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

doi:10.21203/rs.3.rs-4848416/v1

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

https://doi.org/10.21203/rs.3.rs-4848416/v1

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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 Δnuc1 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

Septic arthritis, the most aggressive joint disease carrying high mortality and morbidity risk, is predominantly caused by Staphylococcus aureus¹. 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 dysfunction². 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 nuc2⁵.

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 Ca²⁺-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-products⁶.

NucA functions to degrade extracellular DNA (eDNA), thus promoting the dispersal and destabilization of biofilms. Consequently, in the S. aureus Δnuc1 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 mortality⁷. 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 microbes⁸. 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 neutrophils⁹. 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 cells¹⁰.

Here we have investigated the role of NucA in a well-established mouse model of septic arthritis¹¹. 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.

The S. aureus Newman Δnuc1 mutant is much less pathogenic in the mouse model of septic arthritis. The pathogenicity of S. aureus Newman Wild Type (NWT) and its Δnuc1 mutant was evaluated in a mouse model of S. aureus septic arthritis. Mice received intravenous inoculations with both NWT or its Δnuc1 mutant. Remarkably, Δnuc1-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. 1a). Likewise, Δnuc1-infected mice displayed significantly lower clinical arthritis symptoms than NWT-infected mice. Twenty percent of Δnuc1-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. 1b). 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. 1c). Importantly, both the kidney bacterial load (Fig. 1d) and kidney abscess scores (Fig. 1e) were significantly lower in the mice infected with the Δnuc1 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 Δnuc1 mutant showed almost no sign of bone erosion (Fig. 2a, b). Figure 2c shows representative 3D images of a wrist, a knee, and a shoulder from mice infected either with the Δnuc1 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. 2d, the levels of IL-6, KC (CXCL1), and S100A8/A9 were markedly reduced in Δnuc1-infected mice as compared to NWT-infected mice. While the TNF levels tended to be lower in Δnuc1-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 Δnuc1 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 SA113Δlgt 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 SA113Δlgt 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. 3a). 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. 3a).

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 JE2Δnuc1, JE2Δlgt and JE2Δnuc1Δlgt with S. aureus gDNA. In all mutants in which nuc1 was deleted, the gDNA remained intact, whereas in JE2 and the JE2Δlgt 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. 3b,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 JE2Δnuc1, JE2Δlgt, and JE2Δnuc1Δlgt 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 JE2Δnuc1 stimulation in RAW 264.7 (Fig. 4a). 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. 4b,c), particularly those in which lgt was deleted (JE2Δlgt and JE2Δnuc1Δlgt). Similar results were observed in NWT and Δnuc1. 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 Δnuc1 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 JE2Δnuc1, JE2Δlgt and JE2Δnuc1Δlgt was significantly decreased compared to JE2, indicating that the mutants were better killed (Fig. 5a). 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 Δnuc1 mutants as compared to JE2 (Fig. 5b,c). In the complemented strain JE2Δnuc1 (pRBnuc1) 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. 6a-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. 6b-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. 6a-Supernatant). However, in neutrophils exposed to the supernatants of all nucA expressing strains (JE2 and JE2Δlgt), 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 JE2Δlgt) no compact stained DNA particles were visible; however, a background fluorescence of digested, smaller DNA fragments was visible (Fig. 6b-Supernatant). Upon exposure to the supernatant of nuc1 deletion mutants (JE2Δnuc1 and JE2Δnuc1Δlgt) 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.

