During the Early Stages of Staphylococcus aureus Biofilm Formation, Induced Neutrophil Extracellular Traps Are Degraded by Autologous Thermonuclease

Staphylococcus aureus extracellular DNA (eDNA) plays a crucial role in the structural stability of biofilms during bacterial colonization; on the contrary, host immune responses can be induced by bacterial eDNA. Previously, we observed production of S. aureus thermonuclease during the early stages of biofilm formation in a mammalian cell culture medium.

ABSTRACT

Staphylococcus aureus extracellular DNA (eDNA) plays a crucial role in the structural stability of biofilms during bacterial colonization; on the contrary, host immune responses can be induced by bacterial eDNA. Previously, we observed production of S. aureus thermonuclease during the early stages of biofilm formation in a mammalian cell culture medium. Using a fluorescence resonance energy transfer (FRET)-based assay, we detected thermonuclease activity of S. aureus biofilms grown in Iscove’s modified Dulbecco’s medium (IMDM) earlier than that of widely studied biofilms grown in tryptic soy broth (TSB). The thermonuclease found was Nuc1, confirmed by mass spectrometry and competitive Luminex assay. These results indicate that biofilm development in IMDM may not rely on eDNA for structural stability. A bacterial viability assay in combination with wheat germ agglutinin (WGA) staining confirmed the accumulation of dead cells and eDNA in biofilms grown in TSB. However, in biofilms grown in IMDM, minimal amounts of eDNA were found; instead, polysaccharide intercellular adhesin (PIA) was detected. To investigate if this early production of thermonuclease plays a role in immune modulation by biofilm, we studied the effect of thermonuclease on human neutrophil extracellular trap (NET) formation using a nuc knockout and complemented strain. We confirmed that thermonuclease produced by early-stage biofilms grown in IMDM degraded biofilm-induced NETs. Additionally, neither the presence of biofilms nor thermonuclease stimulated an increase in reactive oxygen species (ROS) production by neutrophils. Our findings indicated that S. aureus, during the early stages of biofilm formation, actively evades the host immune responses by producing thermonuclease.

INTRODUCTION

Staphylococcus aureus is a well-known human pathogen that can cause mild subcutaneous infections but also life-threatening invasive infections (1–3). When infections caused by S. aureus become chronic, biofilm formation is often observed (4–7). The noncellular component of the biofilm, known as bacterial extracellular matrix (ECM), is a complex matrix that consists of polysaccharides, glycolipids, proteins, and extracellular DNA (eDNA) (8–14). eDNA formation in S. aureus biofilms is facilitated by an autolysis process that mimics apoptosis of eukaryotic cells (12) and is thought to play a crucial role in the structural stability of biofilms (14).

In a previous study from our group (15), we observed that during the early stages of biofilm formation, S. aureus produces immune modulators like staphylococcal complement inhibitor (SCIN), chemotaxis inhibitory protein of staphylococci (CHIPS), and formyl peptide receptor-like 1 inhibitor (FLIPr). These immunomodulators facilitate the defense of the developing biofilms against the host early immune responses. In addition to these immune modulators, we observed an early production of thermonuclease when S. aureus was grown in Iscove’s modified Dulbecco’s medium (IMDM) (15). S. aureus can produce the following two types of thermonucleases: Nuc1 and Nuc2 (16, 17). The major type of thermonuclease produced by S. aureus is Nuc1 (18). Thermonuclease Nuc1 production is particularly conserved across all strains of S. aureus, and it has been used as one of the specific markers to identify S. aureus (19, 20). Thermonuclease is an exoenzyme that catalyzes the hydrolysis of both DNA and RNA (21–23).

