Quantitation of endotoxin and lipoteichoic acid virulence using toll receptor reporter gene.

PURPOSE: To apply quantitative Toll-like receptors (TLR) cell assays to compare lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs) from different oral bacterial strains for potential pathogenicity in vitro.

METHODS: The potency of LPS and LTA from different bacteria on activation of TLR reporter genes in HEK-tlr cell lines was examined. P. gingivalis LPS mix, P. gingivalis 1690 LPS, P. gingivalis 1435/50 LPS, E. coli LPS (E. coli K12), B. subtilis LTA, S. aureus LTA, E. hirae LTA and S. pyogenes LTA were examined in both TLR2 and TLR4 HEK cell line reporter assays. Solutions of LPS and LTA from selected bacteria were applied in a dose response fashion to the TLR reporter cells under standard culture conditions for mammalian cells. Reporter gene secreted-embryonic-alkaline-phosphatase (SEAP) was measured, and half maximal effective concentration (EC50) was determined for each sample. Concentration dependent TLR activation was compared to similar responses to LPS and LTA for commercial BODIPY-TR-Cadaverine and LAL biochemical (non cell based) assays.

RESULTS: All LPS from P. gingivalis activated both TLR2 and TLR4 responses. E. coli LPS is a strong activator for TLR4 but not for TLR2 responses. In contrast, both B. subtilis and S. aureus LTA provoked responses only in TLR2, but not in the TLR4 assay. Interestingly, E. hirae LTA and S. pyogenes LTA did not stimulate strong TLR2 responses. Instead, both E. hirae LTA and S. pyogenes LTA mounted a reasonable response in TLR4 reporter gene assay. Both LPS and LTA showed deactivation of fluorescence in BODIPY-TR-Cadaverine while only LPS was active in LAL. As with biochemical assays, an EC50 could be determined for LPS and LTA from various bacterial strains. The EC50 is defined as a concentration of LPS or LTA that provokes a response halfway between the baseline and maximum responses. Lower EC50 means higher potency in promoting TLR responses, and in principle indicates greater toxicity to the host.

CLINICAL SIGNIFICANCE: InvivoGen TLR2 and TLR4 assays distinguish specific types of microbial products, such as LPS and LTA from different bacteria. Application of EC50 determinations creates a means for quantitative and comparisons of LPS and LTA virulence in a cellular-based assay and combinations of TLR reporter cell assays along with biochemical evaluation of LPS#47;LTA in BODIPY-TR-Cadaverine and LPS in LAL assays provides a means to quantitate virulence of plaque samples with respect to both LPS and LTA. These learnings have long-term implications for patient care in that understanding the virulence of patients' plaque provides important information to assess risk of oral diseases.