Elimination of negative feedback in TLR signalling allows rapid and hypersensitive detection of microbial contaminants.

The exquisite specificity of Toll-like receptors (TLRs) to sense microbial molecular signatures is used as a powerful tool to pinpoint microbial contaminants. Various cellular systems, from native human blood cells to transfected cell lines exploit TLRs as pyrogen detectors in biological preparations. However, slow cellular responses and limited sensitivity have hampered the replacement of animal-based tests such as the rabbit pyrogen test or lipopolysaccharide detection by Limulus amoebocyte lysate. Here, we report a novel human cell-based approach to boost detection of microbial contaminants by TLR-expressing cells. By genetic and pharmacologic elimination of negative control circuits, TLR-initiated cellular responses to bacterial molecular patterns were accelerated and significantly elevated. Combining depletion of protein phosphatase PP2ACA and pharmacological inhibition of PP1 in the optimized reporter cells further enhanced the sensitivity to allow detection of bacterial lipoprotein at 30 picogram/ml. Such next-generation cellular monitoring is poised to replace animal-based testing for microbial contaminants.