Staphylococcus aureus thermonuclease NucA is a key virulence factor in septic arthritis

Staphylococcus aureus thermonuclease NucA is a key virulence factor in septic arthritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Staphylococcus aureus thermonuclease NucA is a key virulence factor in septic arthritis Friedrich Götz, Ningna Li, Meghshree Deshmukh, Filiz Sahin, Nourhane Hafza, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4848416/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Apr, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Septic arthritis, primarily caused by Staphylococcus aureus , poses a significant risk of both mortality and morbidity due to its aggressive nature. The nuc1 -encoded thermonuclease NucA of S. aureus degrades extracellular DNA/RNA, allowing the pathogen to escape neutrophil extracellular traps (NETs) and maintain the infection unabated. Here we show that in the mouse model for hematogenous septic arthritis the Δ nuc1 mutant was much less pathogenic and the severity of clinical septic arthritis was markedly reduced, including decreased weight loss, lower kidney bacterial loads and much less IL-6 production. In vitro, S. aureus genomic DNA induced in macrophages a robust TNF-α response which was abrogated when the DNA was degraded by NucA. NucA induced higher IL-6 production in SAOS-2 and higher TNF-α and IL-10 production in neutrophils and shielded S. aureus from phagocyte engulfment and killing. NucA exacerbates septic arthritis possibly by increased internalization by host cells and killing of neutrophiles. Biological sciences/Microbiology/Pathogens Health sciences/Diseases/Infectious diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Septic arthritis, the most aggressive joint disease carrying high mortality and morbidity risk, is predominantly caused by Staphylococcus aureus 1 . In half of the patients, even when they receive immediate treatment, the joint damage caused by septic arthritis is often irreversible, leading to permanent joint dysfunction 2 . It is known that the innate immunity, including neutrophils and complement system, protects from development of septic arthritis 3 , 4 . S. aureus is one of the most successful bacterial pathogens because it expresses many colonization and pathogenicity factors, such as envelope-bound adhesins, or many secreted exoenzymes, including exotoxins, proteases, coagulase, collagenase, hyaluronidase, lipases and, also the thermonuclease (NucA). S. aureus encodes two nucleases: one is secreted and is encoded by nuc1 ; and the other is membrane-anchored with the C-terminus facing the extracellular environment and is encoded by nuc2 5 . The nuc1 gene encodes a pre-pro-protein that is composed of a signal peptide, which is cleaved off by the signal peptidase, and a short pro-region which is processed by a protease releasing the mature and fully active NucA. NucA, also referred to as thermonuclease, is a Ca 2+ -dependent sugar nonspecific endonuclease that catalyzes the hydrolysis of both DNA and RNA at the 5' position of the phosphodiester bond producing nucleoside 3'-phosphates and 3'-phosphooligonucleotide as end-products 6 . NucA functions to degrade extracellular DNA (eDNA), thus promoting the dispersal and destabilization of biofilms. Consequently, in the S. aureus Δ nuc1 mutant biofilm formation was shown to be much more pronounced than in the parent strain. Survival analysis in a hematogenous implant-associated infection mouse model indicated that nuc1 expression is associated with higher mortality 7 . NucA also plays an important role in bacterial escape from neutrophil extracellular traps (NETs). NETs are released at sites of infection by activated neutrophils and consist of nuclear or mitochondrial DNA as a backbone with embedded antimicrobial peptides, histones, and cell-specific proteases, thereby providing an extracellular matrix to entrap and kill various microbes 8 . NucA delayed bacterial clearance in the lung and increased mortality after intranasal infection thus promoting resistance against NET-mediated antimicrobial activity of neutrophils; consequently, the nuc1 -deficient mutant was significantly more susceptible to extracellular killing by activated neutrophils 9 . However, it is not only the degradation of DNA by NucA that is important for the escape from NETs, but also the concomitant production of nucleoside 3'-phosphates and 3'-phosphooligonucleotides, which act as substrates for the adenosine synthase also secreted by S. aureus . The adenosine synthase converts the NucA products to deoxyadenosine, which triggers the caspase-3-mediated death of immune cells 10 . Here we have investigated the role of NucA in a well-established mouse model of septic arthritis 11 . Our data show that NucA causes in the hematogenous septic arthritis mouse model massive bone destruction, rapid weight loss and high proinflammatory cytokine production. In vitro analysis suggest that the increased host cell internalization and killing of neutrophils as well as NETs degrading activity by NucA and its induction of proinflammatory cytokins in certain host cells could be responsible for the high in vivo pathogenicity. Results The S. aureus Newman Δ nuc1 mutant is much less pathogenic in the mouse model of septic arthritis. The pathogenicity of S. aureus Newman Wild Type (NWT) and its Δ nuc1 mutant was evaluated in a mouse model of S. aureus septic arthritis. Mice received intravenous inoculations with both NWT or its Δ nuc1 mutant. Remarkably, Δ nuc1 -infected mice exhibited minimal weight loss until day 7, whereas NWT-infected mice continued to loose weight, reaching 20% by the experiment's termination on day 7 (Fig. 1 a). Likewise, Δ nuc1 -infected mice displayed significantly lower clinical arthritis symptoms than NWT-infected mice. Twenty percent of Δ nuc1 -infected mice developed mild clinical arthritis symptoms up to day 7. In contrast, 40% of NWT-infected mice exhibited clinical arthritis symptoms as early as day 3, and by day 5, all animals had developed severe septic arthritis (Fig. 1 b). Not only was the frequency of arthritis higher, but the severity of septic arthritis was also elevated in mice infected with the NWT strain compared to the mutant strain (Fig. 1 c). Importantly, both the kidney bacterial load (Fig. 1 d) and kidney abscess scores (Fig. 1 e) were significantly lower in the mice infected with the Δ nuc1 mutant compared to those infected with its parental strain. To further confirm our clinical observations, we conducted µCT scans on all joints from mice inoculated with S. aureus . Intravenous injection of S. aureus NWT in NMRI mice resulted in severe bone destruction in 12% of joints after 7 days post-infection, whereas mice infected with the Δ nuc1 mutant showed almost no sign of bone erosion (Fig. 2 a, b). Figure 2 c shows representative 3D images of a wrist, a knee, and a shoulder from mice infected either with the Δ nuc1 mutant or NWT strain. Infection with wild type S. aureus results in a massive increase in the levels of IL-6 and S100A8/A9. On day 7 post infection we collected blood samples from the infected mice and measured the levels of selected pro-inflammatory cytokines, including IL-6, TNF, KC (CXCL1), and S100A8/A9. As shown in Fig. 2 d, the levels of IL-6, KC (CXCL1), and S100A8/A9 were markedly reduced in Δ nuc1 -infected mice as compared to NWT-infected mice. While the TNF levels tended to be lower in Δ nuc1 -infected mice, the difference did not reach statistical significance. Collectively, our results indicate that NucA is a crucial virulence factor in the pathogenicity of S. aureus in an infection model of septic arthritis. We further investigated possible reasons of the lower pathogenicity of the Δ nuc1 mutant in the septic mouse model using different host cells. NucA digestion of gDNA decreased TNF-α production in mouse macrophages. It is well known that the TLR9 receptor in macrophages recognizes unmethylated bacterial DNA and CpG dinucleotides. As staphylococcal macromolecules are frequently contaminated with lipoproteins/lipopeptides that are sensed at by TLR2 at picomolar levels, cytokine induction could be be due to these compounds. To exclude cytokine induction by contaminated lipopeptides, we isolated the gDNA from the S. aureus SA113Δ lgt mutant, which lacks the phosphatidylglycerol:prolipoprotein diacylglyceryl transferase Lgt, in which no lipidation of lipoproteins takes place and therefore no TLR2 response can be triggered 12 , 13 . Indeed, genomic DNA (gDNA) from S. aureus SA113Δ lgt concentration-dependently increased the production of the proinflammatory cytokine TNF-α in mouse macrophages RAW 264.7, while gDNA from mouse macrophages RAW 264.7 did not (Fig. 3 a). The effect was already visible at S. aureus gDNA concentration of 3 ng/ml. The standard CpG oligonucleotides ODN2006 and the non-CpG oligonucleotides ODN2137 were used as positive and negative control, respectively (Fig. 3 a). Of the two nuclease genes ( nuc1 and nuc2 ) in S. aureus , it is the nuc1 encoded NucA that is secreted into the supernatant and is able to completely degrade extracellular DNA and RNA (eDNA and eRNA, respectively). This was demonstrated when we incubated the supernatant of JE2 and its mutants JE2Δ nuc1 , JE2Δ lgt and JE2Δ nuc1 Δ lgt with S. aureus gDNA. In all mutants in which nuc1 was deleted, the gDNA remained intact, whereas in JE2 and the JE2Δ lgt mutant the gDNA was completely degraded ( Supplementary Fig. 1a ). Furthermore, the degradation of eDNA by recombinant NucA, which was expressed and purified from Escherichia coli ( Supplementary Fig. 2 ), suggested that NucA plays a role in controlling DNA/RNA-dependent immune stimulation. Indeed, the stepwise degradation of S. aureus gDNA by increasing amounts of NucA (from 46 pM to 3 nM) correlated with a stepwise decrease in TNF-α production (Fig. 3 b,c). The S. aureus nuc1 deletion mutants have an impact on cytokines production in various cell lines. We investigated whether the immune stimulation of JE2 and its mutants differed. We incubated live S. aureus JE2 and its JE2Δ nuc1 , JE2Δ lgt , and JE2Δ nuc1 Δ lgt mutants for 18 h with RAW 264.7, SAOS-2 cells, and 5 h with neutrophils. There was no difference in IL-6 and TNF-α production between JE2 and JE2Δ nuc1 stimulation in RAW 264.7 (Fig. 4 a). In SAOS-2 cells, IL-6 production was decreased in response to all mutants while in neutrophils the production of TNF-α, IL-10, and IL-1Ra was significantly decreased in response to all the mutants (Fig. 4 b,c), particularly those in which lgt was deleted (JE2Δ lgt and JE2Δ nuc1 Δ lgt) . Similar results were observed in NWT and Δ nuc1 . Compared with NWT, there was less IL-6 production in RAW 264.7 and no significant difference in IL-6 and TNF-α in SAOS-2 when exposed to Δ nuc1 strain ( Supplementary Fig. 3 ), which suggested a similar pathogenicity in Newman and JE2 strains. The results show that at the bacterial level not so much bacterial DNA but the TLR-2 activating lipoproteins are the prominent immune stimulants. As the generated mutants showed hardly any difference in growth and hemolytic activity compared to the parent strain ( Supplementary Fig. 2b,c,d ), we assume that all the effects seen are mainly due to the deletion of nuc1 or lgt genes. JE2Δ nuc1 exhibit decreased internalization or survival in various host cells. In neutrophils the bacterial survival was determined. The survival of JE2Δ nuc1 , JE2Δ lgt and JE2Δ nuc1 Δ lgt was significantly decreased compared to JE2, indicating that the mutants were better killed (Fig. 5 a). We investigated whether live JE2 and its mutants differed in internalization by different host cells such as RAW 264.7 and SAOS-2 cells. After 2 h incubation with S. aureus strains, membrane adherent and extracellular bacteria were killed, and then the CFU of internalized bacteria was determined. In both RAW 264.7 and SAOS-2 cells we observed less CFUs upon incubation with the Δ nuc1 mutants as compared to JE2 (Fig. 5 b,c). In the complemented strain JE2Δ nuc1 (pRB nuc1 ) JE2 phenotype was restored, suggesting that internalization was affected by nuc1 ( Supplementary Fig. 4 ). Effect of live bacteria and supernatant on NET formation and clearing. Neutrophils act as the first defense line of the innate immune system and form neutrophil extracellular traps (NETs) to kill pathogens. Here, neutrophils were exposed to live bacteria (MOI:2) and bacterial supernatants for 2 and 5 h following eDNA-staining with SYTOX Green. Live bacteria caused a time-dependent increase in stained eDNA, starting from 3% after 2 h to 10% after 5 h exposure (Fig. 6 a -Bacteria ). Not much difference in relative fluorescence units (RFU) was observed between exposure to JE2 and its mutants over the timeline. This was also visible in the fluorescence microscopic images after 2 and 5 h of incubation (Fig. 6 b -Bacteria ). The fluorescent labeled eDNA appeared as typical green-fluorescent particles indicating compact chromosomal DNA which is indicative of necrosis. The situation was quite different upon exposure of neutrophils to bacteria-free supernatants. The RFU decreased continuously with increasing time of exposure to all supernatants (Fig. 6 a -Supernatant ). However, in neutrophils exposed to the supernatants of all nucA expressing strains (JE2 and JE2Δ lgt ), the eDNA abundance was lower. In the fluorescence microscope images after 2 and 5 h of incubation with the supernatant of NucA expressing cells (JE2 and JE2Δ lgt ) no compact stained DNA particles were visible; however, a background fluorescence of digested, smaller DNA fragments was visible (Fig. 6 b -Supernatant ). Upon exposure to the supernatant of nuc1 deletion mutants (JE2Δ nuc1 and JE2Δ nuc1 Δ lgt ) neutrophil DNA appeared as diffuse areal, which is indicative of NET. This suggested that NucA-contained supernatant showed lower DNA-abuandance when exposed to neutrophils. Discussion The comparative studies between S. aureus parental strain and the Δ nuc1 mutant revealed that NucA is an important virulence factor in septic arthritis. S. aureus Newman wild type (NWT)-infected mice showed massive weight loss, much increased clinical arthritis frequency, a 3-fold abscess score, a massive bone erosion and very high IL-6 content in the plasma. In contrast, the Δ nuc1 -infected mice showed hardly any signs of septic arthritis, there was almost no weight loss, the clinical arthritis and abscess score were much lower, and the bacterial load in kidneys was decreased (Fig. 1 ). Most remarkable, however, was that Δ nuc1 -infected mice showed almost no bone erosion in the joints (Fig. 2 a-c). The question that arises in this context is the molecular causes of NucA-induced septic arthritis and bone erosion. One reason could be the increased IL-6 content. In the septic arthritis mouse model, NWT induces an almost 100-fold higher IL-6 production in plasma than its Δ nuc1 mutant. Indeed, the proinflammatory IL-6, which is mainly produced by macrophages and T lymphocytes in response to pathogens, is not only a key player in rheumatoid arthritis. It also promotes megakaryocyte maturation and the release of platelets when reaching the bone marrow 14 , 15 . The massive upregulation of S100A8/A9 in NWT-infected mice compared to the Δ nuc1 -infected mice could be a stimulation to IL-6 triggered inflammation and leukocyte recruitment 16 , 17 . The high levels of IL-6 and S100A8/A9 at the end of the mouse experiment reflect the severity of infection. Inflammatory cytokines play a role in bone remodeling process. For example, in IL-6 deficient mice, the bone erosion was lower 18 . The scenario for bone disruption could therefore look like this: NucA causes high IL-6 production which may lead to uncontrolled progression of bone destruction by osteoclasts. In various cell culture studies, we also tried to better understand the role of NucA in septic arthritis. JE2Δ nuc1 mutant triggered no increased IL-6 or TNF-α production in RAW 264.7 (Fig. 4 a,b). What we see is that lipoproteins play a decisive role in immune stimulation in RAW 264.7 cells, which is in agreement with earlier results 13 , 19 , 20 . However, JE2Δ nuc1 mutant induced less IL-6 in SAOS-2 cells and less IL-10, TNF-α and IL-1RA in neutrophils, while NWTΔ nuc1 induced less IL-6 in RAW264.7 cells (Fig. 4 and Supplementary Fig. 3 ). We also found that the internalization of the JE2Δ nuc1 mutant was decreased in both RAW 264.7 and SAOS-2 cells ( Fig. 5bc ), and JE2Δ nuc1 mutant was better killed by neutrophils (Fig. 5 a), suggesting that NucA shields S. aureus from phagocyte engulfment and killing and effectively digests NETs formed by neutrophils. These results suggest that NucA impacts immune stimulation and contributes to an increase in the severity of septic arthritis. Most bacteria release DNA and RNA during proliferation. In S. aureus DNA is released during cell lysis resulting from induction of prophages or activation of proteins with holin-like properties such as CidA and LrgA 21 . The secreted NucA degrades very efficiently eDNA/RNA to allow the reuse of the degradation products. How powerful NucA is in degrading eDNA is illustrated in the Supplementary Fig. 1a . When the supernatants of JE2 and its Δ nuc1 mutant was incubated with gDNA, this was completely degraded within 1 h by the supernatant of JE2 but not the Δ nuc1 mutants. This efficient degradation of eDNA ensures not only the reuse of nucleic acid building blocks but also the escape of staphylococci from a biofilm community and from NETs, providing NucA-expressing bacteria a clear advantage during infection 22 . The receptor for bacterial DNA is TLR-9, which is mainly expressed in immune cells such as dendritic cells and macrophages, but also in other non-immune cells including muscle and epithelial cells 23 . In RAW 264.7 mouse macrophages only large-sized staphylococcal DNA induced TNF-α production in a dose-dependent manner (Fig. 3 a); as soon as the gDNA was degraded by NucA TNF-α production decreased with progressing degradation (Fig. 3 c,d). Similar results were also obtained with Group A Streptococcus (GAS), which produce the DNase Sda1 to prevent IFN-α and TNF-α secretion by murine macrophages 24 . TNF-α contributes to pathogenicity in the initiation and progression of septic arthritis, as TNF-α deficient mice developed less severe forms of arthritis 25 . Moreover, combining antibiotics with a TNF-α inhibitor yielded superior results compared to antibiotics alone. The combination effectively reduced synovitis and joint destruction in a mouse model of septic arthritis 26 . Simultaneously, TNF-α plays a crucial role in the Th1 response and primes phagocytes for effective elimination of pathogens 27 . Anti-TNF-α treatment was shown to compromise immune killing efficacy, leading to increased kidney bacterial load in a mouse model of S. aureus septic arthritis 28 . The mouse model for septic arthritis in our study was performed with S. aureus Newman and its Δ nuc1 mutant. A control with JE2 (a USA300 derivative) was not possible, as approval was not granted due to its resistance to methicillin. We therefore asked how similar or dissimilar are these strains. In a previous study both strains, USA300 and Newman, were compared in a mouse sepsis model and there was no significant difference observed between the strains 29 . Growth kinetics and hemolytic activity also revealed similar phenotypes ( Supplementary Fig. 1b,c,d ). Both Δ nuc1 mutants could be complemented to nuclease production so we are pretty sure that JE2, Newman and their Δ nuc1 mutants behave similarly. In hematogenous septic arthritis, compromised innate immune defenses increase the likelihood of bacteria in the bloodstream invading joints, ultimately leading to the development of septic arthritis 11 . Neutrophils are recognized as vital immune cells guarding against S. aureus septic arthritis. Neutrophil-depleted mice exhibited heightened and more frequent septic arthritis, coupled with compromised bacterial clearance as evidenced by elevated CFU counts in both blood and kidneys 4 . We also compared the effects of live bacteria and the corresponding culture supernatant on neutrophil extracellular traps (NETs) degradation. NETs represent a form of innate immune response that binds microorganisms and prevents them from spreading; and the high local concentration of antimicrobial agents may kill bacteria 30 , 31 . It is well known that the S. aureus thermonuclease (NucA) degrades the released DNA of NETs to escape scavenging and killing 10 , 22 , 32 , 33 . When we exposed neutrophils to live S. aureus for up to 5 h we observed a time-dependent increase in stained eDNA, but there was not much difference between JE2 and its mutants (Fig. 6 a,b -Bacteria ). However, when we incubated neutrophils with the corresponding supernatants, we observed a clear difference between the nuc1 expressing strains JE2 and JE2Δ lgt and the Δ nuc1 mutants. In JE2Δ nuc1 and JE2Δ nuc1 Δ lgt neutrophil eDNA was stained as diffuse areal which is indicative of NET formation, while in nuc1 expressing strains there was almost no eDNA stained (Fig. 6 b -Supernatant ). The question remains: why did we not see a major differences between the wild type and the Δ nuc1 live bacteria? We assume that by washing the bacterial cells with PBS, NucA is washed out and the bacteria are rapidly phagocytized and killed when intubated with neutrophils. Overall, our study suggests that NucA plays a crucial role in the pathogenesis of S. aureus septic arthritis, as evidenced by Δ nuc1 -infected mice showing reduced arthritis severity, bone erosion, and kidney abscess formation, alongside lower bacterial loads. In vitro data further support these findings, demonstrating that NucA degrades bacterial DNA, shields S. aureus from phagocyte killing, and digests neutrophil extracellular traps, ultimately promoting bacterial survival and worsening disease severity. How NucA triggers massive bone erosion is the subject of further research. Materials and Methods Bacterial strains, plasmids, primers, and culture conditions Bacterial strains and plasmids used in this study are described in Supplementary Table 1 . All the primers are listed in Supplementary Table 2 . Escherichia coli BL21 (DE3) was grown in Luria-broth medium (LB), and Staphylococcus aureus strains were cultured in tryptic soy broth (TSB, Millipore, Merck) or basic medium (BM) broth (Luria broth supplemented with 0.1% K 2 HPO 4 and 0.1% glucose) or stored as previously mentioned 34 . To keep the plasmids in the bacteria, E. coli was supplied with ampicillin or kanamycin and S. aureus was supplied with 10 µg/ml chloramphenicol. For growth kinetic comparison, the S. aureus JE2, Newman and their mutants were cultured into TSB medium, and measured with Varioskan LUX Multimode Microplate Reader (Thermo fisher) for 24 h. Deletion of nuc1 and lgt in S. aureus nuc1 is the nuclease1-encoding