Staphylococcus aureus internalization impairs osteoblastic activity and early differentiation process.

Staphylococcus aureus is the most frequent aetiology of bone and joint infections (BJI) and can cause relapsing and chronic infections. One of the main factors involved in the chronicization of staphylococcal BJIs is the internalization of S. aureus into osteoblasts, the bone-forming cells. Previous studies have shown that S. aureus triggers an impairment of osteoblasts function that could contribute to bone loss. However, these studies focused mainly on the extracellular effects of S. aureus. Our study aimed at understanding the intracellular effects of S. aureus on the early osteoblast differentiation process. In our in vitro model of osteoblast lineage infection, we first observed that internalized S. aureus 8325-4 (a reference lab strain) significantly impacted RUNX2 and COL1A1 expression compared to its non-internalized counterpart 8325-4∆fnbAB (with deletion of fnbA and fnbB). Then, in a murine model of osteomyelitis, we reported no significant effect for S. aureus 8325-4 and 8325-4∆fnbAB on bone parameters at 7 days post-infection whereas S. aureus 8325-4 significantly decreased trabecular bone thickness at 14 days post-infection compared to 8325-4∆fnbAB. When challenged with two clinical isogenic strains isolated from initial and relapse phase of the same BJI, significant impairments of bone parameters were observed for both initial and relapse strain, without differences between the two strains. Finally, in our in vitro osteoblast infection model, both clinical strains impacted alkaline phosphatase activity whereas the expression of bone differentiation genes was significantly decreased only after infection with the relapse strain. Globally, we highlighted that S. aureus internalization into osteoblasts is responsible for an impairment of the early differentiation in vitro and that S. aureus impaired bone parameters in vivo in a strain-dependent manner.

Phagocytosis and macrophage polarization on bacterially contaminated dental implant materials and effects on tissue integration.

Bacterial contamination is hard to avoid during dental implant surgery. Macrophages and their polarisation play a decisive role in bacterial colonisation and tissue integration on bacterially contaminated dental implants. The present study investigated the role of macrophages in stimulating tissue coverage overgrowth of contaminating oral bacteria on polished titanium (Ti-P) and acid-etched zirconium dioxide (ZrO2-MA) dental implant materials. Different co-culture models were employed to determine phagocytosis rates of Streptococcus mitis or Staphylococcus aureus contaminating a dental implant surface and the influence of contaminating bacteria and osteoblasts (U2OS) on macrophage polarisation. S. aureus was phagocytized in higher numbers than S. mitis in bi-cultures on smooth Ti-P surfaces. Contaminating S. mitis stimulated near full polarisation of macrophages from a non-Ym1-expressing- to a Ym1-expressing-phenotype on smooth Ti-P, but on ZrO2-MA both phenotypes occurred. In tri-cultures with U2OS-cells on smooth Ti-P, a larger percentage of macrophages remained in their non-Ym1-expressing, "fighting" M1-like phenotype to clear Ti-P surfaces from contaminating bacteria. On ZrO2-MA surfaces, more macrophages tended towards their "fix- and-repair" M2-like phenotype than on Ti-P surfaces. Surface coverage of smooth, bacterially contaminated Ti-P surfaces by U2OS-cells was more effectively stimulated by fighting, M1-like macrophages than on ZrO2-MA surfaces. Comprehensive guidelines are provided for the development of infection-resistant, dental implant materials, including bacteria, tissue and immune cells. These guidelines point to more promising results for clinical application of Ti-P as compared with ZrO2-MA.

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

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-ß1) 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).

EGFR/FAK and c-Src signalling pathways mediate the internalisation of Staphylococcus aureus by osteoblasts.

