Transcriptional Response of T. pyogenes to Luteolin

T. pyogenes ATCC19411, BM07-1, and BMH06-3 were cultured on blood plates for 36 h and suspended in NB medium (Solarbio) containing 8% FBS to the logarithmic phase. The T. pyogenes suspension was diluted to 1 × 10⁵ CFU/mL and cultured with luteolin (final concentrations: 1/8 MIC, 1/4 MIC, and 1/2 MIC). The bacterial dilutions were mixed with the same final concentration of DMSO as a control. Fifty milliliters of bacterial suspension were incubated at 37 °C for 36 h, and bacterial cells were collected by centrifugation at 10,000× g and 4 °C. Total RNA of bacterial cells was extracted by TRIzol Reagent (Ambion, Carlsbad, CA, USA), and cDNA was reverse-transcribed using TransScript^®II All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Then, the transcription level of tatD genes was measured using quantitative Real-Time PCR (qPCR), as described by Guo et al. [24 (link)]. The relative expression quantities of tatD genes were normalized against 16S rRNA, and primer sequences are shown in Table 1. The optimum concentration of luteolin for treating T. pyogenes (ATCC 19411, BM07-1 and BMH06-3) was determined and the other T. pyogenes strains were treated with that concentration of luteolin. Finally, the expression of tatD genes was examined using qPCR in the same manner.