Staphylococcus aureus Infects Osteoclasts and Replicates Intracellularly

Abstract

Osteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. OM is predominantly caused by Staphylococcus aureus, and even with proper treatment, OM has a high rate of recurrence and chronicity. While S. aureus has been shown to infect osteoblasts, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. Here, we demonstrate the ability of S. aureus to intracellularly infect and divide within OCs. OCs were differentiated from bone marrow macrophages (BMMs) by exposure to receptor activator of nuclear factor kappa-B ligand (RANKL). By utilizing an intracellular survival assay and flow cytometry, we found that at 18 h postinfection the intracellular burden of S. aureus increased dramatically in cells with at least 2 days of RANKL exposure, while the bacterial burden decreased in BMMs. To further explore the signals downstream of RANKL, we manipulated factors controlling OC differentiation, NFATc1 and alternative NF-κB, and found that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal and time-lapse microscopy in mature OCs showed a range of intracellular infection that correlated inversely with S. aureus-phagolysosome colocalization. The propensity of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing S. aureus to evade initial immune regulation and proliferate at the periphery of lesions where OCs are most abundant. IMPORTANCE The inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium Staphylococcus aureus. To date, the bone-building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone-resorbing cells, osteoclasts, participate. In this study, we show that S. aureus can infect osteoclasts and proliferate inside these cells, whereas bone-residing macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system.

Multitasking by the OC Lineage during Bone Infection: Bone Resorption, Immune Modulation, and Microbial Niche

“…( A ) Low-power image of a multinucleated OC surrounded by mononuclear pre-OCs in the XY plane. In this image, the degree of co-localization of bacteria (green) and lysosomes (red), indicated in yellow, is particularly variable in the pre-OCs, but similar variation occurs in OCs as well, as previously shown [ 97 ]. ( B ) Images in the XZ plane along the dotted line in (A) show that the bacteria are inside, rather than on top of, the OC.…”

“…Recently, we showed that although OCs and their uncommitted myeloid progenitors take up S. aureus similarly, their handling of intracellular bacteria is quite different. OCs are not only unable to kill S. aureus , but also allow its intracellular proliferation, albeit with significant heterogeneity between cells [ 97 ] ( Figure 3 ). Mycobacterium tuberculosis , an obligate intracellular pathogen capable of infecting bone, has also been found to proliferate within OCs to a much higher level than in precursors [ 98 ].…”

“… Internalization of S. aureus by mature OC and pre-OCs. Murine bone-marrow monocytes were differentiated in RANKL and M-CSF for 3 days, then infected with GFP-labeled S. aureus as previously described [ 97 ]. After 18 h, cultures were fixed and stained with LysoTracker and imaged using a Nikon A1RSi confocal microscope and Nikon NIS-C Elements software.…”

De Novo Purine Biosynthesis Is Required for Intracellular Growth of Staphylococcus aureus and for the Hypervirulence Phenotype of a purR Mutant

“…Dependence on purines has also been demonstrated for Francisella tularensis (4), Brucella abortus (5) and E. coli (6), among other bacteria. S. aureus has been shown to replicate in macrophages (22,24) and osteoclasts (25), but little is known about the nutritional requirements of S. aureus in the intracellular environment. Studies in S. aureus metabolism have identified that mutants in glycolysis (pfkA and pyk) (26) and lactate dehydrogenase (27) have reduced survival in RAW 264.7 macrophages, but no data is available on nucleotide biosynthesis.…”

Antibiotic Tolerance of Staphylococcus aureus Biofilm in Periprosthetic Joint Infections and Antibiofilm Strategies

“…In addition to being able to form biofilms, bacteria can also colonize human cells. In vitro and in vivo studies showed that S. aureus can persist intracellularly in a lot of cells found in the bone environment, including bone cells such as osteoblasts, osteoclasts, and osteocytes, and also in immune cells such as Kupffer cells and macrophages in the case of systemic infection [ 35 , 36 , 37 , 38 , 39 ]. However, continued research is warranted to highlight the impact of internalization in each cell type on the durability and the severity of the infection.…”