSK4. Equine TLR4 is activated by a different panel of ligands to human and murine TLR4, but less is known about species differences at TLR2. We therefore cloned equine TLR2, TLR1 and TLR6, which showed over 80% sequence identity with these receptors from other mammals, and performed a structure-function analysis. TLR2/1 and TLR2/6 from both horses and humans dose-dependently responded to lipoteichoic acid from Staphylococcus aureus, with no significant species difference in EC50 at either receptor pair. The EC50 of Pam2CSK4 was the same for equine and human TLR2/6, indicating amino acid differences between the two species' TLRs do not significantly affect ligand recognition. Species differences were seen between the responses to Pam2CSK4 and Pam3CSK4 at TLR2/1. Human TLR2/1, as expected, responded to Pam3CSK4 with greater potency and efficacy than Pam2CSK4. At equine TLR2/1, however, Pam3CSK4 was less potent than Pam2CSK4, with both ligands having similar efficacies. Molecular modelling indicates that the majority of non-conserved ligand-interacting residues are at the periphery of the TLR2 binding pocket and in the ligand peptide-interacting regions, which may cause subtle effects on ligand positioning. These results suggest that there are potentially important species differences in recognition of lipopeptides by TLR2/1, which may affect how the horse deals with bacterial infections.

Show MeSH
Related in: MedlinePlus