While eDNA stabilizes the biofilm structure, it also induces host immune responses. For example, unmethylated cytosine-phosphate-guanine (CpG) motifs of bacterial eDNA can be recognized by Toll-like receptor 9 (TLR9) (24, 25). In biofilm, thermonuclease is an enzyme that can dissolve and degrade eDNA that interconnects bacterial cells within the biofilm (26). Furthermore, in a study with planktonic S. aureus, thermonuclease was associated with the evasion of neutrophil extracellular traps (NETs), formed by a mechanism of cell death of neutrophils, that trap and kill bacteria (27, 28). Additionally, Berends et al. (27) demonstrated in a murine respiratory tract infection model that S. aureus thermonuclease also plays a role in immune avoidance against NETs in vivo. Taken together, thermonuclease production seems to be an important strategy for S. aureus to modulate the innate host response, allowing a biofilm to accumulate; conversely, thermonuclease is thought to destabilize biofilms as well.

To investigate this controversy, we studied, in the context of a developing biofilm, the consequences of thermonuclease production, particularly Nuc1, on (i) biofilm accumulation and (ii) defense against the innate host immune response. We first determined biofilm accumulation, composition, and thermonuclease production in IMDM, a mammalian cell culture medium, compared to those in the classical bacterial growth medium tryptic soy broth (TSB). Furthermore, we studied the NETosis-inducing abilities of early biofilms and the effect of thermonuclease Nuc1 on NET degradation, reactive oxygen species (ROS) production, and the activation of human TLR9.

RESULTS

Thermonuclease expression by early biofilms.

To determine the production of thermonuclease during the early stages of biofilm formation in both TSB and IMDM, we quantified thermonuclease activity for 2 h, starting with 3-h-old biofilms, using a fluorescence resonance energy transfer (FRET)-based assay. We saw that the thermonuclease activity of strains RN6390, Newman, and M116 was higher when grown in IMDM than when grown in TSB (P ≤ 0.001) (Fig. 1A to C). Strain RWW50 initially showed higher production of thermonuclease in IMDM than in TSB; however, this difference is lost over time (P = 0.05) (Fig. 1D).

FIG 1.

Thermonuclease production of S. aureus biofilms in TSB and IMDM. The mean fluorescence intensity (MFI) from FRET-based measurement of thermonuclease production (y axis) during the 4th and 5th hour (x axis) of biofilm formation of S. aureus RN6390 (A), Newman (B), M116 (C), and RWW50 (D) in TSB and IMDM. Figures have been generated from three independent experiments. Error bars represent SEM.

To demonstrate the timing of thermonuclease production, specifically of Nuc1, during biofilm formation in IMDM, biofilm supernatants from the 1st, 2nd, 4th, and 24th hours of biofilm formation were studied using a competitive Luminex assay (15, 29). Nuc1 was found in the supernatant of biofilms from all isolates (Fig. 2). Nuc1 expression was significantly higher in M116 (P ≤ 0.001) and RWW50 (P ≤ 0.001) (Fig. 2C and D) during the 4th hour of biofilm formation than during the 1st hour of biofilm formation. Furthermore, a positive correlation between thermonuclease production and biofilm mass was found for RN6390 (r = 0.999; P = 0.0002), Newman (r = 0.947; P = 0.053), M116 (r = 0.813; P = 0.187), and RWW50 (r = 0.847; P = 0.152) (Fig. 2). The presence of thermonuclease Nuc1 in the supernatant of the 4th hour of biofilm formation was additionally confirmed with mass spectrometry (Table 1).

FIG 2.

Thermonuclease Nuc1 expression during biofilm formation. With a competitive Luminex assay, we monitored the presence of thermonuclease Nuc1 (red line, left y axis) in the supernatants of the 1st, 2nd, 4th, and 24th hour during biofilm formation of S. aureus RN6390 (A), Newman (B), M116 (C), and RWW50 (D). Presented are biofilm mass (bar, right y axis) and thermonuclease Nuc1 (red line, left y axis). Significant statistical differences are indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). These figures have been generated from three independent experiments. Error bars presented are mean and SEM.

TABLE 1.

Mass spectrometry analysis of thermonuclease Nuc1 at the 4th and 24th hours of biofilm formation

Strain	Hours of biofilm formation	No. of unique peptides detecteda			Sequence coverage (%)a			Protein identification probability (%)a
		I	II	III	I	II	III	I	II	III
Newman	4	5			25.60			100
	24	11	3	1	40.8	16.7	6.58	100	100	92.9
RWW50	4	5	5	11	21.1	24.6	46.9	100	100	100
	24	16	10		47.8	36.8		100	100

^aThree separate experiments (experiment I, II, and III). Thermonuclease accession number is Q5HHM4.