Internalisation of Staphylococcus aureus in osteoblasts plays a critical role in the persistence and recurrence of osteomyelitis, the mechanisms involved in this process remain largely unknown. In the present study, evidence of internalised S. aureus in osteoblasts was found in long bone of haematogenous osteomyelitis in mice after 2 weeks of infection. Meanwhile, eliminating extracellular S. aureus by gentamicin can partially rescue bone loss, whereas the remaining intracellular S. aureus in osteoblasts may be associated with continuous bone destruction. In osteoblastic MC3T3 cells, intracellular S. aureus was detectable as early as 15 min after infection, and the internalisation rates increased with the extension of infection time. Additionally, S. aureus invasion stimulated the expression of phosphor-focal adhesion kinase (FAK), phosphor-epidermal growth factor receptor (EGFR) and phosphor-c-Src in a time-dependent way, and blocking EGFR/FAK or c-Src signalling significantly reduced the internalisation rate of S. aureus in osteoblasts. Our findings provide new insights into the mechanism of S. aureus internalisation in osteoblast and raise the potential of targeting EGFR/FAK and c-Src as adjunctive therapeutics for treating chronic S. aureus osteomyelitis.

Melt electrohydrodynamic 3D printed poly (ε-caprolactone)/polyethylene glycol/roxithromycin scaffold as a potential anti-infective implant in bone repair.

Implanted scaffold or bone substitute is a common method to treat bone defects. However, the possible bone infection caused by orthopaedic surgery has created a challenging clinical problem and generally invalidate bone repair and regeneration. In this study, a poly (ε-caprolactone) (PCL)/polyethylene glycol (PEG)/roxithromycin (ROX) composite scaffold was prepared via melt electrohydrodynamic (EHD) 3D printing. Fourier transform infrared spectroscopy (FTIR) spectroscopy was performed to verify the existence of PEG and ROX in the scaffolds. By water contact angle measurement, the addition of both PEG and ROX was found to improve the hydrophilicity of the scaffolds. By in vitro drug release assay, the PCL/PEG/ROX scaffolds showed an initial burst drug release and subsequent long-term sustained release behaviour, which is favourable for the prevention and treatment of bone infections. The antibacterial assays against E. coli and S. aureus demonstrated that the composite scaffold with ROX possessed effective antibacterial activity, especially for S. aureus, the main cause of bone infection. The immunostaining and MTT assay with human osteoblast-like cells (MG63) indicated that cells showed good viability and growth on the scaffolds. Therefore, the melt EHD 3D printed PCL/PEG/ROX scaffold could be a promising anti-infective implant for bone tissue engineering.

Staphylococcus aureus Infects Osteoclasts and Replicates Intracellularly.

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.

Staphylococcus aureus induces DNA damage in host cell.

Staphylococcus aureus causes serious medical problems in human and animals. Here we show that S. aureus can compromise host genomic integrity as indicated by bacteria-induced histone H2AX phosphorylation, a marker of DNA double strand breaks (DSBs), in human cervix cancer HeLa and osteoblast-like MG-63 cells. This DNA damage is mediated by alpha phenol-soluble modulins (PSMα1-4), while a specific class of lipoproteins (Lpls), encoded on a pathogenicity island in S. aureus, dampens the H2AX phosphorylation thus counteracting the DNA damage. This DNA damage is mediated by reactive oxygen species (ROS), which promotes oxidation of guanine forming 7,8-dihydro-8-oxoguanine (8-oxoG). DNA damage is followed by the induction of DNA repair that involves the ATM kinase-signaling pathway. An examination of S. aureus strains, isolated from the same patient during acute initial and recurrent bone and joint infections (BJI), showed that recurrent strains produce lower amounts of Lpls, induce stronger DNA-damage and prompt the G2/M transition delay to a greater extent that suggest an involvement of these mechanisms in adaptive processes of bacteria during chronicization. Our findings redefine our understanding of mechanisms of S. aureus-host interaction and suggest that the balance between the levels of PSMα and Lpls expression impacts the persistence of the infection.

Effect of the three Enterococcus faecalis strains on apoptosis in MC3T3 cells.