gene and lgt is the lipoprotein diacylglyceryl transferase enzyme-encoding gene 13 , 35 . For deleting nuc1 and lgt genes, the knockout plasmid pBASE6 was employed. The disruption primers were designed to contain upstream and downstream of the target genes regions. Fragments were generated by PCR and subcloned into EcoRV digested vector pBASE6, resulting in pBASE6Δ nuc1 and pBASE6Δ lgt . The resulting plasmids were transformed into E. coil DC10B for amplification. Then, plasmids were isolated and checked for DNA sequences. The correct plasmids pBASE6Δ nuc1 and pBASE6Δ lgt were transformed into an intermediate host, S. aureus RN4220, by electroporation to restrict foreign DNA and then into S. aureus JE2 or S. aureus Newman (NWT). The process for deletion of lgt and nuc1 from S. aureus was followed as described previously, yielding JE2Δ nuc1 , NewmanΔ nuc1 , JE2Δ lgt , and JE2Δ nuc1 Δ lgt 36 . Construction of complementary strain For complementation, the plasmid pRB473- nuc1 was introduced. The nuc1 fragment was amplified, ligated to pRB473 plasmids and transformed into E. coli DC10B. Positive colonies were selected and verified via DNA sequencing. Then correct plasmid was purified and transformed into JE2Δ nuc1 and Δ nuc1 , yielding JE2Δ nuc1 (pRB nuc1 ) and Δ nuc1 (pRB nuc1 ). Hemolytic activity and nuclease activity assay The bacteria were grown in TSB medium overnight. The OD578 of overnight culture was adjusted and spotted on the Blood Agar (TSA with Sheep Blood) plates (Thermo Fisher) at 37 o C and then the hemolysis zone was measured. The supernatants of overnight culture were checked for nuclease activity using DNase Test Agar with Toluidine Blue (Merck, Millipore). The DNase Test Agar plates were incubated at 37 o C. Mouse model for S. aureus septic arthritis To compare the pathogenicity of S. aureus Newman wild-type strain (NWT) and its Δ nuc1 mutant, a septic arthritis mouse model was used. NMRI female mice, aged 8 weeks, were purchased from Envigo (Venray, Netherlands). All mice were housed at the animal facility at the University of Gothenburg. Mice were kept under standard temperature and light conditions and were fed laboratory chow and water ad libitum. Mice (n = 5) in respective groups were intravenously injected with a 200 µl arthritic dose of either S. aureus Newman strain (2.8×10 6 CFU/mouse) or Δ nuc1 mutant (2.8×10 6 CFU/mouse). Mice were monitored for weight loss and clinical signs of arthritis from day 0 to day 7. On day 7, mice were sacrificed to collect samples including blood, kidneys, joints. Kidneys were collected aseptically and scored on a scale of 0 (no abscess), 1 (mild abscess), 2 (moderate abscess) to 3 (severe abscess). The kidneys were then minced and diluted with sterile PBS. Dilutions were plated on horse blood agar plates, incubated at 37 o C for 24 hours, and colonies obtained were counted using a colony counter (Stuart Scientific, Made in the UK). Microcomputed tomography (µCT) All four paws were scanned by SkyScan 1176 µCT (Bruker, Antwerp, Belgium). The scanning was conducted at 55 kV/ 455 µA, with a 0.2-mm aluminum filter. The exposure time was 47 ms. The X-ray projections were obtained at 0.7° intervals with a scanning angular rotation of 180°. The NRecon software (version 1.6.9.8; Bruker) was used to reconstruct 3D images which were further evaluated by using CT vox (version 2.7.0; Bruker). Each joint was evaluated by two researchers (M.D. and T.J.) using a scoring system from 0 to 3 (0: healthy joint; 1: mild bone destruction; 2: moderate bone destruction; and 3: marked bone destruction) as previously described 37 . NucA expression and purification NucA is an extracellular enzyme, which is secreted as mature Nuc1. The nucA gene was cloned into vector pET28a with C-terminal His-tag and this construct was transformed into E. coli BL21(DE3). The transformant carrying pET28a-NucA-6xHis was grown in LB at 37 o C supplemented with 50 µg/ml ampicillin. When OD600 reached 0.6–0.8, the bacteria were induced with 1 mM IPTG at 18 o C overnight for overexpressing NucA. The overnight culture was collected, resuspended in buffer A (50 mM Tris-HCl pH 8.0, 300 mM NaCl), and lysed by an ultra-sonicator with a pulse every 4 s for 4 min. The lysate was centrifuged at 14,000 g for 1 h. The supernatant of lysate was collected and then loaded onto the Ni-NTA column. Fractions containing NucA were collected with Buffer B (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 250 nM imidazole) and analyzed by SDS-PAGE. NucA enzyme was dialyzed with PBS, concentrated, flash-frozen in liquid nitrogen, and stored at -80 o C until use. Genomic bacterial DNA (gDNA) degradation assay with NucA S. aureus SA113Δ lgt was cultured in TSB medium for overnight. The bacterial pellet was collected and resuspended in TE buffer supplemented with lysostaphin and RNase at 37 o C for 30 min. Bacterial gDNA was then purified via phenol-chloroform-isoamyl alcohol, precipitated with isopropanol, washed with ethanol, and then dissolved in H 2 O 38 . gDNA was incubated with varying concentrations of recombinant NucA for 1 h at 37 o C. The samples were then added to RAW 264.7 cells for stimulation and loaded on the agarose gel for visualization. Preparation of bacteria and bacterial supernatant BM medium was used for inoculating S. aureus at 37 o C with shaking from fresh BM agar plates. The cultures were harvested after 16 h by centrifuging and washed with PBS. To get bacterial dosage (MOI, multiplicity of infection), bacteria were calculated to OD/CFU. Bacterial supernatants were collected and filtered with a 0.2 µm pyrogen-free round column. The supernatants were kept on ice until use and adjusted to equal concentrations according to bacterial number which is described in former study 13 . The supernatant was tested for its ability to degrade gDNA and its activity on neutrophiles. Neutrophils Isolation Venous blood was freshly collected by EDTA-tubes (Sarstedt, Germany) from several healthy individuals. 6 mL blood was layered on 6 mL of Lympholyte poly-cell separation medium (Cedarlane, Burlington, Canada). Centrifugation was done without the break, at 500×g for 40 min at room temperature. PMN layer was collected and washed twice with 12 mL PBS and centrifuged at 400×g for 10 min at room temperature with settings of acceleration 5 and deceleration 4. Cells were resuspended in RPMI medium without phenol red (Sigma-Aldrich, Darmstadt, Germany). Cell counts were obtained by the Trypan Blue exclusion method, utilizing a Neubauer counting chamber. Cell culture and immune stimulation assay The murine macrophages cell line RAW 264.7 was cultured in Dulbecco’s modified Eagle’s medium (gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 o C with 5% CO 2 . The human osteoblast-like cell line SAOS-2 was grown in McCoy's 5A Medium (Sigma) supplemented with 15% FBS, 1% Glucose and 1% penicillin-streptomycin. Prior to stimulation, RAW 264.7 and SAOS-2 cells were seeded in 96-well plates and incubated overnight until reaching confluency, while neutrophils were directly seeded in plates before adding all stimulants. Immune stimulation was performed for 18 h at 37 o C and 5% CO 2 , except neutrophils were stimulated for 5 h. The cellular supernatants were then collected and stored at -20 o C until determining the cytokines production. Detection of cytokines by ELISA The cytokines collected from cellular supernatant of different cell lines were measured with the uncoated ELISA kit (Invitrogen) according to instructions. The plasma levels of IL-6, TNF-α, keratinocyte chemoattractant (KC), and S100A8/A9 in blood collected from NMRI mice infected intravenously with Newman WT (NWT) and Δ nuc1 were quantified using DuoSet ELISA kits (R&D Systems Europe) according to the manufacturer’s instruction. Invasion assay RAW 264.7 and SAOS-2 cells were seeded in 24-well plates with 500µl cultural medium until reaching confluency. Cells were washed with PBS, and the prewarmed cultural medium without antibiotics was added to each well. Bacteria were grown till the log phase before infection. The cells were then incubated with bacteria for 1.5 h to yield an MOI of 20:1. After incubation, gentamycin and lysostaphin were added to kill the extracellular bacteria for 1h. The cells were lysed with 0.1% Triton X-100 supplied with 0.05% Trypsin and lysates were plated to determine internalized bacteria 39 . Bacterial killing assay S. aureus JE2 and its mutants were grown overnight. Then the bacteria were collected, regrown till the log phase, washed with PBS and opsonized with 10% human pooled serum in RPMI for 1 h at 37 o C. Neutrophils were seeded in 24 well plates and incubated with bacteria (100%) at a MOI = 0.1. After 1.5 h, the neutrophils were lysed with ice-cold ddH 2 O and centrifuging for 15 min at 4 o C. The lysates were then plated on agar plates with serial dilution and CFUs were counted the next day. The S. aureus survival (killing) was calculated by comparing the counted CFU with original added CFU. Sytox Green Assay Isolated neutrophils were prepared to be 2×10 5 cells/mL, then 1 µM Sytox Green (Thermo Fisher, Waltham, USA) was added. 1% Triton X-100 solution was used for extracellular DNA normalization. Cells were stimulated for 5 h with MOI:2 live bacteria and 5% overnight bacterial supernatant. Fluorescent intensity was measured every 30 minutes at Ex 485 nm/Em 520 nm, and the cells were incubated at 37°C with 5% CO 2 by the microplate reader (FluoStar Omega, BMG Labtech, Ortenberg, Germany). Microscopy pictures were taken with EVOS Fl (Thermo Fisher) fluorescent microscope at 2 and 5 hours of incubation. Declarations Ethical Statement The Ethics Committee of Animal Research of Gothenburg approved all experiments conducted on mice. The mouse experiments were performed in accordance with the Swedish Board of Agriculture's regulations and recommendations on animal experiments. Blood was collected from healthy adult volunteers and written informed consent was given. The institutional review board of the University of Tübingen approved the study and all adult subjects provided informed consent. This study was done in accordance with the ethics committee of the medical faculty of the University of Tübingen that approved the study (Approval number 015/2014 BO2). Statistics and reproducibility All the data are analyzed using GraphPad Prism (version 8.0; GraphPad Software). The data are represented in mean ± standard error of the mean (SEM). Statistical significances: not significant p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Details of statistical analyses for each experiment are provided in ‘Materials and Methods’. Competing interests The authors declare no competing interests. Authors contributions FG, NL designed the study and the experiments; TJ, MVD designed and carried out the septic arthritis model; FS, SE, AN and NL carried out neutrophil experiments; NL, NH constructed deletion mutants; NL, AVA and AW carried out cell lines experiments; FG, NL wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by funding from the Deutsche Forschungsgemeinschaft the Germany’s Excellence Strategy – EXC 2124–390838134 ‘Controlling Microbes to Fight Infections’ to F.G., grants from the Swedish state under the agreement between the Swedish Government and the county councils, the ALF-agreement grant number ALFGBG-823941 to T.J., N.L. was supported by the Chinese Scholarship Council. We are grateful to Stefanie Krajewski, Clinical Research Laboratory, Department of Thoracic, Cardiac and Vascular Surgery, University Hospital Tübingen, 72076 Tübingen, Germany, for providing us with SAOS-2 cells. S.E. received funding from the German Research Council (EH471/5 − 1). Data availability The data generated and annlysed in this study’s are available from the corresponding author upon reasonable request. References Mathews, C.J., Weston, V.C., Jones, A., Field, M., Coakley, G.: Bacterial septic arthritis in adults. Lancet. 