Contribution of eDNA to biofilm mass in the early stages of biofilm formation.

If eDNA is an important factor in the accumulation of bacterial cells during biofilm formation, how is it possible that the early stages of biofilm formation in IMDM do not seem to be hampered by the presence of thermonuclease? To understand this, we investigated the biofilm content of 4- and 24-h biofilms grown in TSB and IMDM. After 4 h of biofilm formation, dead cells and eDNA were minimally present in each growth medium (Fig. 3A to C). However, after 24 h of biofilm formation, eDNA was observed, in particular, in the biofilm formed in TSB medium (Fig. 3A to C). Interestingly, in biofilms grown in IMDM, accumulation of N-acetylglucosamine, presumably the consequence of an increase of the polysaccharide intercellular adhesin (PIA), was visible after 4 h of biofilm formation and much more abundant after 24 h than in biofilms grown in TSB (Fig. 3A, B, and D).

FIG 3.

Presence of extracellular DNA and PIA in biofilms of S. aureus. Four- and twenty-four-hour biofilms of S. aureus Newman (A) and RWW50 (USA300) (B) grown in TSB and IMDM were stained with SYTO 9 (DNA/life cells, green), PI (extracellular DNA/dead cells, red), and lectin WGA (N-acetylglucosamine/PIA, blue). Figures are a representative result of three separate experiments. For relative quantification, the same staining procedure was followed, only the different stains were applied in separate wells. Results are reported as the mean ratio of the PI (C) and WGA (D) signal to the SYTO9 signal (in %) of three separate experiments. Error bars represent SEM.

Biofilm activates ROS-independent NET formation.

It was described previously in a planktonic setting that thermonuclease can degrade NETs to ensure the survival of S. aureus (27, 28). Prior to preforming a NETosis assay in a biofilm setup, we determined the thermonuclease production of a wild-type (WT) (UAMS-1), a thermonuclease (nuc) mutant (UAMS-1471), and a nuc complementation strain (UAMS-1552) during the 3rd and the 4th hour of biofilm formation using the FRET assay (see Fig. S1 in the supplemental material). In order to investigate whether thermonuclease production by young biofilms had an effect on NETosis, we added freshly isolated neutrophils to 3-h-old washed biofilms. After a coincubation period of 90 min, we saw massive NET formation with the nuc mutant (Fig. 4C and E), while minimal amounts were seen with the wild-type (Fig. 4B and E) or the nuc complementation strain (Fig. 4D and E). A 5-min coincubation of neutrophils with freshly washed, 3-h-old biofilms of Newman and RWW50 gave visible NET formation (Fig. 5A, C, and E). However, if biofilms were pretreated with 5 μg/ml of exogenous thermonuclease Nuc1 (eNuc1), almost no NETs were observed (Fig. 5B, D, and E).

FIG 4.

Three-hour-old biofilms produce enough thermonuclease to degrade NETs. Massive NET (red diffuse) formation can be observed when 3-h-old biofilms of the nuc mutant (Δnuc) are coincubated with human neutrophils (green, alive; red [localized], dead) (C, E). Thermonuclease produced by the biofilms degrades NETs induced by 3-h-old biofilms of the wild-type (WT) (B, E) and the nuc complemented (nuc⁺) (D, E) strains. No spontaneous NET formation was observed in the control neutrophils only (A). Figures have been generated from three independent experiments. Scale bar = 100 μm. Error bars represent SEM.

FIG 5.

Inductions of NETosis by early biofilm. Exogenous thermonuclease Nuc1 degrades the NETs (red diffuse) formed after coincubation of neutrophils with 3-h-old washed biofilms of Newman (A, B, E) and USA300 (RWW50) (C, D, E). Figures have been generated from three independent experiments. Scale bar = 50 μm. Error bars represent SEM.