OBJECTIVE: Osteoblast apoptosis is critical for the development and repairing of bone destruction in persistent apical periodontitis (PAP). Enterococcus faecalis is considered as a frequently isolated pathogen of PAP. This study aimed to explore the effect of E. faecalis on apoptosis in osteoblastic MC3T3-E1 cells via an in vitro model. MATERIALS AND METHODS: MC3T3 cells were incubated with live clinically isolated strains of E. faecalis at a multiplicity of infection (MOI) of 1,000:1 for 2 hr. Flow cytometry analysis using annexin V-FITC and PI staining, JC-1 staining and TUNEL assay were conducted to detect the apoptosis in the infected cells. Western blotting and quantitative real-time PCR were used to determine the expression of caspase-3, Bcl-2 and Bax. RESULTS: The proliferation of the infected cells was inhibited. Decreased mitochondrial membrane potential (ΔΨm) and enhanced DNA fragmentation of the infected cells were observed. The relative expression of Bax and cleavage caspase-3 was upregulated, and the expression of Bcl-2 and Bcl-2/Bax was downregulated in the infected cells. CONCLUSION: Together, the clinically isolated strains of E. faecalis can induce apoptosis in MC3T3 osteoblasts, which may be attributed to the regulation of interaction between members of the Bcl-2 family.

Regulation of ghrelin receptor by microbial and inflammatory signals in human osteoblasts

Abstract: Recently, it has been suggested that the anti-inflammatory hormone ghrelin (GHRL) and its receptor GHS-R may play a pivotal role in periodontal health and diseases. However, their exact regulation and effects in periodontitis are not known. The aim of this in-vitro study was to investigate the effect of microbial and inflammatory insults on the GHS-R1a expression in human osteoblast-like cells. MG-63 cells were exposed to interleukin (IL)-1β and Fusobacterium nucleatum in the presence and absence of GHRL for up to 2 d. Subsequently, gene expressions of GHS-R1a, inflammatory mediators and matrix metalloproteinase were analyzed by real-time PCR. GHS-R protein synthesis and NF-κB p65 nuclear translocation were assessed by immunocytochemistry and immunofluorescence microscopy, respectively. IL-1β and F. nucleatum caused a significant upregulation of GHS-R1a expression and an increase in GHS-R1a protein. Pre-incubation with a MEK1/2 inhibitor diminished the IL-1β-induced GHS-R1a upregulation. IL-1β and F. nucleatum also enhanced the expressions of cyclooxygenase 2, CC-chemokine ligand 2, IL-6, IL-8, and matrix metalloproteinase 1, but these stimulatory effects were counteracted by GHRL. By contrast, the stimulatory actions of IL-1β and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1β and F. nucleatum regulate the GHS-R/GHRL system in osteoblast-like cells. Furthermore, we demonstrate for the first time that the proinflammatory and proteolytic actions of IL-1β and F. nucleatum on osteoblast-like cells are inhibited by GHRL. Our study suggests that microbial and inflammatory insults upregulate GHS-R1a, which may represent a protective negative feedback mechanism in human bone.

B. Abortus Modulates Osteoblast Function Through the Induction of Autophagy.

Osteoarticular brucellosis is the most common localization of human active disease. Osteoblasts are specialized mesenchymal-derived cells involved in bone formation and are considered as professional mineralizing cells. Autophagy has been involved in osteoblast metabolism. The present study demonstrates that Brucella abortus infection induces the activation of the autophagic pathway in osteoblast cells. Autophagy was revealed by upregulation of LC3II/LC3I ratio and Beclin-1 expression as well as inhibition of p62 expression in infected cells. Induction of autophagy was also corroborated by using the pharmacological inhibitors wortmannin, a PI 3-kinase inhibitor, and leupeptin plus E64 (inhibitors of lysosomal proteases). Autophagy induction create a microenvironment that modifies osteoblast metabolism by the inhibition of the deposition of organic and mineral matrix, the induction of matrix metalloproteinase (MMP)-2, osteopontin, and RANKL secretion leading to bone loss. Accordingly, autophagy is also involved in the down-modulation of the master transcription factor in bone formation osterix during B. abortus infection. Taking together our results indicate that B. abortus induces the activation of autophagy pathway in osteoblast cells and this activation is involved in the modulation of osteoblast function and bone formation.