The comparative studies between S. aureus parental strain and the Δnuc1 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 Δnuc1-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 Δnuc1-infected mice showed almost no bone erosion in the joints (Fig. 2a-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 Δnuc1 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 Δnuc1-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 lower¹⁸. 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. JE2Δnuc1 mutant triggered no increased IL-6 or TNF-α production in RAW 264.7 (Fig. 4a,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, JE2Δnuc1 mutant induced less IL-6 in SAOS-2 cells and less IL-10, TNF-α and IL-1RA in neutrophils, while NWTΔnuc1 induced less IL-6 in RAW264.7 cells (Fig. 4 and Supplementary Fig. 3). We also found that the internalization of the JE2Δnuc1 mutant was decreased in both RAW 264.7 and SAOS-2 cells (Fig. 5bc), and JE2Δnuc1 mutant was better killed by neutrophils (Fig. 5a), 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 LrgA²¹. 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 Δnuc1 mutant was incubated with gDNA, this was completely degraded within 1 h by the supernatant of JE2 but not the Δnuc1 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 infection²².

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 cells²³. In RAW 264.7 mouse macrophages only large-sized staphylococcal DNA induced TNF-α production in a dose-dependent manner (Fig. 3a); as soon as the gDNA was degraded by NucA TNF-α production decreased with progressing degradation (Fig. 3c,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 macrophages²⁴. TNF-α contributes to pathogenicity in the initiation and progression of septic arthritis, as TNF-α deficient mice developed less severe forms of arthritis²⁵. 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 pathogens²⁷. 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 arthritis²⁸.

The mouse model for septic arthritis in our study was performed with S. aureus Newman and its Δnuc1 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 strains²⁹. Growth kinetics and hemolytic activity also revealed similar phenotypes (Supplementary Fig. 1b,c,d). Both Δnuc1 mutants could be complemented to nuclease production so we are pretty sure that JE2, Newman and their Δnuc1mutants 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 arthritis¹¹. 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 kidneys⁴. 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. 6a,b-Bacteria). However, when we incubated neutrophils with the corresponding supernatants, we observed a clear difference between the nuc1 expressing strains JE2 and JE2Δlgt and the Δnuc1 mutants. In JE2Δnuc1 and JE2Δnuc1Δlgt 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. 6b-Supernatant). The question remains: why did we not see a major differences between the wild type and the Δnuc1 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 Δnuc1-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.

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% K₂HPO₄ and 0.1% glucose) or stored as previously mentioned³⁴. 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 pBASE6Δnuc1 and pBASE6Δlgt. The resulting plasmids were transformed into E. coil DC10B for amplification. Then, plasmids were isolated and checked for DNA sequences. The correct plasmids pBASE6Δnuc1 and pBASE6Δlgt 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 JE2Δnuc1, NewmanΔnuc1, JE2Δlgt, and JE2Δnuc1Δlgt³⁶.

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 JE2Δnuc1 and Δnuc1, yielding JE2Δnuc1(pRBnuc1) and Δnuc1(pRBnuc1).

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^oC 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^oC.

Mouse model for S. aureus septic arthritis

To compare the pathogenicity of S. aureus Newman wild-type strain (NWT) and its Δnuc1 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 (2.8×10⁶ CFU/mouse) or Δnuc1 mutant (2.8×10⁶ 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^oC 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 described³⁷.

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^oC supplemented with 50 µg/ml ampicillin. When OD600 reached 0.6–0.8, the bacteria were induced with 1 mM IPTG at 18^oC 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^oC until use.

Genomic bacterial DNA (gDNA) degradation assay with NucA

S. aureus SA113Δlgt was cultured in TSB medium for overnight. The bacterial pellet was collected and resuspended in TE buffer supplemented with lysostaphin and RNase at 37^oC for 30 min. Bacterial gDNA was then purified via phenol-chloroform-isoamyl alcohol, precipitated with isopropanol, washed with ethanol, and then dissolved in H₂O³⁸. gDNA was incubated with varying concentrations of recombinant NucA for 1 h at 37^oC. 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^oC 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 study¹³. 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^oC with 5% CO₂. 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^oC and 5% CO₂, except neutrophils were stimulated for 5 h. The cellular supernatants were then collected and stored at -20^oC 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 Δnuc1 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 bacteria³⁹.

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^oC. 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 ddH₂O and centrifuging for 15 min at 4^oC. 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 2×10⁵ 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% CO₂ 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.

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.

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

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