375 , 846–855 (2010) Kaandorp, C.J., Krijnen, P., Moens, H.J., Habbema, J.D., van Schaardenburg, D.: The outcome of bacterial arthritis: a prospective community-based study. Arthritis Rheum. 40 , 884–892 (1997) Na, M., et al.: Deficiency of the Complement Component 3 but Not Factor B Aggravates Staphylococcus aureus Septic Arthritis in Mice. Infect. Immun. 84 , 930–939 (2016) Verdrengh, M., Tarkowski, A.: Role of neutrophils in experimental septicemia and septic arthritis induced by Staphylococcus aureus . Infect. Immun. 65 , 2517–2521 (1997) Kiedrowski, M.R., et al.: Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease. 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PLoS Pathog. 8 , e1003016 (2012) Fatima, F., et al.: Radiological features of experimental staphylococcal septic arthritis by micro computed tomography scan. PLoS One. 12 , e0171222 (2017) Dalpke, A., Frank, J., Peter, M., Heeg, K.: Activation of toll-like receptor 9 by DNA from different bacterial species. Infect. Immun. 74 , 940–946 (2006) Nguyen, M.T., et al.: The νSaα Specific Lipoprotein Like Cluster ( lpl ) of S. aureus USA300 Contributes to Immune Stimulation and Invasion in Human Cells. PLoS Pathog. 11 , e1004984 (2015) Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryfiles.pdf Cite Share Download PDF Status: Published Journal Publication published 10 Apr, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Statistical analyses were performed using the Mann-Whitney U test (\u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003ec\u003c/strong\u003e), where the data were represented the mean ± SEM (standard error of the mean). Statistical significances: not significant p\u0026gt;0.05; *p\u0026lt;0.05; **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4848416/v1/35e5a8bfd100740a6de41b51.jpeg"},{"id":65131082,"identity":"3cb28136-27f8-4344-a2c1-9d2470718922","added_by":"auto","created_at":"2024-09-24 02:42:11","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":191301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMonitoring bone erosion by microcomputed tomography (μCT) and cytokine level in mice infected with NWT or Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enuc1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) CT frequency of the joints in NMRI mice intravenously injected with \u003cem\u003eS. aureus\u003c/em\u003e NWT or its Δ\u003cem\u003enuc1 \u003c/em\u003emutant. (\u003cstrong\u003eb\u003c/strong\u003e) Bone erosion frequency of joints evaluated using μCT in NMRI mice. (\u003cstrong\u003ec\u003c/strong\u003e) Representative images of micro-computed tomography (µCT) scanning of the mice joints (hand, knee, shoulder) after infection. Upper panel: NWT; lower panel: Δ\u003cem\u003enuc1\u003c/em\u003e; arrow indicates bone erosion. (\u003cstrong\u003ed\u003c/strong\u003e) Levels of IL-6, TNF, KC, and S100A8/A9 levels in plasma derived from NMRI mice intravenously injected with NWT or Δ\u003cem\u003enuc1\u003c/em\u003e. Statistical analyses were performed using the Mann-Whitney U test \u003cstrong\u003e(a\u003c/strong\u003e and \u003cstrong\u003ec)\u003c/strong\u003e, where the data were represented in mean ± SEM. Statistical significances: not significant p\u0026gt;0.05; *p\u0026lt;0.05; **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4848416/v1/acbfa93a5a8d53fb835528ff.jpeg"},{"id":65131251,"identity":"455a1efc-99ec-408b-b842-384d561130b8","added_by":"auto","created_at":"2024-09-24 02:50:11","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":196497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInduction of TNF-α by macrophages upon exposure to diverse DNA. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Stimulation of mouse macrophage RAW 264.7 cells with SA113Δ\u003cem\u003elgt \u003c/em\u003egDNA, mammal gDNA, ODN2006 (CpG DNA), and ODN2137 (non-CpG DNA) at varying concentrations for 18 h, followed by TNF- α measurements. (\u003cstrong\u003eb\u003c/strong\u003e) Agarose gel analysis showed the degradation of SA113Δ\u003cem\u003elgt\u003c/em\u003e gDNA by gradually decreasing NucA concentration. (\u003cstrong\u003ec\u003c/strong\u003e) Undigested and digested SA113Δ\u003cem\u003elgt\u003c/em\u003e gDNA were incubated with RAW 264.7 cells for 18 h. Released TNF-α in cellular supernatants was determined by ELISA. Data represents the mean ± SEM from three independent experiments; not significant p\u0026gt;0.05; *p\u0026lt;0.05; ****p\u0026lt;0.0001, one-way ANOVA with Dunnett’s posttest.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4848416/v1/a2dce7c2fe562965132a5877.jpeg"},{"id":65131086,"identity":"b858b269-6050-4a67-a513-fb302b7eedee","added_by":"auto","created_at":"2024-09-24 02:42:11","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":232301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInduction of cytokines by various host cells upon exposure to live JE2, JE2Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enuc1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, JE2Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elgt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and JE2Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enuc1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eΔ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elgt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eThe PBS-washed bacteria were incubated with (\u003cstrong\u003ea\u003c/strong\u003e) RAW 264.7 at a MOI=30 and (\u003cstrong\u003eb\u003c/strong\u003e)SAOS-2 cells at a MOI=3 and (\u003cstrong\u003ec\u003c/strong\u003e)with neutrophils at a MOI=50. Cellular supernatants were collected after 18 h for RAW 264.7and SAOS-2 cells, and after 5 h for neutrophils to measure various cytokines by ELISAassay. For neutrophils, the experiments weredisplayed from n = 10 donors. Triplet experiments were conducted; error bars indicate ± SEM; not significant p\u0026gt;0.05; *p\u0026lt;0.05; **p\u0026lt;0.01; and ****p\u0026lt;0.0001, one-way ANOVA with Dunnett’s posttest.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4848416/v1/625fe1badc2e0f0de1754cdb.jpeg"},{"id":65131083,"identity":"23e930d7-6cb6-44f1-be69-8188423c42df","added_by":"auto","created_at":"2024-09-24 02:42:11","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":133215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInternalization studies and bacteria killing with various cells. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) For bacterial killing in neutrophils, JE2 and its mutants were opsonized with 10% human pooled serum and then incubated with neutrophils at a MOI=0.1 for checking survival. For investigating internalization, JE2 and its mutants were incubated with (\u003cstrong\u003eb\u003c/strong\u003e) RAW 264.7 and (\u003cstrong\u003ec\u003c/strong\u003e) SAOS-2 cells at a MOI:20 for 1.5 h, after which cells were lysed and bacterial CFUs counted. Each experiment was performed at least three times; error bars indicate mean ± SEM. Statistical analyses were performed using one-way ANOVA with Dunnett’s posttest. Statistical significances: not significant p\u0026gt;0.05, *p\u0026lt;0.05; **p\u0026lt;0.01; and***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4848416/v1/dcc9f2a275c795250584da40.jpeg"},{"id":65131250,"identity":"2066a7b4-12c6-4ccc-9571-c5eaff4222eb","added_by":"auto","created_at":"2024-09-24 02:50:11","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":290625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNET formation upon exposure to live bacteria and supernatants of JE2 and its mutants. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Fluorescence microscopy of neutrophils exposed to JE2, JE2Δ\u003cem\u003enuc1\u003c/em\u003e, JE2Δ\u003cem\u003elgt\u003c/em\u003e, and JE2Δ\u003cem\u003enuc1\u003c/em\u003eΔ\u003cem\u003elgt\u003c/em\u003e with live bacteria (MOI:2) or overnight supernatant (5% volume) for 5 h. Relative fluorescent units normalized to Triton-X lysed neutrophils are shown. (\u003cstrong\u003eb\u003c/strong\u003e) Exemplary fluorescence images of Sytox Green staining at the 2 and 5 h of incubation. Scale bar = 500 µm. The graph displays the average values ±SEM obtained from n = 4 donors; *p\u0026lt;0.05 and ****p\u0026lt;0.0001, one-way between-groups ANOVA with Dunnett’s posttest.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4848416/v1/e6dcd80a870a9538039ba2bc.jpeg"},{"id":80372358,"identity":"e9192b26-5ac9-4225-95c2-704721f0e59f","added_by":"auto","created_at":"2025-04-11 07:08:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2682126,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4848416/v1/6aebad57-f49b-49b7-877e-08f78d9624b9.pdf"},{"id":65131249,"identity":"bb2c5b03-2904-4f69-8841-061d55844d81","added_by":"auto","created_at":"2024-09-24 02:50:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":535003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementaryfiles.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4848416/v1/7512765dc0d75ef6dcdfa914.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Staphylococcus aureus thermonuclease NucA is a key virulence factor in septic arthritis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSeptic arthritis, the most aggressive joint disease carrying high mortality and morbidity risk, is predominantly caused by \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In half of the patients, even when they receive immediate treatment, the joint damage caused by septic arthritis is often irreversible, leading to permanent joint dysfunction\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It is known that the innate immunity, including neutrophils and complement system, protects from development of septic arthritis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. aureus\u003c/em\u003e is one of the most successful bacterial pathogens because it expresses many colonization and pathogenicity factors, such as envelope-bound adhesins, or many secreted exoenzymes, including exotoxins, proteases, coagulase, collagenase, hyaluronidase, lipases and, also the thermonuclease (NucA). \u003cem\u003eS. aureus\u003c/em\u003e encodes two nucleases: one is secreted and is encoded by \u003cem\u003enuc1\u003c/em\u003e; and the other is membrane-anchored with the C-terminus facing the extracellular environment and is encoded by \u003cem\u003enuc2\u003c/em\u003e\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003enuc1\u003c/em\u003e gene encodes a pre-pro-protein that is composed of a signal peptide, which is cleaved off by the signal peptidase, and a short pro-region which is processed by a protease releasing the mature and fully active NucA. NucA, also referred to as thermonuclease, is a Ca\u003csup\u003e2+\u003c/sup\u003e-dependent sugar nonspecific endonuclease that catalyzes the hydrolysis of both DNA and RNA at the 5' position of the phosphodiester bond producing nucleoside 3'-phosphates and 3'-phosphooligonucleotide as end-products\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNucA