Streptolysin S induces pronounced calcium-ion influx-dependent expression of immediate early genes encoding transcription factors

Abstract

Anginosus group streptococci (AGS) are opportunistic human pathogens of the oral cavity. The β-hemolytic subgroup of Streptococcus anginosus subsp. anginosus secretes streptolysin S (SLS) and exhibits not only hemolytic activity but also cytotoxicity toward cultured human cell lines. However, the detailed mechanism of action of SLS and the cellular responses of host cells have not yet been fully clarified. To determine the pathogenic potential of SLS-producing β-hemolytic S. anginosus subsp. anginosus, the SLS-dependent response induced in the human oral squamous cell carcinoma HSC-2 cells was investigated to determine the pathogenic potential of SLS-producing β-hemolytic S. anginosus subsp. anginosus. This study revealed that the Ca²⁺ influx and the expression of immediate early genes (IEGs) encoding transcription factors such as early growth responses (EGRs) and activator protein-1 (AP-1) were greatly increased in HSC-2 cells incubated with the culture supernatant of SLS-producing β-hemolytic S. anginosus subsp. anginosus. Moreover, this SLS-dependent increase in expression was significantly suppressed by Ca²⁺ chelation, except for jun. These results suggest that SLS caused Ca²⁺ influx into the cells following greatly enhanced expression of IEG-encoding transcription factors. The results of this study may help in understanding the pathogenicity of SLS-producing AGS.

Figure 1

SLS-dependent cytotoxicity toward the human oral squamous cell carcinoma cell line HSC-2. The viability of HSC-2 cells incubated with the culture supernatant prepared from S. anginosus subsp. anginosus strain NCTC10713^T or its sagA-genes (sagA1 and sagA2) deletion mutant (∆sagAs) was evaluated by CCK-8 assay (Dojindo) (a) and by CellTox™ Green assay (Promega) (b). The “w/o” indicates HSC-2 cells incubated with the co-cultivation medium as the control. Triplicate samples were assayed, and the result is presented as the mean ± SD. The significance of the SLS-dependent cytotoxicity was evaluated by Students’ t-test (**p < 0.01, n.s. not significant). a.u. arbitrary unit.

Figure 2

SLS-dependent influx of extracellular Ca²⁺ into HSC-2 cells. The influx of Ca²⁺ into HSC-2 cells incubated with the culture supernatant prepared from the tested strains was evaluated using Fura-2 Calcium Kit (Dojindo). HSC-2 cells treated with 0.1 μM ionomycin were used as the Ca²⁺-permeabilized control for this assay. The “w/o” indicates HSC-2 cells incubated with the co-cultivation medium as the control without cultivating the tested strains. Triplicate samples were assayed, and the result is presented as the mean ± SD. The significance of the SLS-dependent increase of Ca²⁺ influx was evaluated by Students’ t-test (**p < 0.01, n.s. not significant).

Figure 3

Volcano plot of the SLS-dependent induction (magenta) and reduction (cyan) of the gene expression in HSC-2 cells incubated with the culture supernatant prepared from S. anginosus subsp. anginosus strain NCTC10713^T. The SLS-dependent induction or reduction of the expression of genes in HSC-2 cells was evaluated using the results of RNA-seq analysis outsourced to Macrogen Japan. The genes focused on in this study are indicated with black symbols (IEGs) and green symbols (cytokine genes) with the product name.

Figure 4

SLS-dependent and Ca²⁺-influx-induced expression of the egr genes, egr1 (a), egr2 (b), egr3 (c), and egr4 (d). HSC-2 cells were incubated with the culture supernatant prepared from S. anginosus subsp. anginosus strain NCTC10713^T or its sagA-genes (sagA1 and sagA2) deletion mutant (∆sagAs). The contribution of Ca²⁺ influx to the expression of the genes was also evaluated in the presence of 5 mM EGTA. Triplicate samples were assayed, and the representative result is presented as the mean ± SD. These results were described as the relative expression against the control sample of HSC-2 cells incubated with co-cultivation medium. The significance of the increase of SLS-dependent and Ca²⁺-influx-dependent expression was evaluated by Welch’s or Students’ t-test (**p < 0.01, *p < 0.05).

Figure 5

SLS-dependent and Ca²⁺-influx-induced expression of fos (a), fosB (b), jun (c), junB (d), and junD (e). HSC-2 cells were incubated with the culture supernatant prepared from S. anginosus subsp. anginosus strain NCTC10713^T or its sagA-genes (sagA1 and sagA2) deletion mutant (∆sagAs). The contribution of Ca²⁺ influx to the expression of the genes was also evaluated in the presence of 5 mM EGTA. Triplicate samples were assayed, and the representative result is presented as the mean ± SD. These results were described as the relative expression against the control sample of HSC-2 cells incubated with co-cultivation medium. The significance of the increase of SLS-dependent and Ca²⁺-influx-dependent expression was evaluated by Welch’s or Students’ t-test (**p < 0.01, *p < 0.05, n.s. not significant).

Figure 6

SLS-dependent and Ca²⁺-influx-induced expression of the genes encoding IL-6 (a) and CXCL8 (b). HSC-2 cells were incubated with the culture supernatant prepared from S. anginosus subsp. anginosus strain NCTC10713^T or its sagA-genes (sagA1 and sagA2) deletion mutant (∆sagAs). The contribution of Ca²⁺ influx to the expression of the genes was also evaluated in the presence of 5 mM EGTA. Triplicate samples were assayed, and the representative result is presented as the mean ± SD. These results were described as the relative expression against the control sample of HSC-2 cells incubated with co-cultivation medium. The significance of the increase of SLS-dependent and Ca²⁺-influx-dependent expression was evaluated by Welch’s or Students’ t-test (**p < 0.01).