Commensal Microbiota Enhance Both Osteoclast and Osteoblast Activities.

Recent studies suggest that the commensal microbiota affects not only host energy metabolism and development of immunity but also bone remodeling by positive regulation of osteoclast activity. However, the mechanism of regulation of bone cells by the commensal microbiota has not been elucidated. In this study, 8-week-old specific pathogen-free (SPF) and germ-free (GF) mice were compared in terms of alveolar bones and primary osteoblasts isolated from calvarias. Micro-CT analysis showed that SPF mice had larger body size associated with lower bone mineral density and bone volume fraction in alveolar bones compared with GF mice. Greater numbers of osteoclasts in alveolar bone and higher serum levels of tartrate-resistant acid phosphatase 5b were observed in SPF mice. Tissue extracts from SPF alveolar bone showed higher levels of cathepsin K, indicating higher osteoclast activity. SPF alveolar extracts also showed elevated levels of γ-carboxylated glutamic acid⁻osteocalcin as a marker of mature osteoblasts compared with GF mice. Polymerase chain reaction (PCR) array analysis of RNA directly isolated from alveolar bone showed that in SPF mice, expression of mRNA of osteocalcin, which also acts as an inhibitor of bone mineralization, was strongly enhanced compared with GF mice. Cultured calvarial osteoblasts from SPF mice showed reduced mineralization but significantly enhanced expression of mRNAs of osteocalcin, alkaline phosphatase, insulin-like growth factor-I/II, and decreased ratio of osteoprotegerin/receptor activator of nuclear factor-kappa B ligand compared with GF mice. Furthermore, PCR array analyses of transcription factors in cultured calvarial osteoblasts showed strongly upregulated expression of Forkhead box g1. In contrast, Gata-binding protein 3 was strongly downregulated in SPF osteoblasts. These results suggest that the commensal microbiota prevents excessive mineralization possibly by stimulating osteocalcin expression in osteoblasts, and enhances both osteoblast and osteoclast activity by regulating specific transcription factors.

Targeting intracellular Staphylococcus aureus to lower recurrence of orthopaedic infection.

Staphylococcus aureus is often found in orthopaedic infections and may be protected from commonly prescribed antibiotics by forming biofilms or growing intracellularly within osteoblasts. To investigate the effect of non-antibiotic compounds in conjunction with antibiotics to clear intracellular and biofilm forming S. aureus causing osteomyelitis. SAOS-2 osteoblast-like cell lines were infected with S. aureus BB1279. Antibiotics (vancomycin, VAN; and dicloxacillin, DICLOX), bacterial efflux pump inhibitors (piperine, PIP; carbonyl cyanide m-chlorophenyl hydrazone, CCCP), and bone morphogenetic protein (BMP-2) were evaluated individually and in combination to kill intracellular bacteria. We present direct evidence that after gentamicin killed extracellular planktonic bacteria and antibiotics had been stopped, seeding from the infected osteoblasts grew as biofilms. VAN was ineffective in treating the intracellular bacteria even at 10× MIC; however in presence of PIP or CCCP the intracellular S. aureus was significantly reduced. Bacterial efflux pump inhibitors (PIP and CCCP) were effective in enhancing permeability of antibiotics within the osteoblasts and facilitated killing of intracellular S. aureus. Confocal laser scanning microscopy (CLSM) showed increased uptake of propidium iodide within osteoblasts in presence of PIP and CCCP. BMP-2 had no effect on growth of S. aureus either alone or in combination with antibiotics. Combined application of antibiotics and natural agents could help in the treatment of osteoblast infected intracellular bacteria and biofilms associated with osteomyelitis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1086-1092, 2018.

Response of MG63 osteoblasts on bacterial challenge is dependent on the state of differentiation.