functions to degrade extracellular DNA (eDNA), thus promoting the dispersal and destabilization of biofilms. Consequently, in the \u003cem\u003eS. aureus\u003c/em\u003e Δ\u003cem\u003enuc1\u003c/em\u003e mutant biofilm formation was shown to be much more pronounced than in the parent strain. Survival analysis in a hematogenous implant-associated infection mouse model indicated that \u003cem\u003enuc1\u003c/em\u003e expression is associated with higher mortality\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. NucA also plays an important role in bacterial escape from neutrophil extracellular traps (NETs). NETs are released at sites of infection by activated neutrophils and consist of nuclear or mitochondrial DNA as a backbone with embedded antimicrobial peptides, histones, and cell-specific proteases, thereby providing an extracellular matrix to entrap and kill various microbes\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. NucA delayed bacterial clearance in the lung and increased mortality after intranasal infection thus promoting resistance against NET-mediated antimicrobial activity of neutrophils; consequently, the \u003cem\u003enuc1\u003c/em\u003e-deficient mutant was significantly more susceptible to extracellular killing by activated neutrophils\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, it is not only the degradation of DNA by NucA that is important for the escape from NETs, but also the concomitant production of nucleoside 3'-phosphates and 3'-phosphooligonucleotides, which act as substrates for the adenosine synthase also secreted by \u003cem\u003eS. aureus\u003c/em\u003e. The adenosine synthase converts the NucA products to deoxyadenosine, which triggers the caspase-3-mediated death of immune cells\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere we have investigated the role of NucA in a well-established mouse model of septic arthritis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Our data show that NucA causes in the hematogenous septic arthritis mouse model massive bone destruction, rapid weight loss and high proinflammatory cytokine production. \u003cem\u003eIn vitro\u003c/em\u003e analysis suggest that the increased host cell internalization and killing of neutrophils as well as NETs degrading activity by NucA and its induction of proinflammatory cytokins in certain host cells could be responsible for the high \u003cem\u003ein vivo\u003c/em\u003e pathogenicity.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e \u003cb\u003eNewman Δ\u003c/b\u003e\u003cb\u003enuc1\u003c/b\u003e \u003cb\u003emutant is much less pathogenic in the mouse model of septic arthritis.\u003c/b\u003e The pathogenicity of \u003cem\u003eS. aureus\u003c/em\u003e Newman Wild Type (NWT) and its Δ\u003cem\u003enuc1\u003c/em\u003e mutant was evaluated in a mouse model of \u003cem\u003eS. aureus\u003c/em\u003e septic arthritis. Mice received intravenous inoculations with both NWT or its Δ\u003cem\u003enuc1\u003c/em\u003e mutant. Remarkably, Δ\u003cem\u003enuc1\u003c/em\u003e-infected mice exhibited minimal weight loss until day 7, whereas NWT-infected mice continued to loose weight, reaching 20% by the experiment's termination on day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Likewise, Δ\u003cem\u003enuc1\u003c/em\u003e-infected mice displayed significantly lower clinical arthritis symptoms than NWT-infected mice. Twenty percent of Δ\u003cem\u003enuc1\u003c/em\u003e-infected mice developed mild clinical arthritis symptoms up to day 7. In contrast, 40% of NWT-infected mice exhibited clinical arthritis symptoms as early as day 3, and by day 5, all animals had developed severe septic arthritis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Not only was the frequency of arthritis higher, but the severity of septic arthritis was also elevated in mice infected with the NWT strain compared to the mutant strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Importantly, both the kidney bacterial load (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) and kidney abscess scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) were significantly lower in the mice infected with the Δ\u003cem\u003enuc1\u003c/em\u003e mutant compared to those infected with its parental strain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further confirm our clinical observations, we conducted \u0026micro;CT scans on all joints from mice inoculated with \u003cem\u003eS. aureus\u003c/em\u003e. Intravenous injection of \u003cem\u003eS. aureus\u003c/em\u003e NWT in NMRI mice resulted in severe bone destruction in 12% of joints after 7 days post-infection, whereas mice infected with the Δ\u003cem\u003enuc1\u003c/em\u003e mutant showed almost no sign of bone erosion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec shows representative 3D images of a wrist, a knee, and a shoulder from mice infected either with the Δ\u003cem\u003enuc1\u003c/em\u003e mutant or NWT strain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInfection with wild type\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e \u003cb\u003eresults in a massive increase in the levels of IL-6 and S100A8/A9.\u003c/b\u003e On day 7 post infection we collected blood samples from the infected mice and measured the levels of selected pro-inflammatory cytokines, including IL-6, TNF, KC (CXCL1), and S100A8/A9. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, the levels of IL-6, KC (CXCL1), and S100A8/A9 were markedly reduced in Δ\u003cem\u003enuc1\u003c/em\u003e-infected mice as compared to NWT-infected mice. While the TNF levels tended to be lower in Δ\u003cem\u003enuc1\u003c/em\u003e-infected mice, the difference did not reach statistical significance. Collectively, our results indicate that NucA is a crucial virulence factor in the pathogenicity of \u003cem\u003eS. aureus\u003c/em\u003e in an infection model of septic arthritis. We further investigated possible reasons of the lower pathogenicity of the Δ\u003cem\u003enuc1\u003c/em\u003e mutant in the septic mouse model using different host cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNucA digestion of gDNA decreased TNF-α production in mouse macrophages.\u003c/b\u003e It is well known that the TLR9 receptor in macrophages recognizes unmethylated bacterial DNA and CpG dinucleotides. As staphylococcal macromolecules are frequently contaminated with lipoproteins/lipopeptides that are sensed at by TLR2 at picomolar levels, cytokine induction could be be due to these compounds. To exclude cytokine induction by contaminated lipopeptides, we isolated the gDNA from the \u003cem\u003eS. aureus\u003c/em\u003e SA113Δ\u003cem\u003elgt\u003c/em\u003e mutant, which lacks the phosphatidylglycerol:prolipoprotein diacylglyceryl transferase Lgt, in which no lipidation of lipoproteins takes place and therefore no TLR2 response can be triggered\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Indeed, genomic DNA (gDNA) from \u003cem\u003eS. aureus\u003c/em\u003e SA113Δ\u003cem\u003elgt\u003c/em\u003e concentration-dependently increased the production of the proinflammatory cytokine TNF-α in mouse macrophages RAW 264.7, while gDNA from mouse macrophages RAW 264.7 did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The effect was already visible at \u003cem\u003eS. aureus\u003c/em\u003e gDNA concentration of 3 ng/ml. The standard CpG oligonucleotides ODN2006 and the non-CpG oligonucleotides ODN2137 were used as positive and negative control, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOf the two nuclease genes (\u003cem\u003enuc1\u003c/em\u003e and \u003cem\u003enuc2\u003c/em\u003e) in \u003cem\u003eS. aureus\u003c/em\u003e, it is the \u003cem\u003enuc1\u003c/em\u003e encoded NucA that is secreted into the supernatant and is able to completely degrade extracellular DNA and RNA (eDNA and eRNA, respectively). This was demonstrated when we incubated the supernatant of JE2 and its mutants JE2Δ\u003cem\u003enuc1\u003c/em\u003e, JE2Δ\u003cem\u003elgt\u003c/em\u003e and JE2Δ\u003cem\u003enuc1\u003c/em\u003eΔ\u003cem\u003elgt\u003c/em\u003e with \u003cem\u003eS. aureus\u003c/em\u003e gDNA. In all mutants in which \u003cem\u003enuc1\u003c/em\u003e was deleted, the gDNA remained intact, whereas in JE2 and the JE2Δ\u003cem\u003elgt\u003c/em\u003e mutant the gDNA was completely degraded (\u003cb\u003eSupplementary Fig.\u0026nbsp;1a\u003c/b\u003e). Furthermore, the degradation of eDNA by recombinant NucA, which was expressed and purified from \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e), suggested that NucA plays a role in controlling DNA/RNA-dependent immune stimulation. Indeed, the stepwise degradation of \u003cem\u003eS. aureus\u003c/em\u003e gDNA by increasing amounts of NucA (from 46 pM to 3 nM) correlated with a stepwise decrease in TNF-α production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,c).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003eS. aureus nuc1\u003c/b\u003e \u003cb\u003edeletion mutants have an impact on cytokines production in various cell lines.\u003c/b\u003e We investigated whether the immune stimulation of JE2 and its mutants differed. We incubated live \u003cem\u003eS. aureus\u003c/em\u003e JE2 and its JE2Δ\u003cem\u003enuc1\u003c/em\u003e, JE2Δ\u003cem\u003elgt\u003c/em\u003e, and JE2Δ\u003cem\u003enuc1\u003c/em\u003eΔ\u003cem\u003elgt\u003c/em\u003e mutants for 18 h with RAW 264.7, SAOS-2 cells, and 5 h with neutrophils. There was no difference in IL-6 and TNF-α production between JE2 and JE2Δ\u003cem\u003enuc1\u003c/em\u003e stimulation in RAW 264.7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In SAOS-2 cells, IL-6 production was decreased in response to all mutants while in neutrophils the production of TNF-α, IL-10, and IL-1Ra was significantly decreased in response to all the mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c), particularly those in which \u003cem\u003elgt\u003c/em\u003e was deleted (JE2Δ\u003cem\u003elgt\u003c/em\u003e and JE2Δ\u003cem\u003enuc1\u003c/em\u003eΔ\u003cem\u003elgt)\u003c/em\u003e. Similar results were observed in NWT and Δ\u003cem\u003enuc1\u003c/em\u003e. Compared with NWT, there was less IL-6 production in RAW 264.7 and no significant difference in IL-6 and TNF-α in SAOS-2 when exposed to Δ\u003cem\u003enuc1\u003c/em\u003e strain (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e), which suggested a similar pathogenicity in Newman and JE2 strains. The results show that at the bacterial level not so much bacterial DNA but the TLR-2 activating lipoproteins are the prominent immune stimulants. As the generated mutants showed hardly any difference in growth and hemolytic activity compared to the parent strain (\u003cb\u003eSupplementary Fig.\u0026nbsp;2b,c,d\u003c/b\u003e), we assume that all the effects seen are mainly due to the deletion of \u003cem\u003enuc1\u003c/em\u003e or \u003cem\u003elgt\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eJE2Δ\u003c/b\u003e \u003cb\u003enuc1\u003c/b\u003e \u003cb\u003eexhibit decreased internalization or survival in various host cells.