Different Modulatory Effects of Four Methicillin-Resistant Staphylococcus aureus Clones on MG-63 Osteoblast-Like Cells

Abstract

Staphylococcus aureus is a Gram-positive bacterium responsible for a variety of mild to life-threatening infections including bone infections such as osteomyelitis. This bacterium is able to invade and persist within non-professional phagocytic cells such as osteoblasts. In the present study, four different S. aureus strains, namely, 2SA-ST239-III (ST239), 5SA-ST5-II (ST5), 10SA-ST228-I (ST228), and 14SA-ST22-IVh (ST22), were tested for their ability to modulate cell viability in MG-63 osteoblast-like cells following successful invasion and persistence. Methicillin-sensitive S. aureus (MSSA) ATCC-12598-ST30 (ST30) was used as control strain. Despite being proven that ST30, ST239, and ST22 have a similar ability to internalize and persist in MG-63 osteoblast-like cells under our experimental conditions, we demonstrated that the observed decrease in cell viability was due to the different behavior of the considered strains, rather than the number of intracellular bacteria. We focused our attention on different biochemical cell functions related to inflammation, cell metabolism, and oxidative stress during osteoblast infections. We were able to show the following: (1) ST30 and ST239 were the only two clones able to persist and maintain their number in the hostile environment of the cell during the entire period of infection; (2) ST239 was the only clone able to significantly increase gene expression (3 and 24 h post-infection (p.i.)) and protein secretion (24 h p.i.) of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in MG-63 osteoblast-like cells; (3) the same clone determined a significant up-regulation of the transforming growth factorbeta 1 (TGF-β1) and of the metabolic marker glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs at 24 h p.i.; and (4) neither the MSSA nor the four methicillin-resistant S. aureus (MRSA) strains induced oxidative stress phenomena in MG-63 cells, although a high degree of variability was observed for the different clones with regard to the expression pattern of nuclear factor E2-related factor 2 (Nrf2) and its downstream gene heme oxygenase 1 (HO-1) activation. Our results may pave the way for an approach to S. aureus-induced damage, moving towards individualized therapeutic strategies that take into account the differences between MSSA and MRSA as well as the distinctive features of the different clones. This approach is based on a change of paradigm in antibiotic therapy involving a case-based use of molecules able to counteract pro-inflammatory cytokines activity such as selective cytokine signaling inhibitors (IL-6, TNF-α).

Keywords: Staphylococcus aureus; cytokines; eukaryotic host­–pathogen interaction; immune system; inflammation; internalization; osteoblast-like cells.

Figure 1

(A) Colony-forming units (CFUs)/mL counts measured following cell lysis for the five different S. aureus strains at 3 and 24 h post-infection (p.i.). (B) Percentage of spot categories (0 Scheme 1. to 5, and over 6 spots) for each strain at 24 h p.i. Values are reported as means ± SD of three independent experiments. Significant comparisons between each category are indicated by lines. *** Significantly different, p < 0.001. (C) Representative image showing MG-63 cells with no spots inside the cell (0 spots), a single MG-63 cells with three spots (1–5 spots), and a single MG-63 cell with 6 spots (>5 spots). CH1 = brightfield; CH2 = 505–560 nm; CH6 = side scatter (SSC) at 785 nm.

Figure 2

Changes in cell viability of MG-63 osteoblast-like cells infected at a multiplicity of infection (MOI) of 100:1 with five different S. aureus strains detected after (A) 3 h and (B) 24 h. Values are reported as absorbance measured at 569 nm and represent the means ± SD of at least five independent experiments. Blue-dotted line indicates the absorbance measured at 569 nm for uninfected MG-63 cells. Significant comparisons between each experimental condition are indicated by lines. * Significantly different, p < 0.05; ** significantly different, p < 0.01; *** significantly different, p < 0.001.

Figure 3

Gene expression of interleukin 6 (IL-6) (A,B) and tumor necrosis factor alpha (TNF-α) (C,D) in uninfected (No Bacteria) MG-63 osteoblast-like cells and in MG-63 cells infected at an MOI of 100:1 with five different S. aureus strains detected at 3 and 24 h p.i. N.D. = not detectable; A.U. = almost undetectable. The abundance of each mRNA is expressed relative to the abundance of β-actin-mRNA. Values are means ± SD of three independent experiments. Significant compariScheme 0. ** significantly different, p < 0.05;** significantly different, p < 0.01; *** significantly different, p < 0.001.

Figure 4

Gene expression of transforming growth factor beta 1 (TFG-β1) (A,B) and glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (C,D) in uninfected (No Bacteria) MG-63 osteoblast-like cells and in MG-63 cells infected at an MOI of 100:1 with five different S. aureus strains detected at 3 and 24 h. The abundance of each mRNA is expressed relative to the abundance of β-actin-mRNA. Values are means ± SD of three independent experiments. Significant comparisons between each experimental condition are indicated by lines. * Significantly different, p < 0.05; ** significantly different, p < 0.01; *** significantly different, p < 0.001.

Figure 5

Modulation of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and transforming growth factor beta 1 (TGF-β1) secretion by bacteria. Supernatants from uninfected (No Bacteria) MG-63 osteoblast-like cells and MG-63 cells infected at an MOI of 100:1 with three different S. aureus strains for 24 h were analyzed using a Custom Multi-Analyte ELISArray Kit. Each treatment was analyzed at least in triplicate. The production of each cytokine is expressed as the percent variation with respect to the production recorded in uninfected (control) cells. (A) IL-6, (B) TNF-α, and (C) TGF-β1. Values are means ± SD of three to four independent experiments. Significant comparisons between each experimental condition are indicated by lines. * Significantly different, p < 0.05; ** significantly different, p < 0.01; *** significantly different, p < 0.001.

Figure 6

Gene expression of nuclear factor E2-related factor 2 (Nrf2) (A,B) and heme oxygenase 1 (HO-1) (C,D) in uninfected (No Bacteria) MG-63 osteoblast-like cells and in MG-63 cells infected at an MOI of 100:1 with five different S. aureus strains detected at 3 and 24 h. The abundance of each mRNA is expressed relative to the abundance of β-actin-mRNA. Values are means ± SD of three independent experiments. Significant comparisons between each experimental condition are indicated by lines. * Significantly different, p < 0.05; ** significantly different, p < 0.01; *** significantly different, p < 0.001.