Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

Abstract

Recent evidence suggests that several cattle breeds may be more resistant to infection with the zoonotic pathogen Mycobacterium bovis. Our data presented here suggests that the response to mycobacterial antigens varies in macrophages generated from Brown Swiss (BS) and Holstein Friesian (HF) cattle, two breeds belonging to the Bos taurus family. Whole genome sequencing of the Brown Swiss genome identified several potential candidate genes, in particular Toll-like Receptor-2 (TLR2), a pattern recognition receptor (PRR) that has previously been described to be involved in mycobacterial recognition. Further investigation revealed single nucleotide polymorphisms (SNP) in TLR2 that were identified between DNA isolated from cells of BS and HF cows. Interestingly, one specific SNP, H326Q, showed a different genotype frequency in two cattle subspecies, Bos (B.) taurus and Bos indicus. Cloning of the TLR2 gene and subsequent gene-reporter and chemokine assays revealed that this SNP, present in BS and Bos indicus breeds, resulted in a significantly higher response to mycobacterial antigens as well as tri-acylated lipopeptide ligands in general. Comparing wild-type and H326Q containing TLR2 responses, wild-type bovine TLR2 response showed clear, diminished mycobacterial antigen responses compared to human TLR2, however bovine TLR2 responses containing H326Q were found to be partially recovered compared to human TLR2. The creation of human:bovine TLR2 chimeras increased the response to mycobacterial antigens compared to the full-length bovine TLR2, but significantly reduced the response compared to the full-length human TLR2. Thus, our data, not only present evidence that TLR2 is a major PRR in the mammalian species-specific response to mycobacterial antigens, but furthermore, that there are clear differences between the response seen in different cattle breeds, which may contribute to their enhanced or reduced susceptibility to mycobacterial infection.

Prospects of toll‐like receptors in dairy cattle breeding

“…However, as different geographic and microbial environments may require a different immune response, studies should focus not only on high-producing dairy breeds but also on genetic variants in the immune response of traditional unselected breeds (Jann et al, 2009;Novak, 2014). It would also be interesting to identify alleles that may represent the 'wild-type' and compare them to alleles in high-producing dairy cattle (especially Holstein Friesian) to see whether intensive genetic selection for milk production resulted in an immunologically less-fitter status (Bartens et al, 2021). Bartens et al (2021) described the H326Q mutation (rs68343167), which results in an amino acid change of the positively charged histidine (H) to the uncharged glutamine (Q) at position 326.…”

“…It would also be interesting to identify alleles that may represent the 'wild-type' and compare them to alleles in high-producing dairy cattle (especially Holstein Friesian) to see whether intensive genetic selection for milk production resulted in an immunologically less-fitter status (Bartens et al, 2021). Bartens et al (2021) described the H326Q mutation (rs68343167), which results in an amino acid change of the positively charged histidine (H) to the uncharged glutamine (Q) at position 326. The cells containing the H326Q mutation showed significantly increased NF-κB activity and TLR2 signalling strength.…”

“…TLRs are transmembrane proteins characterised by an extracellular N‐terminal ectodomain containing leucine‐rich repeats (LRR) involved in PAMPs recognition, a transmembrane domain, and an intracellular C‐terminal domain known as the Toll/interleukin‐1 receptor (TIR) domain, that initiates downstream signalling (Bartens et al, 2021; Kaisho & Akira, 2006; Kawai & Akira, 2010; Kawasaki & Kawai, 2014; Ruiz‐Larrañaga et al, 2011; Sharma et al, 2015). The ectodomain displays a horseshoe‐like structure (Kawasaki & Kawai, 2014; Mucha et al, 2009), and the ligands, depending on their category, bind to both the concave and convex side of the horseshoe (Novak, 2014).…”

“…The variability of LRR motifs in TLRs contributes to the specificity of ligand binding (Novak, 2014), and irregularities, such as mutations, may affect PAMP binding onto the TLR horseshoe (Mucha et al, 2009). The inner (usually intracellular) C‐terminal region of TLR molecules, the TIR domain, is responsible for the signal transduction between TLR and downstream proteins or kinases (Bai et al, 2022; Kaisho & Akira, 2006) and, together with the transmembrane domain, is highly conserved compared to the ectodomain (Bartens et al, 2021; Novak, 2014).…”

“…Toll‐like receptors (TLRs) are a structurally conserved type I membrane‐bound class of pattern recognition receptor that can differentiate between a wide range of PAMPs (Blagitz et al, 2015; Russell et al, 2012). Ten functional bovine TLRs (TLR1 to TLR10) have been identified and mapped in cattle, with each TLR evolving to recognise specific PAMPs (Bartens et al, 2021; Elmagharaby et al, 2018; Mišeikienė et al, 2020; Russell et al, 2012).…”