\u003c/b\u003e In neutrophils the bacterial survival was determined. The survival of JE2Δ\u003cem\u003enuc1\u003c/em\u003e, JE2Δ\u003cem\u003elgt\u003c/em\u003e and JE2Δ\u003cem\u003enuc1\u003c/em\u003eΔ\u003cem\u003elgt\u003c/em\u003e was significantly decreased compared to JE2, indicating that the mutants were better killed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). We investigated whether live JE2 and its mutants differed in internalization by different host cells such as RAW 264.7 and SAOS-2 cells. After 2 h incubation with \u003cem\u003eS. aureus\u003c/em\u003e strains, membrane adherent and extracellular bacteria were killed, and then the CFU of internalized bacteria was determined. In both RAW 264.7 and SAOS-2 cells we observed less CFUs upon incubation with the Δ\u003cem\u003enuc1\u003c/em\u003e mutants as compared to JE2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb,c). In the complemented strain JE2Δ\u003cem\u003enuc1\u003c/em\u003e (pRB\u003cem\u003enuc1\u003c/em\u003e) JE2 phenotype was restored, suggesting that internalization was affected by \u003cem\u003enuc1\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of live bacteria and supernatant on NET formation and clearing.\u003c/b\u003e Neutrophils act as the first defense line of the innate immune system and form neutrophil extracellular traps (NETs) to kill pathogens. Here, neutrophils were exposed to live bacteria (MOI:2) and bacterial supernatants for 2 and 5 h following eDNA-staining with SYTOX Green. Live bacteria caused a time-dependent increase in stained eDNA, starting from 3% after 2 h to 10% after 5 h exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cb\u003e-Bacteria\u003c/b\u003e). Not much difference in relative fluorescence units (RFU) was observed between exposure to JE2 and its mutants over the timeline. This was also visible in the fluorescence microscopic images after 2 and 5 h of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e-Bacteria\u003c/b\u003e). The fluorescent labeled eDNA appeared as typical green-fluorescent particles indicating compact chromosomal DNA which is indicative of necrosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe situation was quite different upon exposure of neutrophils to bacteria-free supernatants. The RFU decreased continuously with increasing time of exposure to all supernatants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cb\u003e-Supernatant\u003c/b\u003e). However, in neutrophils exposed to the supernatants of all \u003cem\u003enucA\u003c/em\u003e expressing strains (JE2 and JE2Δ\u003cem\u003elgt\u003c/em\u003e), the eDNA abundance was lower. In the fluorescence microscope images after 2 and 5 h of incubation with the supernatant of NucA expressing cells (JE2 and JE2Δ\u003cem\u003elgt\u003c/em\u003e) no compact stained DNA particles were visible; however, a background fluorescence of digested, smaller DNA fragments was visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e-Supernatant\u003c/b\u003e). Upon exposure to the supernatant of \u003cem\u003enuc1\u003c/em\u003e deletion mutants (JE2Δ\u003cem\u003enuc1\u003c/em\u003e and JE2Δ\u003cem\u003enuc1\u003c/em\u003eΔ\u003cem\u003elgt\u003c/em\u003e) neutrophil DNA appeared as diffuse areal, which is indicative of NET. This suggested that NucA-contained supernatant showed lower DNA-abuandance when exposed to neutrophils.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe comparative studies between \u003cem\u003eS. aureus\u003c/em\u003e parental strain and the Δ\u003cem\u003enuc1\u003c/em\u003e mutant revealed that NucA is an important virulence factor in septic arthritis. \u003cem\u003eS. aureus\u003c/em\u003e Newman wild type (NWT)-infected mice showed massive weight loss, much increased clinical arthritis frequency, a 3-fold abscess score, a massive bone erosion and very high IL-6 content in the plasma. In contrast, the Δ\u003cem\u003enuc1\u003c/em\u003e-infected mice showed hardly any signs of septic arthritis, there was almost no weight loss, the clinical arthritis and abscess score were much lower, and the bacterial load in kidneys was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Most remarkable, however, was that Δ\u003cem\u003enuc1\u003c/em\u003e-infected mice showed almost no bone erosion in the joints (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c).\u003c/p\u003e \u003cp\u003eThe question that arises in this context is the molecular causes of NucA-induced septic arthritis and bone erosion. One reason could be the increased IL-6 content. In the septic arthritis mouse model, NWT induces an almost 100-fold higher IL-6 production in plasma than its Δ\u003cem\u003enuc1\u003c/em\u003e mutant. Indeed, the proinflammatory IL-6, which is mainly produced by macrophages and T lymphocytes in response to pathogens, is not only a key player in rheumatoid arthritis. It also promotes megakaryocyte maturation and the release of platelets when reaching the bone marrow\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The massive upregulation of S100A8/A9 in NWT-infected mice compared to the Δ\u003cem\u003enuc1\u003c/em\u003e-infected mice could be a stimulation to IL-6 triggered inflammation and leukocyte recruitment\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The high levels of IL-6 and S100A8/A9 at the end of the mouse experiment reflect the severity of infection. Inflammatory cytokines play a role in bone remodeling process. For example, in IL-6 deficient mice, the bone erosion was lower\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The scenario for bone disruption could therefore look like this: NucA causes high IL-6 production which may lead to uncontrolled progression of bone destruction by osteoclasts.\u003c/p\u003e \u003cp\u003eIn various cell culture studies, we also tried to better understand the role of NucA in septic arthritis. JE2Δ\u003cem\u003enuc1\u003c/em\u003e mutant triggered no increased IL-6 or TNF-α production in RAW 264.7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). What we see is that lipoproteins play a decisive role in immune stimulation in RAW 264.7 cells, which is in agreement with earlier results\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, JE2Δ\u003cem\u003enuc1\u003c/em\u003e mutant induced less IL-6 in SAOS-2 cells and less IL-10, TNF-α and IL-1RA in neutrophils, while NWTΔ\u003cem\u003enuc1\u003c/em\u003e induced less IL-6 in RAW264.7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003eand Supplementary Fig.\u0026nbsp;3\u003c/b\u003e). We also found that the internalization of the JE2Δ\u003cem\u003enuc1\u003c/em\u003e mutant was decreased in both RAW 264.7 and SAOS-2 cells (\u003cb\u003eFig.\u0026nbsp;5bc\u003c/b\u003e), and JE2Δ\u003cem\u003enuc1\u003c/em\u003e mutant was better killed by neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), suggesting that NucA shields \u003cem\u003eS. aureus\u003c/em\u003e from phagocyte engulfment and killing and effectively digests NETs formed by neutrophils. These results suggest that NucA impacts immune stimulation and contributes to an increase in the severity of septic arthritis.\u003c/p\u003e \u003cp\u003eMost bacteria release DNA and RNA during proliferation. In \u003cem\u003eS. aureus\u003c/em\u003e DNA is released during cell lysis resulting from induction of prophages or activation of proteins with holin-like properties such as CidA and LrgA\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The secreted NucA degrades very efficiently eDNA/RNA to allow the reuse of the degradation products. How powerful NucA is in degrading eDNA is illustrated in the \u003cb\u003eSupplementary Fig.\u0026nbsp;1a\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWhen the supernatants of JE2 and its Δ\u003cem\u003enuc1\u003c/em\u003e mutant was incubated with gDNA, this was completely degraded within 1 h by the supernatant of JE2 but not the Δ\u003cem\u003enuc1\u003c/em\u003e mutants. This efficient degradation of eDNA ensures not only the reuse of nucleic acid building blocks but also the escape of staphylococci from a biofilm community and from NETs, providing NucA-expressing bacteria a clear advantage during infection\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe receptor for bacterial DNA is TLR-9, which is mainly expressed in immune cells such as dendritic cells and macrophages, but also in other non-immune cells including muscle and epithelial cells\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In RAW 264.7 mouse macrophages only large-sized staphylococcal DNA induced TNF-α production in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea); as soon as the gDNA was degraded by NucA TNF-α production decreased with progressing degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d). Similar results were also obtained with Group A \u003cem\u003eStreptococcus\u003c/em\u003e (GAS), which produce the DNase Sda1 to prevent IFN-α and TNF-α secretion by murine macrophages\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. TNF-α contributes to pathogenicity in the initiation and progression of septic arthritis, as TNF-α deficient mice developed less severe forms of arthritis\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Moreover, combining antibiotics with a TNF-α inhibitor yielded superior results compared to antibiotics alone. The combination effectively reduced synovitis and joint destruction in a mouse model of septic arthritis \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Simultaneously, TNF-α plays a crucial role in the Th1 response and primes phagocytes for effective elimination of pathogens\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Anti-TNF-α treatment was shown to compromise immune killing efficacy, leading to increased kidney bacterial load in a mouse model of \u003cem\u003eS. aureus\u003c/em\u003e septic arthritis\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe mouse model for septic arthritis in our study was performed with \u003cem\u003eS. aureus\u003c/em\u003e Newman and its Δ\u003cem\u003enuc1\u003c/em\u003e mutant. A control with JE2 (a USA300 derivative) was not possible, as approval was not granted due to its resistance to methicillin. We therefore asked how similar or dissimilar are these strains. In a previous study both strains, USA300 and Newman, were compared in a mouse sepsis model and there was no significant difference observed between the strains\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Growth kinetics and hemolytic activity also revealed similar phenotypes (\u003cb\u003eSupplementary Fig.\u0026nbsp;1b,c,d\u003c/b\u003e). Both Δ\u003cem\u003enuc1\u003c/em\u003e mutants could be complemented to nuclease production so we are pretty sure that JE2, Newman and their Δ\u003cem\u003enuc1\u003c/em\u003emutants behave similarly.