Complement Inactivation Strategy of Staphylococcus aureus Using Decay-Accelerating Factor and the Response of Infected HaCaT Cells

Abstract

Staphylococcus aureus is a species of Gram-positive staphylococcus. It can cause sinusitis, respiratory infections, skin infections, and food poisoning. Recently, it was discovered that S. aureus infects epithelial cells, but the interaction between S. aureus and the host is not well known. In this study, we confirmed S. aureus to be internalized by HaCaT cells using the ESAT-6-like protein EsxB and amplified within the host over time by escaping host immunity. S. aureus increases the expression of decay-accelerating factor (CD55) on the surfaces of host cells, which inhibits the activation of the complement system. This mechanism makes it possible for S. aureus to survive in host cells. S. aureus, sufficiently amplified within the host, is released through the initiation of cell death. On the other hand, the infected host cells increase their surface expression of UL16 binding protein 1 to inform immune cells that they are infected and try to be eliminated. These host defense systems seem to involve the alteration of tight junctions and the induction of ligand expression to activate immune cells. Taken together, our study elucidates a novel aspect of the mechanisms of infection and immune system evasion for S. aureus.

Keywords: ESAT-6-like protein EsxB; Staphylococcus aureus; UL16 binding protein 1; decay-accelerating factor; internalization; tight junction.

Figure 1

EsxB-mediated internalization of S. aureus. (A) The colony forming unit (CFU) of intracellular S. aureus according to the incubation time of S. aureus and HaCaT cells. (B) The CFU of intracellular S. aureus according to the dose of S. aureus administered to HaCaT cells. (C to E) HaCaT cells were cocultured with S. aureus for 6 h, washed, and further incubated in a fresh medium with or without gentamycin for the indicated times. Intracellular CFU (C), cell viability (D), and extracellular CFU from the culture media (E) were counted. (F) The amount of EsxA and EsxB was examined using indirect ELISA with culture supernatants from HaCaT cells incubated with S. aureus for the indicated times. (G to H) HaCaT cells were pretreated with anti-EsxA and anti-EsxB antibodies for 30 min, and then were incubated with S. aureus for 6 h. Intracellular CFU were counted (G), and a cell viability assay was performed (H) after 24 h of further incubation in a fresh medium containing gentamycin. The data displayed are the mean ± SD of three independent experiments. Statistical analysis was conducted using a one-way ANOVA (A to F) or unpaired two-tailed t test (G). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 or None.

Figure 2

Decay-accelerating factor (CD55) was increased by S. aureus infection. HaCaT cells were treated with the indicated doses of S. aureus (A) for 24 h or with 1 × 10⁸ CFU/mL of S. aureus for the indicated times (B). In an alternative experiment, HaCaT cells were treated with heat-killed S. aureus for 24 h (C). The mRNA expression of CIP was examined by real-time PCR and normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (D) The CD55 protein level was examined via Western blotting after S. aureus treatment. (E) Secreted CD55 was examined using indirect ELISA from the culture supernatants of HaCaT cells treated with S. aureus. The CD55 mRNA levels (F) and protein levels (G) of HaCaT cells pretreated with neutralization antibodies and then treated with S. aureus were examined by real-time PCR. The data displayed are the mean ± SD of three independent experiments. Statistical analysis was conducted with a one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 or None.

Figure 3

S. aureus infection inhibited the complement system. Mice (n = 5) were injected with 5 × 10⁷/kg of S. aureus, and blood was collected after 24 h. (A) The serum C3C level was examined using a commercially available C3C ELISA kit. (B) C3 cleavages were examined by Western blotting. Lane 1: HaCaT cell culture media; Lane 2: NHS (1:20 diluted); Lane 3: NHS incubated with S. aureus; Lane 4: Coculture media from HaCaT cells and NHS; Lane 5: Coculture media from S. aureus-infected HaCaT cells and NHS. (C) The serum MAC level was examined using a commercially available MAC ELISA kit. (D) A bactericidal assay was performed with normal mouse sera and S. aureus-infected mouse sera. (E) A complement dependent cytotoxicity (CDC) assay was performed with HaCaT cells and NHS. (F) A CDC assay was performed with neutralization antibody–treated HaCaT cells and NHS. The data displayed are the mean ± SD. Statistical analysis was conducted with an unpaired two-tailed t test. * p < 0.05, ** p < 0.01 vs. None.

Figure 4

S. aureus-mediated cell death in HaCaT cells. HaCaT cells were treated with 1 × 10⁸ CFU/mL of S. aureus for the indicated times. Real-time PCR was performed to examine the mRNA levels of caspase-3 (A), Bcl2 (B), caspase-1 (C), caspase-7 (D), Rip1 (E), and MLKL (F). The data displayed are the mean ± SD of three independent experiments. Statistical analysis was conducted with a one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 h. (G) Protein levels were examined via Western blotting. (H) A fluorescence-activated cell sorting (FACS) analysis was performed to examine the type of cell death that occurred in S. aureus-infected HaCaT cells.

Figure 5

S. aureus-infected HaCaT cells produced increased ULBP-1. The mRNA levels of NKG2D ligands were examined by real-time PCR according to the S. aureus dose (A) and treatment time (B). The data displayed are the mean ± SD of three independent experiments. Statistical analysis was conducted with a one-way ANOVA. *** p < 0.001 vs. 0 h or None. (C) The ULBP-1 protein level was examined via Western blotting. (D) A cell viability test was performed after coculturing HaCaT cells and THP-1 cells. (E) A cell viability test was performed after coculturing HaCaT cells pretreated with a neutralization antibody and THP-1 cells. (F) A cell viability test was performed after coculturing HaCaT cells and NK cells. (G) A cell viability test was performed after coculturing HaCaT cells pretreated with neutralization antibody and NK cells. (a) Indicates activated THP-1 cells; (I) indicates cells infected by S. aureus. The data displayed are the mean ± SD of two independent experiments. Statistical analysis was conducted with an unpaired two-tailed t test. * p < 0.05, ** p < 0.01.

Figure 6

S. aureus-infected HaCaT cells increased tight junctions. (A) Tight junction–related gene expression was examined by real-time PCR after S. aureus infection for 6 h. The tight junction protein level was examined via FACS analysis (B) and Western blotting (C). (D) Paracellular flux was examined by measuring the amount of fluorescein that passed in a Transwell chamber. (E) Intracellular S. aureus CFU were examined in a Transwell chamber. The data displayed are the mean ± SD. Statistical analysis was conducted with a one-way ANOVA (A) or unpaired two-tailed t test (D,E). * p < 0.05, ** p < 0.01.