Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed 1 and Species-Specific Innate Immune Functionality 2 3

Abstract

17 18 Recent evidence suggests that several cattle breeds may be more resistant to infection with the zoonotic 19 pathogen Mycobacterium bovis. Our data presented here suggests that the response to mycobacterial 20 antigens varies in macrophages generated from Brown Swiss (BS) and Holstein Frisian (HF) cattle, two 21 breeds belonging to the Bos taurus family. Whole genome sequencing of the Brown Swiss genome 22 identified several potential candidate genes, in particular Toll-like Receptor-2 (TLR2) a pattern 23 recognition receptor (PRR) that has previously been described to be involved in mycobacterial 24 recognition. Further investigation revealed single nucleotide polymorphisms (SNP) in TLR2 that were 25 identified between DNA isolated from cells of BS and HF cows. Interestingly, one specific SNP, 26 H326Q, showed a different genotype frequency in two cattle subspecies, Bos (B.) taurus and Bos 27 indicus. Cloning of the TLR2 gene and subsequent gene-reporter and chemokine assays revealed that 28 this SNP, present in BS and Bos indicus breeds, resulted in a significantly higher response to 29 mycobacterial antigens as well as tri-acylated lipopeptide ligands in general. Comparing wild-type and 30 H326Q containing TLR2 responses, wild-type bovine TLR2 response showed clear, diminished 31 mycobacterial antigen responses compared to human TLR2, however bovine TLR2 responses 32 containing H326Q were found to be partially recovered compared to human TLR2. The creation of 33 human:bovine TLR2 chimeras increased the response to mycobacterial antigens compared to the full-34 length bovine TLR2, but significantly reduced the response compared to the full-length human TLR2. 35 Thus, our data, not only present evidence that TLR2 is a major PRR in the mammalian species-specific 36 response to mycobacterial antigens, but furthermore, that there are clear differences between the 37 response seen in different cattle breeds, which may contribute to their enhanced or reduced 38 susceptibility to mycobacterial infection. 39 40 41