Sustainable Arabidopsis-based Endotoxin Assay

Overview

Harvesting Limulus polyphemus (horseshoe crabs) to produce the Limulus amoebocyte lysate endotoxin assay for medical devices, pharmaceuticals, and drinking water is ravaging coastal ecosystems. This project develops a more sustainable and cost-effective quantitative endotoxin assay. The PAMP-Triggered Immunity response of Arabidopsis thaliana to pathogen-associated molecular patterns (PAMPs) was harnessed for quantitative determination of endotoxin presence based on induction of the FRK1 gene. Transgenic FRK1-LUC A. thaliana were used to express luciferase (LUC) upon activation of FRK1 by exposure to gram-negative bacteria. Luciferase enzyme control was first tested via plate reader, and luminescence produced by varying enzyme quantities was recorded. Next, E. coli ranging from 6*10^5 to 10^3 CFU/mL were infiltrated into the leaf apoplastic space of FRK1-LUC and wild-type plants. Luminescence of infiltrated leaf discs was measured after adding luciferin substrate to reconstitute functional luciferase. The results showed a direct relationship between bacteria concentration and luminescence. The FRK1-LUC luminescence versus endotoxin concentration results yielded a formula of y = 1518e^0.0196x. (R^2 = 0.937). Data suggest this assay achieves a sensitivity down to 18 endotoxin units/mL (p < 0.001, SEM = 1.76%). To determine specificity, the SeeSAR software was used to calculate binding affinities of endotoxin (LPS) and flg22 with several receptors. Results indicate high specificity in LORE-LPS binding, signifying the luminesce results were caused by LPS concentration. This method’s sensitivity and specificity combined with its elimination of environmental impacts and low cost make it a promising new bacterial endotoxin assay for pharmaceutical and drinking water testing.