The present in vitro study examines molecular processes that are relevant during bone homeostasis after Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis infection with a focus on the differentiation level of osteoblasts. Regenerative processes are often hindered by the recurrence of bacterial infections, which can ultimately provoke a severe destruction of bone tissue. To obtain more detailed insights into such a complex scenario, we have used undifferentiated MG63 osteoblast-like cells as an experimental paradigm to examine the impact of two oral pathogens, A. actinomycetemcomitans and P. gingivalis, on proliferation, cytotoxicity and osteogenic differentiation. Cell culture experiments were performed to analyze cellular behavior. The level of genes interfering with bone tissue integrity (matrix metalloproteinases and their tissue inhibitors) and osteogenic markers (alkaline phosphatase, Runx2, human ß-defensin-2) was compared in undifferentiated versus differentiated MG63 cells using real-time polymerase chain reaction. Functional activity of matrix metalloproteinases was quantified by zymography. Western blot analysis was used to verify the phosphorylation state of mitogen-activated protein kinases p38 and extracellular-signal-regulated kinases 1/2. When co-cultured with undifferentiated MG63 cells, oral pathogens provoked distinct cellular effects. Only A. actinomycetemcomitans reduced cell proliferation, increased cell death, and induced osteogenic differentiation. A comparison of matrix metalloproteinase network stability in the presence of oral pathogens revealed a partial sensitivity towards P. gingivalis but not A. actinomycetemcomitans. So, beside the proof of concept that MG63 cells co-cultured with oral pathogens can serve as an in vitro model for mimicking destructive and regenerative events after bacterial infections, our data indicate that double infections might counterbalance otherwise positive effects.

Bone responses in health and infectious diseases: A focus on osteoblasts.

Historically, bone was thought to be immunologically inactive with the sole function of supporting locomotion and ensuring stromaness functions as a major lymphoid organ. However, a myriad of pathogens (bacteria such as staphylococcus as well as viruses including alphaviruses, HIV or HCV) can invade the bone. These pathogens can cause apoptosis, autophagy and necrosis of osteoblasts and lead to lymphopenia and immune paralysis. There are now several detailed studies on how osteoblasts contribute to innate immune and inflammatory responses; indeed, osteoblasts in concert with resident macrophages can engage an armory of defense mechanisms capable of detecting and controlling pathogen evasion mechanisms. Osteoblasts can express the so-called pattern recognition receptors such as TOLL-like receptors involved in the detection for example of lipids and unique sugars (polysaccharides and polyriboses) expressed by bacteria or viruses (e.g. LPS and RNA respectively). Activated osteoblasts can produce interferon type I, cytokines, chemokines and interferon-stimulated proteins through autocrine and paracrine mechanisms to control for viral replication and to promote phagocytosis or lysis of bacteria for example by defensins. Uncontrolled and sustained innate immune activation of infected osteoblasts will also lead to an imbalance in the production of osteoclastogenic factors such as RANKL and osteoprotegerin involved in bone repair.

PKR induces the expression of NLRP3 by regulating the NF-κB pathway in Porphyromonas gingivalis-infected osteoblasts.

The double-stranded RNA-dependent kinase (PKR), which is activated by double stranded RNA, induces inflammation by regulating NF-κB signaling. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome also modulates inflammation in response to infection. Porphyromonas gingivalis (P.gingivalis) is an oral bacterium which is implicated in the pathogenesis of periodontal diseases. We previously reported that PKR is a key modulator of bone metabolism and inflammation in the periodontal tissue. PKR was also reported to induce inflammation in response to microbes by regulating the NLRP3 inflammasome, suggesting that PKR could affect inflammation along with NLRP3 in periodontal diseases. In this study, we investigated the effects of PKR on NLRP3 expression and NF-κB activity in P. gingivalis infected osteoblasts. We first constructed a SNAP26b-tagged P.gingivalis (SNAP-P. g.) and traced its internalization into the cell. SNAP-P. g. increased the activity of PKR and NF-κB and also induced NLRP3 expression in osteoblasts. Inhibition of NF-κB attenuated SNAP-P. g.-induced NLRP3 expression. The knockdown of PKR using shRNA decreased both the activity of NF-κB and the expression of NLRP3 induced by SNAP-P.g.. We therefore concluded that in osteoblasts, P. gingivalis activated PKR, which in turn increased NLRP3 expression by activating NF-κB. Our results suggest that PKR modulates inflammation by regulating the expression of the NLRP3 inflammasome through the NF-κB pathway in periodontal diseases.

