Staphylococcus aureus Decreases SUMOylation Host Response to Promote Intramacrophage Survival

Abstract

Staphylococcus aureus is a commensal bacterium that causes severe infections in soft tissue and the bloodstream. During infection, S. aureus manipulates host cell response to facilitate its own replication and dissemination. Here, we show that S. aureus significantly decreases the level of SUMOylation, an essential post-translational modification, in infected macrophages 24 h post-phagocytosis. The reduced level of SUMOylation correlates with a decrease in the SUMO-conjugating enzyme Ubc9. The over-expression of SUMO proteins in macrophages impaired bacterial intracellular proliferation and the inhibition of SUMOylation with ML-792 increased it. Together, these findings demonstrated for the first time the role of host SUMOylation response toward S. aureus infection.

Keywords: SUMOylation; Staphylococcus aureus; Ubc9; infection; intracellular survival; macrophage.

Figure 1

S. aureus strain NSA739 persists intracellularly in Raw264.7 macrophages up to 48 h post-gentamicin treatment (pGt). (a) Macrophages were infected with S. aureus NSA739 strain expressing the GFP protein at an MOI of 10 for 1 h, and the number of gentamicin-protected bacteria was determined at different time points by plating intracellular bacteria for CFU enumeration. (b) Representative images of fluorescence microscopy of GFP-labeled S. aureus NSA739. Macrophages seeded on coverslips were infected with GFP-labeled bacteria at a MOI of 10 and analyzed at 5, 24, and 48 h pGt using a 63-oil objective. Quantification was performed using intracellular bacteria at T0 pGt as 100% and is the result of independent counting of 100 cells from each of three independent experiments. n.i, non-infected control cells.

Figure 2

S. aureus decreases SUMOylation after long-term macrophage infection. Immunoblot analysis of SUMO1 (a), SUMO2/3 (b), and GAPDH levels in lysates of macrophages infected with S. aureus for different time points up to 5 h pGt. SUMO1 and SUMO2/3 smears were quantified from four independent experiments using Image lab software (ChemiDoc) and normalized to GAPDH (right panels). The fold change graph represents the percentage of SUMOylated proteins by SUMO1 obtained in the infected cells compared to the amount of SUMOylated proteins in the non-infected control macrophages. The data presented are the mean ± SD of three independent biological experiments. n.s, not significant (Kruskal–Wallis test followed by Dunn’s post hoc test). (c,d) Immunoblot analysis of SUMO1 (c), SUMO2/3 (d) and GAPDH levels in lysates of macrophages infected with S. aureus, heat-killed S. aureus and S. epidermidis for 24 h pGt. n.i., non-infected control cells. SUMO1 and SUMO2/3 smears were quantified from four independent experiments using Image lab software (ChemiDoc) and normalized to GAPDH (bottom panels). The fold change graph represents the percentage of SUMOylated proteins by SUMO1 obtained in the infected cells compared to the quantity of SUMOylated proteins in the non-infected control cells (right panels). *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test.

Figure 3

SUMOylation over-expression decreases the intracellular survival of S. aureus. (a) Immunoblot analysis of SUMO1 (left panel), or SUMO2/3 (right panel) over-expressing macrophages versus control macrophages (GFP vector). GAPDH levels in lysates were used to standardize protein amounts. SUMO1 and SUMO2/3 smears were detected using Image lab software (ChemiDoc) and normalized to GAPDH (bottom panels). (b) Intracellular S. aureus number recovered from macrophages overexpressing SUMO was counted after cell lysis and is presented as the ratio of intracellular bacteria at 5 h and 24 h post-gentamicin compared to cells transfected with an empty GFP vector, considered as 100%. Cell mortality was assessed using trypan blue exclusion dye 0.4% (w/v) to evaluate the number of blue (dead) cells. (c) Representative confocal images of infected macrophages with S. aureus for 24 h pGt. Arrows show intracellular bacteria. The percentage of intracellular bacteria in macrophages overexpressing SUMO1, SUMO3, or GFP vector is represented as the ratio of intracellular bacteria at 24 h vs. T0 pGt and is the result of independent counting of 100 cells from each of three independent experiments. DIC: differential interference contrast. *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test.

Figure 4

SUMOylation inhibition increases the intracellular survival of S. aureus. (a) Immunoblot analysis of Raw264.7 macrophages were pretreated with ML-792 at 0.5 µM or DMSO as a control. SUMO1 and SUMO2/3 smears were quantified and normalized to GAPDH (bottom panels). (b) Macrophages pretreated with ML-792 at 0.5 µM or DMSO were infected with S. aureus. The number of intracellular bacteria recovered from macrophages after 5 h or 24 h post-gentamicin was counted and is presented as the ratio of intracellular bacteria compared to cells pretreated with DMSO, which is considered as 100%. Cell mortality was assessed using trypan blue exclusion dye 0.4% (w/v) to evaluate numbers of blue (dead) cells. (c) Representative confocal images from cells infected with S. aureus NSA739 for 24 h pGt using a 63-oil objective. Arrows show intracellular bacteria. The percentage of intracellular bacteria in pretreated cells with ML-792 is represented as the ratio of intracellular bacteria from ML-792 pretreated cells over DMSO-pretreated cells at 24 h pGt. The quantification is the result of independent counting of 100 cells from each of three independent experiments. DIC: differential interference contrast. *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test.

Figure 5

S. aureus decreases the Ubc9 protein level in a proteasome-independent pathway. Immunoblot analysis of Ubc9 and GAPDH levels in lysates of macrophages infected with S. aureus and S. epidermidis for 5 h (a) and 24 h (b) post-gentamicin treatment. n.i., non-infected control cells. h, hour post-gentamicin treatment. Ubc9 bands were quantified from three independent experiments and normalized to GAPDH levels. The graph represents fold change compared to non-infected cells at 5 h and 24 h post-gentamicin. n.s.: non-significant; * p ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test. (c,d) Immunoblot analysis of Ubc9 (c), ubiquitin-conjugated proteins (d) in macrophages pretreated or not with MG132 for 3 h prior to infection with S. aureus. Bands were quantified from three independent experiments using Image lab software (ChemiDoc) and normalized to GAPDH. The fold change graph represents the percentage of Ubc9 and ubiqutiin-conjugated proteins obtained in the infected cells compared to the quantity of proteins in the cells treated with DMSO (right panels). *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test.