Staphylococcus aureus Infects Osteoclasts and Replicates Intracellularly

S. aureus proliferates within OCs in vitro in a RANKL-induced Osteoclastogenesis-dependent manner. BMMs were treated with RANKL for up to 3 days and subjected to the gentamicin protection assay. (A) CFU from lysates of infected cells after OC differentiation for 0, 2, or 3 days. Cells were lysed immediately after gentamicin exposure (1.5 hpi) or after an additional 16.5 h in osteoclastogenic media (D2, D3) or control media (D0). n = 3 technical replicates, representative of >5 biological replicates. (B) CFU from lysates of infected human CD14⁺ monocytes isolated from peripheral blood and differentiated into OCs for 3 days before infection. n = 3 biological replicates. (C) CFU from lysates of infected BMMs after exposure for 2 days to either PBS, RANKL, IFN-γ, or IL-4. n = 3 technical replicates, representative of 3 biological replicates. (D) BMMs harvested from NFATc1 conditional knockout animals (cKO) show no basal (D0) or induced (D2) NFATc1 protein by Western blotting compared to littermate control mice BMMs (Ctrl). (E) TRAP staining after 3 days of RANKL exposure demonstrates failure of NFATc1-cKO BMMs to form TRAP+ multinuclear OCs. (F) CFU from lysates of Ctrl or NFATc1 cKO BMMs differentiated into OCs for 0 or 2 days and subjected to the gentamicin protection assay. All bars represent 18 h postinfection (hpi). White bars, Ctrl; black bars, NFATc1 cKO cells. (G) CFU from lysates of wild-type (WT) BMMs transfected with empty vector (pMX-EV [white bars]) or NFATc1-overexpressing vector (pMX-NFAT [black bars]) at 18 hpi, showing positive effect of NFATc1 at D2. (H) Rosiglitazone treatment of WT BMMs increases NFATc1 induction more than RANKL alone as measured by Western blotting of nuclear extracts. H3, histone 3 antibody. (I) CFU from lysates of WT BMMs treated with Rosiglitazone (+ROSI [black bars]) or untreated (-ROSI [white bars]) at 18 hpi. n = 3 biological replicates. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-way ANOVA with Tukey’s posthoc test (A, C, F, G, and I) or Student’s t test (B).

Alternative NF-κB in OCs promotes intracellular S. aureus replication in vitro. (A and B) BMMs harvested from NIK knockout mice (Nik^−/− [black bars]) (A) and RelB knockout mice (Relb^−/− [black bars]) (B) were differentiated into OCs for 0, 2, or 3 days and subjected to the gentamicin protection assay. CFU of intracellular S. aureus proliferation were examined at 18 hpi. (C) TRAP-stained images of WT or Relb^−/− BMMs transduced with empty vector (EV) or NFATc1-overexpressing virus (NFAT) and differentiated for 48 h. (D) Western blot of NFATc1 protein expression from WT or Relb^−/− (KO) BMMs transduced with EV or NFAT and differentiated with RANKL for 0 (D0) or 2 (D2) days. (E) WT or Relb^−/− (KO) cells transduced with EV or NFAT virus, differentiated in RANKL for 3 days, infected with S. aureus, and subjected to the gentamicin protection assay. Bars represent CFU of lysates at 18hpi. n = 3 biological replicates. Statistical significance: in panels A and B, ****, P < 0.0001 by two-way ANOVA with Tukey’s posthoc test; in panel E,**, P < 0.01; ****, P < 0.0001 by one-way ANOVA with Tukey’s posthoc test.

S. aureus proliferates within individual OCs to different magnitudes. (A) Images depict representative OCs infected with GFP+ S. aureus (green) and lysosomes labeled with LysoTracker (red). Outlines indicate cell boundaries. White arrows track the formation of intracellular GFP+ S. aureus within vesicles over the indicated time frames. Insets represent higher magnified images of enumerated white boxes. Bars = 20 μm and 5 μm within image and insert panels, respectively. (B) Offset histogram of flow cytometric data from infected cells differentiated in RANKL for 0 or 2 days and then infected with GFP+ S. aureus at an MOI of 1:1. Infected cells were detected by an increased signal in the FITC channel. The threshold for FITC+ is depicted by the dashed line, based on cells infected with GFP-labeled bacteria. n = 3 biological replicates. (C) Infected cells (GFP+) are shown as a percentage of total cells, as measured via flow cytometry. (D) Mean fluorescence intensity (MFI) as measured from the FITC+ fraction of cells via flow cytometry. **, P < 0.01; ****, P < 0.0001 by two-way ANOVA with Tukey’s posthoc test. (E) Transmission electron micrograph of dividing S. aureus in a membrane-bound compartment inside an OC.