Crystal Structure of an Invasivity-Associated Domain of SdrE in S. aureus.

The surface protein SdrE, a microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family protein expressed on the surface of Staphylococcus aureus (S. aureus), can recognize human complement regulator Factor H and C4BP, thus making it a potentially promising vaccine candidate. In this study, SdrE278-591 was found to directly affect S. aureus host cell invasion. Additionally, the crystal structure of SdrE278-591 at a resolution of 1.25 Å was established, with the three-dimensional structure revealing N2-N3 domains which fold in a manner similar to an IgG fold. Furthermore, a putative ligand binding site located at a conserved charged groove formed by the interface between N2 and N3 domains was identified, with ß2 suspected to occupy the ligand recognizing site and undergo a structural rearrangement to allow ligand binding. Overall, these findings have further contributed to the understanding of SdrE as a key factor for S. aureus invasivity and will enable a better understanding of bacterial infection processes.

TLR9 mediates S. aureus killing inside osteoblasts via induction of oxidative stress.

BACKGROUND: Staphylococcus aureus is the principle causative pathogen of osteomyelitis and implant-associated bone infections. It is able to invade and to proliferate inside osteoblasts thus avoiding antibiotic therapy and the host immune system. Therefore, development of alternative approaches to stimulate host innate immune responses could be beneficial in prophylaxis against S. aureus infection. TLR9 is the intracellular receptor which recognizes unmethylated bacterial CpG-DNA and activates immune cells. Synthetic CpG-motifs containing oligodeoxynucleotide (CpG-ODNs) mimics the stimulatory effect of bacterial DNA. RESULTS: Osteoblast-like SAOS-2 cells were pretreated with CpG-ODN type-A 2216, type-B 2006, or negative CpG-ODN 2243 (negative control) 4 h before infection with S. aureus isolate EDCC 5055 (=DSM 28763). Intracellular bacteria were streaked on BHI plates 4 h and 20 h after infection. ODN2216 as well as ODN2006 but not ODN2243 were able to significantly inhibit the intracellular bacterial growth because about 31 % as well as 43 % of intracellular S. aureus could survive the pretreatment of SAOS-2 cells with ODN2216 or ODN2006 respectively 4 h and 20 h post-infection. RT-PCR analysis of cDNAs from SAOS-2 cells showed that pretreatment with ODN2216 or ODN2006 stimulated the expression of TLR9. Pretreatment of SAOS-2 cells with ODN2216 or ODN2006 but not ODN2243 managed to induce reactive oxygen species (ROS) production inside osteoblasts as measured by flow cytometry analysis. Moreover, treating SAOS-2 cells with the antioxidant Diphenyleneiodonium (DPI) obviously reduced S. aureus killing ability of TLR9 agonists mediated by oxidative stress. CONCLUSIONS: In this work we demonstrated for the first time that CPG-ODNs have inhibitory effects on S. aureus survival inside SAOS-2 osteoblast-like cell line. This effect was attributed to stimulation of TLR9 and subsequent induction of oxidative stress. Pretreatment of infected SAOS-2 cells with ROS inhibitors resulted in the abolishment of the CPG-ODNs killing effects.

Toll-Like Receptor 2 Stimulation of Osteoblasts Mediates Staphylococcus Aureus Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL.