\u003c/p\u003e \u003cp\u003eIn hematogenous septic arthritis, compromised innate immune defenses increase the likelihood of bacteria in the bloodstream invading joints, ultimately leading to the development of septic arthritis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Neutrophils are recognized as vital immune cells guarding against \u003cem\u003eS. aureus\u003c/em\u003e septic arthritis. Neutrophil-depleted mice exhibited heightened and more frequent septic arthritis, coupled with compromised bacterial clearance as evidenced by elevated CFU counts in both blood and kidneys\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. We also compared the effects of live bacteria and the corresponding culture supernatant on neutrophil extracellular traps (NETs) degradation. NETs represent a form of innate immune response that binds microorganisms and prevents them from spreading; and the high local concentration of antimicrobial agents may kill bacteria\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It is well known that the \u003cem\u003eS. aureus\u003c/em\u003e thermonuclease (NucA) degrades the released DNA of NETs to escape scavenging and killing\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhen we exposed neutrophils to live \u003cem\u003eS. aureus\u003c/em\u003e for up to 5 h we observed a time-dependent increase in stained eDNA, but there was not much difference between JE2 and its mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,b\u003cb\u003e-Bacteria\u003c/b\u003e). However, when we incubated neutrophils with the corresponding supernatants, we observed a clear difference between the \u003cem\u003enuc1\u003c/em\u003e expressing strains JE2 and JE2Δ\u003cem\u003elgt\u003c/em\u003e and the Δ\u003cem\u003enuc1\u003c/em\u003e mutants. In JE2Δ\u003cem\u003enuc1\u003c/em\u003e and JE2Δ\u003cem\u003enuc1\u003c/em\u003eΔ\u003cem\u003elgt\u003c/em\u003e neutrophil eDNA was stained as diffuse areal which is indicative of NET formation, while in \u003cem\u003enuc1\u003c/em\u003e expressing strains there was almost no eDNA stained (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e-Supernatant\u003c/b\u003e). The question remains: why did we not see a major differences between the wild type and the Δ\u003cem\u003enuc1\u003c/em\u003e live bacteria? We assume that by washing the bacterial cells with PBS, NucA is washed out and the bacteria are rapidly phagocytized and killed when intubated with neutrophils.\u003c/p\u003e \u003cp\u003eOverall, our study suggests that NucA plays a crucial role in the pathogenesis of \u003cem\u003eS. aureus\u003c/em\u003e septic arthritis, as evidenced by Δ\u003cem\u003enuc1\u003c/em\u003e-infected mice showing reduced arthritis severity, bone erosion, and kidney abscess formation, alongside lower bacterial loads. \u003cem\u003eIn vitro\u003c/em\u003e data further support these findings, demonstrating that NucA degrades bacterial DNA, shields \u003cem\u003eS. aureus\u003c/em\u003e from phagocyte killing, and digests neutrophil extracellular traps, ultimately promoting bacterial survival and worsening disease severity. How NucA triggers massive bone erosion is the subject of further research.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial strains, plasmids, primers, and culture conditions\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBacterial strains and plasmids used in this study are described in Supplementary \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e. All the primers are listed in \u003cstrong\u003eSupplementary Table\u0026nbsp;2\u003c/strong\u003e. \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3) was grown in Luria-broth medium (LB), and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strains were cultured in tryptic soy broth (TSB, Millipore, Merck) or basic medium (BM) broth (Luria broth supplemented with 0.1% K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e and 0.1% glucose) or stored as previously mentioned\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. To keep the plasmids in the bacteria, \u003cem\u003eE. coli\u003c/em\u003e was supplied with ampicillin or kanamycin and \u003cem\u003eS. aureus\u003c/em\u003e was supplied with 10 \u0026micro;g/ml chloramphenicol. For growth kinetic comparison, the \u003cem\u003eS. aureus\u003c/em\u003e JE2, Newman and their mutants were cultured into TSB medium, and measured with Varioskan LUX Multimode Microplate Reader (Thermo fisher) for 24 h.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDeletion of\u003c/strong\u003e \u003cstrong\u003enuc1\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003elgt\u003c/strong\u003e \u003cstrong\u003ein\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003enuc1\u003c/em\u003e is the nuclease1-encoding gene and \u003cem\u003elgt\u003c/em\u003e is the lipoprotein diacylglyceryl transferase enzyme-encoding gene\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. For deleting \u003cem\u003enuc1\u003c/em\u003e and \u003cem\u003elgt\u003c/em\u003e genes, the knockout plasmid pBASE6 was employed. The disruption primers were designed to contain upstream and downstream of the target genes regions. Fragments were generated by PCR and subcloned into EcoRV digested vector pBASE6, resulting in pBASE6\u0026Delta;\u003cem\u003enuc1\u003c/em\u003e and pBASE6\u0026Delta;\u003cem\u003elgt\u003c/em\u003e. The resulting plasmids were transformed into \u003cem\u003eE. coil\u003c/em\u003e DC10B for amplification. Then, plasmids were isolated and checked for DNA sequences. The correct plasmids pBASE6\u0026Delta;\u003cem\u003enuc1\u003c/em\u003e and pBASE6\u0026Delta;\u003cem\u003elgt\u003c/em\u003e were transformed into an intermediate host, \u003cem\u003eS. aureus\u003c/em\u003e RN4220, by electroporation to restrict foreign DNA and then into \u003cem\u003eS. aureus\u003c/em\u003e JE2 or \u003cem\u003eS. aureus\u003c/em\u003e Newman (NWT). The process for deletion of \u003cem\u003elgt\u003c/em\u003e and \u003cem\u003enuc1\u003c/em\u003e from \u003cem\u003eS. aureus\u003c/em\u003e was followed as described previously, yielding JE2\u0026Delta;\u003cem\u003enuc1\u003c/em\u003e, Newman\u0026Delta;\u003cem\u003enuc1\u003c/em\u003e, JE2\u0026Delta;\u003cem\u003elgt\u003c/em\u003e, and JE2\u0026Delta;\u003cem\u003enuc1\u003c/em\u003e\u0026Delta;\u003cem\u003elgt\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eConstruction of complementary strain\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eFor complementation, the plasmid pRB473-\u003cem\u003enuc1\u003c/em\u003e was introduced. The \u003cem\u003enuc1\u003c/em\u003e fragment was amplified, ligated to pRB473 plasmids and transformed into \u003cem\u003eE. coli\u003c/em\u003e DC10B. Positive colonies were selected and verified via DNA sequencing. Then correct plasmid was purified and transformed into JE2\u0026Delta;\u003cem\u003enuc1\u003c/em\u003e and \u0026Delta;\u003cem\u003enuc1\u003c/em\u003e, yielding JE2\u0026Delta;\u003cem\u003enuc1\u003c/em\u003e(pRB\u003cem\u003enuc1\u003c/em\u003e) and \u0026Delta;\u003cem\u003enuc1\u003c/em\u003e(pRB\u003cem\u003enuc1\u003c/em\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eHemolytic activity and nuclease activity assay\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe bacteria were grown in TSB medium overnight. The OD578 of overnight culture was adjusted and spotted on the Blood Agar (TSA with Sheep Blood) plates (Thermo Fisher) at 37\u003csup\u003eo\u003c/sup\u003eC and then the hemolysis zone was measured. The supernatants of overnight culture were checked for nuclease activity using DNase Test Agar with Toluidine Blue (Merck, Millipore). The DNase Test Agar plates were incubated at 37\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMouse model for\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003eseptic arthritis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo compare the pathogenicity of \u003cem\u003eS. aureus\u003c/em\u003e Newman wild-type strain (NWT) and its \u0026Delta;\u003cem\u003enuc1\u003c/em\u003e mutant, a septic arthritis mouse model was used. NMRI female mice, aged 8 weeks, were purchased from Envigo (Venray, Netherlands). All mice were housed at the animal facility at the University of Gothenburg. Mice were kept under standard temperature and light conditions and were fed laboratory chow and water ad libitum. Mice (n\u0026thinsp;=\u0026thinsp;5) in respective groups were intravenously injected with a 200 \u0026micro;l arthritic dose of either \u003cem\u003eS. aureus\u003c/em\u003e Newman strain (2.8\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mouse) or \u0026Delta;\u003cem\u003enuc1\u003c/em\u003e mutant (2.8\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mouse). Mice were monitored for weight loss and clinical signs of arthritis from day 0 to day 7. On day 7, mice were sacrificed to collect samples including blood, kidneys, joints. Kidneys were collected aseptically and scored on a scale of 0 (no abscess), 1 (mild abscess), 2 (moderate abscess) to 3 (severe abscess). The kidneys were then minced and diluted with sterile PBS. Dilutions were plated on horse blood agar plates, incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 24 hours, and colonies obtained were counted using a colony counter (Stuart Scientific, Made in the UK).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eMicrocomputed tomography (\u0026micro;CT)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAll four paws were scanned by SkyScan 1176 \u0026micro;CT (Bruker, Antwerp, Belgium). The scanning was conducted at 55 kV/ 455 \u0026micro;A, with a 0.2-mm aluminum filter. The exposure time was 47 ms. The X-ray projections were obtained at 0.7\u0026deg; intervals with a scanning angular rotation of 180\u0026deg;. The NRecon software (version 1.6.9.8; Bruker) was used to reconstruct 3D images which were further evaluated by using CT vox (version 2.7.0; Bruker). Each joint was evaluated by two researchers (M.D. and T.J.) using a scoring system from 0 to 3 (0: healthy joint; 1: mild bone destruction; 2: moderate bone destruction; and 3: marked bone destruction) as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eNucA expression and purification\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNucA is an extracellular enzyme, which is secreted as mature Nuc1. The \u003cem\u003enucA\u003c/em\u003e gene was cloned into vector pET28a with C-terminal His-tag and this construct was transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). The transformant carrying pET28a-NucA-6xHis was grown in LB at 37\u003csup\u003eo\u003c/sup\u003eC supplemented with 50 \u0026micro;g/ml ampicillin. When OD600 reached 0.6\u0026ndash;0.8, the bacteria were induced with 1 mM IPTG at 18\u003csup\u003eo\u003c/sup\u003eC overnight for overexpressing NucA. The overnight culture was collected, resuspended in buffer A (50 mM Tris-HCl pH 8.0, 300 mM NaCl), and lysed by an ultra-sonicator with a pulse every 4 s for 4 min. The lysate was centrifuged at 14,000 g for 1 h. The supernatant of lysate was collected and then loaded onto the Ni-NTA column. Fractions containing NucA were collected with Buffer B (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 250 nM imidazole) and analyzed by SDS-PAGE. NucA enzyme was dialyzed with PBS, concentrated, flash-frozen in liquid nitrogen, and stored at -80\u003csup\u003eo\u003c/sup\u003eC until use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenomic bacterial DNA (gDNA) degradation assay with NucA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e SA113\u0026Delta;\u003cem\u003elgt\u003c/em\u003e was cultured in TSB medium for overnight. The bacterial pellet was collected and resuspended in TE buffer supplemented with lysostaphin and RNase at 37\u003csup\u003eo\u003c/sup\u003eC for 30 min. Bacterial gDNA was then purified via phenol-chloroform-isoamyl alcohol, precipitated with isopropanol, washed with ethanol, and then dissolved in H\u003csub\u003e2\u003c/sub\u003eO\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. gDNA was incubated with varying concentrations of recombinant NucA for 1 h at 37\u003csup\u003eo\u003c/sup\u003eC. The samples were then added to RAW 264.7 cells for stimulation and loaded on the agarose gel for visualization.