Functional expression of TLR5 of different vertebrate species and diversification in intestinal pathogen recognition

Abstract

Toll-like receptor 5 (TLR5) is activated by bacterial flagellins and plays a crucial role in the first-line defence against pathogenic bacteria and in immune homeostasis, and is highly conserved in vertebrate species. However, little comparative information is available on TLR5 functionality. In this study, we compared TLR5 activation using full-length and chimeric TLR5 of various vertebrate species (human, chicken, mouse, pig, cattle). Chimeric TLR5 receptors, consisting of human transmembrane and intracellular domains, linked to extracellular domains of animal origin, were generated and expressed. The comparison of chimeric TLR5s and their full-length counterparts revealed significant functional disparities. While porcine and chicken full-length TLR5s showed a strongly reduced functionality in human cells, all chimeric receptors were functional when challenged with TLR5 ligand Salmonella FliC. Using chimeric receptors as a tool allowed for the identification of ectodomain-dependent activation potential and partially host species-specific differences in response to various enteric bacterial strains and their purified flagellins. We conclude that both the extra- and intracellular determinants of TLR5 receptors are crucial for compatibility with the species expression background and hence for proper receptor functionality. TLR5 receptors with a common intracellular domain provide a useful system to investigate bacteria- and host-specific differences in receptor activation.

Physical Extraction and Fast Protein Liquid Chromatography for Purifying Flagella Filament From Uropathogenic Escherichia coli for Immune Assay

“…Several proinflammatory cytokines, including TNF-α, IL-1β, IL-6, KC, and IL-12 were induced in response to the purified FliC in a manner consistent with previous literature (Wyant et al, 1999; Eaves-Pyles et al, 2001; Hayashi et al, 2001; Moors et al, 2001; Kinnebrew et al, 2012). We also observed significant responses to lower doses of FliC (e.g., 0.05 μg), a finding consistent with prior studies that have reported activation of innate immune responses, including NF-κβ, TLR5, and host antimicrobial peptides following exposure to this level of FliC (Smith et al, 2003; Faber et al, 2018; Mowbray et al, 2018). We note that our use of a fliC mutant in these assays reflects a crucial control to conclude that the proinflammatory responses induced by FliC are not an artifact secondary to LPS contamination, which is an important consideration in studies of this type (Eaves-Pyles et al, 2001).…”

Toxoplasma gondii profilin does not stimulate an innate immune response through bovine or human TLR5

“…22 This was not examined any further as part of this investigation. However, similar data regarding poTLR5 expression in HEK cells were published recently, 32 indicating that poTLR5 is non-responsive in HEK cells, despite the fact that the main adaptor, MyD88, shares a 94% homology between human and porcine MyD88 (Yamakawa and Werling, unpublished data).…”

Contribution of Heptose Metabolites and the cag Pathogenicity Island to the Activation of Monocytes/Macrophages by Helicobacter pylori

“…To quantitate luciferase in NF-κB reporter cells, the Steady-Glo Luciferase Assay System (Promega) was used. Briefly, the lysis buffer-substrate mixture of the system was added to each well and the lysis was allowed to proceed as recommended by the manufacturer’s instructions [see also ( 54 )]. Lysed cells were analyzed for photon counts within 10 min after the lysis using a Clariostar multi-well reader (BMG Labtech) in luminescence mode (acquisition of photons for 10 s, no filter); output is quantitated as counts per second.…”