Severe Staphylococcus aureus (S. aureus) infections pose an immense threat to population health and constitute a great burden for the health care worldwide. Inter alia, S. aureus septic arthritis is a disease with high mortality and morbidity caused by destruction of the infected joints and systemic bone loss, osteoporosis. Toll-Like receptors (TLRs) are innate immune cell receptors recognizing a variety of microbial molecules and structures. S. aureus recognition via TLR2 initiates a signaling cascade resulting in production of various cytokines, but the mechanisms by which S. aureus causes rapid and excessive bone loss are still unclear. We, therefore, investigated how S. aureus regulates periosteal/endosteal osteoclast formation and bone resorption. S. aureus stimulation of neonatal mouse parietal bone induced ex vivo bone resorption and osteoclastic gene expression. This effect was associated with increased mRNA and protein expression of receptor activator of NF-kB ligand (RANKL) without significant change in osteoprotegerin (OPG) expression. Bone resorption induced by S. aureus was abolished by OPG. S. aureus increased the expression of osteoclastogenic cytokines and prostaglandins in the parietal bones but the stimulatory effect of S. aureus on bone resorption and Tnfsf11 mRNA expression was independent of these cytokines and prostaglandins. Stimulation of isolated periosteal osteoblasts with S. aureus also resulted in increased expression of Tnfsf11 mRNA, an effect lost in osteoblasts from Tlr2 knockout mice. S. aureus stimulated osteoclastogenesis in isolated periosteal cells without affecting RANKL-stimulated resorption. In contrast, S. aureus inhibited RANKL-induced osteoclast formation in bone marrow macrophages. These data show that S. aureus enhances bone resorption and periosteal osteoclast formation by increasing osteoblast RANKL production through TLR2. Our study indicates the importance of using different in vitro approaches for studies of how S. aureus regulates osteoclastogenesis to obtain better understanding of the complex mechanisms of S. aureus induced bone destruction in vivo.

Knockdown of TNFR1 Suppresses Expression of TLR2 in the Cellular Response to Staphylococcus aureus Infection.

Osteomyelitis is a common manifestation of invasive Staphylococcus aureus infection characterized by widespread bone loss and destruction. Phagocytes possess various receptors to detect pathogens, including the Toll-like receptors (TLRs). Previous studies have demonstrated that the S. aureus protein SpA binds directly to pre-osteoblastic cells via tumor necrosis factor receptor-1 (TNFR-1). In our present study, we investigated the relationship between TLR2 and TNFR-1 in S. aureus-infected osteoblasts. Our results showed that cell viability decreased, and apoptosis, expression of TLR2, and the secretion of inflammatory cytokines (TNF-α and IL-6) increased with increasing concentrations of S. aureus. The JNK pathway was also activated in response to S. aureus infection. Knockdown of TNFR1 not only inhibited the JNK pathway but also reduced TLR2 protein and RANKL levels in S. aureus-infected cells. Inhibition of the JNK pathway reduced the protein level of TLR2 and reduced TNF-α and IL-6 secretion in S. aureus-infected cells.

Orthopedic implant infections: Incompetence of Staphylococcus epidermidis, Staphylococcus lugdunensis, and Enterococcus faecalis to invade osteoblasts.

Septic failure is still the major complication of prosthetic implants. Entering host cells, bacteria hide from host immune defenses, shelter from extracellular antibiotics, and cause chronic infection. Staphylococcus aureus, the leading etiologic agent of orthopedic implant infections, is able to enter bone cells and induce osteoblast apoptosis, osteoclast recruitment, and highly destructive osteomyelitis. Staphylococcus epidermidis, Staphylococcus lugdunensis, and Enterococcus faecalis are opportunistic pathogens causative of implant-related infections. This study investigated the ability to internalize into osteoblastic MG63 cells of 22 S. epidermidis, 9 S. lugdunensis, and 21 E. faecalis clinical isolates from orthopedic implant infections. Isolates were categorized in clusters by ribotyping. Internalization assay was carried out by means of a microtiter plate-based method. S. epidermidis, S. lugdunensis, and E. faecalis strains turned out incompetent to enter osteoblasts, exhibiting negligible internalization into MG63 cells, nearly three orders of magnitude lower than that of S. aureus. Osteoblast invasion does not appear as a pathogenetic mechanism utilized by S. epidermidis, S. lugdunensis, or E. faecalis for infecting orthopedic implants. Moreover, it can be inferred that intracellularly active antimicrobials should not be necessary against implant infections caused by the three bacterial species. Finally, implications with the uptake of biomaterial microparticles by nonphagocytic cells are enlightened. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 788-801, 2016.