Staphylococcus aureus can use an alternative pathway to be internalized by osteoblasts in absence of β1 integrins

Abstract

Staphylococcus aureus main internalization mechanism in osteoblasts relies on a tripartite interaction between bacterial fibronectin-binding proteins, extracellular matrix soluble fibronectin, and osteoblasts' β1 integrins. Caveolins, and particularly caveolin-1, have been shown to limit the plasma membrane microdomain mobility, and consequently reduce the uptake of S. aureus in keratinocytes. In this study, we aimed to deepen our understanding of the molecular mechanisms underlying S. aureus internalization in osteoblasts. Mechanistically, S. aureus internalization requires endosomal recycling of β1 integrins as well as downstream effectors such as Src, Rac1, and PAK1. Surprisingly, in β1 integrin deficient osteoblasts, S. aureus internalization is restored when Caveolin-1 is absent and requires αvβ3/5 integrins as backup fibronectin receptors. Altogether, our data support that β1 integrins regulate the level of detergent-resistant membrane at the plasma membrane in a an endosomal and Caveolin-1 dependent manner.

Keywords: Staphylococcus aureus; Caveolin-1; Detergent-resistant membrane; Internalization; Osteoblasts; β1 integrin.

Fig. 1

Internalization of Staphylococcus aureus 8325-4 inside OBβ1 osteoblasts is dependent of Fibronectin-binding proteins, fibronectin and β1 integrin. (a) Internalization of S. aureus 8325-4 and DU5883 (its isogenic counterpart deficient for FnBPs) inside OBβ1^+/+ and OBβ1^-/- (OBβ1 deleted for β1 integrin). 3 independent experiments in technical triplicates (3 wells for each condition for each experiment) were performed. Non-parametric Mann-Whitney tests were performed. ** or **** means p < 0.01 and p < 0.0001, respectively. NS means that no significative difference was observed. (b) Internalization of S. aureus 8325-4 inside OBβ1^+/+ pretreated or not with anti-β1 integrin antibodies before the infection. (c) Internalization of S. aureus 8325-4 inside OBβ1^+/+ pretreated or not with anti-fibronectin antibodies before the infection. Results were presented as number of intracellular colony-forming units (CFU) for 50 000 cells. As validation experiments, only one experiment in technical triplicate was performed for (b) and (c), so no statistical analysis was performed.

Fig. 2

Internalization of Staphylococcus aureus 8325-4 inside OBβ1 osteoblasts is dependent of Src kinase, Rac1, PAK1 and endosomal recycling. (a) Internalization of S. aureus 8325-4 inside OBβ1^+/+ pretreated or not with Src inhibitor before the infection. (b) Internalization of S. aureus 8325-4 inside OBβ1^+/+ pretreated or not with Rac1 and PAK1 inhibitors prior to the infection. (c) Internalization of S. aureus 8325-4 inside OBβ1^+/+ pretreated or not with primaquine before the infection. Results were presented as number of intracellular colony-forming units (CFU) for 50 000 cells. At least 3 independent experiments in technical triplicates (3 wells for each condition for each experiment) were performed. Non-parametric Mann-Whitney tests were performed. ** and **** means p < 0.01 and p < 0.0001, respectively. (d) Presence of GM1 (marker of DRMs) at the surface OBβ1^+/+ pretreated with primaquine and OBβ1^-/-. Results were presented as percentage of CT-FITC staining compared to the non-treated OBβ1^+/+ cells. Three independent experiments but without technical duplicate or triplicate were performed for (d), so no statistical analysis was performed.

Fig. 3

Absence of Cav1 in OBβ1^−/− (OBβ1^−/− Cav1^KOosteoblasts) rescues the internalization of Staphylococcus aureus 8325-4. (a) Internalization of S. aureus 8325-4 inside OBβ1^−/−Cav1^KO and OBβ1^−/−. Results were presented as number of intracellular colony-forming units (CFU) for 50 000 cells. Two independent experiments in technical triplicates (3 wells for each condition for each experiment) were performed. Non-parametric Mann-Whitney test was performed. ** means p < 0.01. (b) Presence of GM1 (marker of DRMs) at the surface of OBβ1^−/−Cav1^KO and OBβ1^−/−. For (b), results were presented as percentage of CT-FITC staining compared to the control cells (OBβ1^+/+). Three experiments but without technical duplicate or triplicate were performed, thus no statistical analysis was performed.

Fig. 4

Internalization of Staphylococcus aureus 8325-4 inside OBβ1^−/− Cav1^KO cells is dependent on Fibronectin-binding proteins, αvβ3/5 integrin, Src kinase, Rac1, PAK1 and endosomal recycling. (a) Internalization of S. aureus 8325-4 and DU5883 inside OBβ1^+/+ and OBβ1^−/−Cav1^KO. (b) Internalization of S. aureus 8325-4 inside OBβ1^+/+ and OBβ1^−/−Cav1^KO pretreated or not with Cilengitide (a specific inhibitor of αv integrins) before the infection. (c) Internalization of S. aureus 8325-4 inside OBβ1^+/+ and OBβ1^−/− Cav1^KO pretreated or not with Src, PAK1 and Rac1 inhibitors or primaquine before the infection. Results were presented as number of intracellular colony-forming units (CFU) for 50 000 cells. At least 2 independent experiments in technical triplicates (3 wells for each condition for each experiment) were performed. Non-parametric Mann-Whitney tests were performed with Prism GraphPad. *, **, *** or **** means p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively. NS means that no significative difference was observed.