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003ePreparation of bacteria and bacterial supernatant\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBM medium was used for inoculating \u003cem\u003eS. aureus\u003c/em\u003e at 37\u003csup\u003eo\u003c/sup\u003eC with shaking from fresh BM agar plates. The cultures were harvested after 16 h by centrifuging and washed with PBS. To get bacterial dosage (MOI, multiplicity of infection), bacteria were calculated to OD/CFU. Bacterial supernatants were collected and filtered with a 0.2 \u0026micro;m pyrogen-free round column. The supernatants were kept on ice until use and adjusted to equal concentrations according to bacterial number which is described in former study\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The supernatant was tested for its ability to degrade gDNA and its activity on neutrophiles.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eNeutrophils Isolation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eVenous blood was freshly collected by EDTA-tubes (Sarstedt, Germany) from several healthy individuals. 6 mL blood was layered on 6 mL of Lympholyte poly-cell separation medium (Cedarlane, Burlington, Canada). Centrifugation was done without the break, at 500\u0026times;g for 40 min at room temperature. PMN layer was collected and washed twice with 12 mL PBS and centrifuged at 400\u0026times;g for 10 min at room temperature with settings of acceleration 5 and deceleration 4. Cells were resuspended in RPMI medium without phenol red (Sigma-Aldrich, Darmstadt, Germany). Cell counts were obtained by the Trypan Blue exclusion method, utilizing a Neubauer counting chamber.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell culture and immune stimulation assay\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe murine macrophages cell line RAW 264.7 was cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37\u003csup\u003eo\u003c/sup\u003eC with 5% CO\u003csub\u003e2\u003c/sub\u003e. The human osteoblast-like cell line SAOS-2 was grown in McCoy\u0026apos;s 5A Medium (Sigma) supplemented with 15% FBS, 1% Glucose and 1% penicillin-streptomycin. Prior to stimulation, RAW 264.7 and SAOS-2 cells were seeded in 96-well plates and incubated overnight until reaching confluency, while neutrophils were directly seeded in plates before adding all stimulants. Immune stimulation was performed for 18 h at 37\u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e, except neutrophils were stimulated for 5 h. The cellular supernatants were then collected and stored at -20\u003csup\u003eo\u003c/sup\u003eC until determining the cytokines production.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eDetection of cytokines by ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe cytokines collected from cellular supernatant of different cell lines were measured with the uncoated ELISA kit (Invitrogen) according to instructions. The plasma levels of IL-6, TNF-\u0026alpha;, keratinocyte chemoattractant (KC), and S100A8/A9 in blood collected from NMRI mice infected intravenously with Newman WT (NWT) and \u0026Delta;\u003cem\u003enuc1\u003c/em\u003e were quantified using DuoSet ELISA kits (R\u0026amp;D Systems Europe) according to the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eInvasion assay\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eRAW 264.7 and SAOS-2 cells were seeded in 24-well plates with 500\u0026micro;l cultural medium until reaching confluency. Cells were washed with PBS, and the prewarmed cultural medium without antibiotics was added to each well. Bacteria were grown till the log phase before infection. The cells were then incubated with bacteria for 1.5 h to yield an MOI of 20:1. After incubation, gentamycin and lysostaphin were added to kill the extracellular bacteria for 1h. The cells were lysed with 0.1% Triton X-100 supplied with 0.05% Trypsin and lysates were plated to determine internalized bacteria\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial killing assay\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e JE2 and its mutants were grown overnight. Then the bacteria were collected, regrown till the log phase, washed with PBS and opsonized with 10% human pooled serum in RPMI for 1 h at 37\u003csup\u003eo\u003c/sup\u003eC. Neutrophils were seeded in 24 well plates and incubated with bacteria (100%) at a MOI\u0026thinsp;=\u0026thinsp;0.1. After 1.5 h, the neutrophils were lysed with ice-cold ddH\u003csub\u003e2\u003c/sub\u003eO and centrifuging for 15 min at 4\u003csup\u003eo\u003c/sup\u003eC. The lysates were then plated on agar plates with serial dilution and CFUs were counted the next day. The \u003cem\u003eS. aureus\u003c/em\u003e survival (killing) was calculated by comparing the counted CFU with original added CFU.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSytox Green Assay\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eIsolated neutrophils were prepared to be 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL, then 1 \u0026micro;M Sytox Green (Thermo Fisher, Waltham, USA) was added. 1% Triton X-100 solution was used for extracellular DNA normalization. Cells were stimulated for 5 h with MOI:2 live bacteria and 5% overnight bacterial supernatant. Fluorescent intensity was measured every 30 minutes at Ex 485 nm/Em 520 nm, and the cells were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e by the microplate reader (FluoStar Omega, BMG Labtech, Ortenberg, Germany). Microscopy pictures were taken with EVOS Fl (Thermo Fisher) fluorescent microscope at 2 and 5 hours of incubation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthical Statement\u003c/h2\u003e \u003cp\u003e The Ethics Committee of Animal Research of Gothenburg approved all experiments conducted on mice. The mouse experiments were performed in accordance with the Swedish Board of Agriculture's regulations and recommendations on animal experiments. Blood was collected from healthy adult volunteers and written informed consent was given. The institutional review board of the University of T\u0026uuml;bingen approved the study and all adult subjects provided informed consent. This study was done in accordance with the ethics committee of the medical faculty of the University of T\u0026uuml;bingen that approved the study (Approval number 015/2014 BO2).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistics and reproducibility\u003c/strong\u003e \u003cp\u003eAll the data are analyzed using GraphPad Prism (version 8.0; GraphPad Software). The data are represented in mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical significances: not significant p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Details of statistical analyses for each experiment are provided in \u0026lsquo;Materials and Methods\u0026rsquo;.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthors contributions\u003c/h2\u003e \u003cp\u003eFG, NL designed the study and the experiments; TJ, MVD designed and carried out the septic arthritis model; FS, SE, AN and NL carried out neutrophil experiments; NL, NH constructed deletion mutants; NL, AVA and AW carried out cell lines experiments; FG, NL wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by funding from the Deutsche Forschungsgemeinschaft the Germany\u0026rsquo;s Excellence Strategy \u0026ndash; EXC 2124\u0026ndash;390838134 \u0026lsquo;Controlling Microbes to Fight Infections\u0026rsquo; to F.G., grants from the Swedish state under the agreement between the Swedish Government and the county councils, the ALF-agreement grant number ALFGBG-823941 to T.J., N.L. was supported by the Chinese Scholarship Council. We are grateful to Stefanie Krajewski, Clinical Research Laboratory, Department of Thoracic, Cardiac and Vascular Surgery, University Hospital T\u0026uuml;bingen, 72076 T\u0026uuml;bingen, Germany, for providing us with SAOS-2 cells. S.E. received funding from the German Research Council (EH471/5\u0026thinsp;\u0026minus;\u0026thinsp;1).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data generated and annlysed in this study\u0026rsquo;s are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMathews, C.J., Weston, V.C., Jones, A., Field, M., Coakley, G.: Bacterial septic arthritis in adults. Lancet. \u003cb\u003e375\u003c/b\u003e, 846\u0026ndash;855 (2010)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaandorp, C.J., Krijnen, P., Moens, H.J., Habbema, J.D., van Schaardenburg, D.: The outcome of bacterial arthritis: a prospective community-based study. 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PLoS One. \u003cb\u003e12\u003c/b\u003e, e0171222 (2017)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDalpke, A., Frank, J., Peter, M., Heeg, K.: Activation of toll-like receptor 9 by DNA from different bacterial species. Infect. Immun. \u003cb\u003e74\u003c/b\u003e, 940\u0026ndash;946 (2006)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen, M.T., et al.: The νSaα Specific Lipoprotein Like Cluster (\u003cem\u003elpl\u003c/em\u003e) of \u003cem\u003eS. aureus\u003c/em\u003e USA300 Contributes to Immune Stimulation and Invasion in Human Cells. PLoS Pathog. \u003cb\u003e11\u003c/b\u003e, e1004984 (2015)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4848416/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4848416/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeptic arthritis, primarily caused by \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, poses a significant risk of both mortality and morbidity due to its aggressive nature. The \u003cem\u003enuc1\u003c/em\u003e-encoded thermonuclease NucA of \u003cem\u003eS. aureus\u003c/em\u003e degrades extracellular DNA/RNA, allowing the pathogen to escape neutrophil extracellular traps (NETs) and maintain the infection unabated. Here we show that in the mouse model for hematogenous septic arthritis the Δ\u003cem\u003enuc1\u003c/em\u003e mutant was much less pathogenic and the severity of clinical septic arthritis was markedly reduced, including decreased weight loss, lower kidney bacterial loads and much less IL-6 production. In vitro, \u003cem\u003eS. aureus\u003c/em\u003e genomic DNA induced in macrophages a robust TNF-α response which was abrogated when the DNA was degraded by NucA. NucA induced higher IL-6 production in SAOS-2 and higher TNF-α and IL-10 production in neutrophils and shielded \u003cem\u003eS. aureus\u003c/em\u003e from phagocyte engulfment and killing. NucA exacerbates septic arthritis possibly by increased internalization by host cells and killing of neutrophiles.\u003c/p\u003e","manuscriptTitle":"Staphylococcus aureus thermonuclease NucA is a key virulence factor in septic arthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-24 02:42:06","doi":"10.21203/rs.3.rs-4848416/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a9944054-f920-4e8e-8767-7d6e1d8e4fe3","owner":[],"postedDate":"September 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36498403,"name":"Biological sciences/Microbiology/Pathogens"},{"id":36498404,"name":"Health sciences/Diseases/Infectious diseases"}],"tags":[],"updatedAt":"2025-04-11T07:08:16+00:00","versionOfRecord":{"articleIdentity":"rs-4848416","link":"https://doi.org/10.1038/s42003-025-07920-4","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-04-10 04:00:00","publishedOnDateReadable":"April 10th, 2025"},"versionCreatedAt":"2024-09-24 02:42:06","video":"","vorDoi":"10.1038/s42003-025-07920-4","vorDoiUrl":"https://doi.org/10.1038/s42003-025-07920-4","workflowStages":[]},"version":"v1","identity":"rs-4848416","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4848416","identity":"rs-4848416","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}