During the coincubation of neutrophils with the 3-h-old biofilms of these strains, we found that reactive oxygen species (ROS) levels from these neutrophils were not significantly different than spontaneous ROS production by neutrophils by themselves (see Fig. S2 in the supplemental material). These findings indicate that S. aureus biofilm-induced NET formation is ROS independent. Meanwhile, the addition of eNuc1 alone had no effect on ROS activation (Fig. S2).

Upon encountering bacteria, neutrophils can be activated via TLR9 (30). TLR9 recognizes specifically the unmethylated cytosine-phosphate-guanine (CpG) dinucleotide part of bacterial or viral DNA (30, 31). After coincubation of S. aureus biofilms with HEK-Blue hTLR9 cells (32, 33), which are human embryonic kidney 293 cells that express human TLR9, no activation of human TLR9 was observed (see Fig. S3 in the supplemental material). However, when these HEK-Blue hTLR9 cells were pretreated with a TLR9 agonist, TLR9 activation was significantly higher than that of the unstimulated control cell line (P < 0.01) (Fig. S3).

Biofilm-associated S. aureus survives in the presence of human neutrophils during the early stages of biofilm formation.

Considering that S. aureus biofilms can already inhibit ROS and NET production during the early stages of biofilm formation, it comes into question whether these S. aureus biofilms can survive exposure to neutrophils. Therefore, we monitored the metabolic activity of biofilm-associated S. aureus cells coincubated with human neutrophils using isothermal microcalorimetry. Interestingly, we saw that the metabolic rates of the biofilm-associated S. aureus cells were not significantly affected by the presence of neutrophils (Fig. 6). This confirms previous results that show that biofilm-associated S. aureus cells are able to withstand human neutrophils (28, 34). Although the metabolic rates of the biofilms formed by the nuc mutant (Δnuc) were lower than those of biofilms formed by the wild-type (WT) strain during coincubation with neutrophils, the differences were not statistically significant. Overall, no significant differences in metabolic rates were found between the WT, Δnuc, and complemented nuc (nuc⁺) strains during coincubation with neutrophils. Finally, addition of exogenous thermonuclease Nuc1 to biofilms of the Δnuc mutant, before coincubation with neutrophils, did not result in significant differences in survival compared to that of untreated biofilms of the same strain (Fig. 6).

FIG 6.

Effect of neutrophils and thermonuclease on the survival of biofilm-associated bacterial cells. Neutrophils were added to 3-h-old biofilms of wild-type (WT), nuc mutant (Δnuc) (with or without an addition of exogenous thermonuclease Nuc1 [eNuc1]), and complementary nuc (nuc⁺) UAMS S. aureus strains. The effect of neutrophils on bacterial survival was monitored using isothermal microcalorimetry. The metabolic rates of the bacteria and neutrophils are depicted as heat flow versus time. Figures have been generated from three independently performed experiments. Error bars represent SEM.

DISCUSSION

Previous studies in conventional growth media showed that thermonuclease transcription and production could be detected after approximately 10 h of incubation of biofilm in brain heart infusion (BHI) broth (35) and after 6 h in TSB (36). In addition, our previous study in IMDM (15), a mammalian cell culture medium, showed that thermonuclease was produced as early as the 4th hour of biofilm formation. Using three different methods—FRET-based assay, Luminex competitive assay, and mass-spectrometry—we confirmed that thermonuclease was indeed already produced during the early stages of biofilm formation.

It is widely accepted that an important role of thermonuclease is to release the bacteria from the eDNA-containing biofilm matrix, which will lead to the destabilization and remodeling of biofilms (36, 37). However, most of the experiments leading to this assumption were performed in classical growth media, such as TSB or BHI broth. When we grew biofilms in IMDM, we observed a steady accumulation of biofilm, even though the amount of thermonuclease, specifically the Nuc1, increased as well. These observations are in line with previous studies, which showed that early biofilms were insensitive to DNase I (36, 38). Our observations further indicate that biofilm formation is independent of eDNA in IMDM. Analysis of biofilm composition formed in IMDM and TSB revealed that, indeed, after 4 h of growth, very minimal amounts of eDNA were seen in any of the biofilms. However, biofilms formed in IMDM did contain N-acetylglucosamine-containing components, presumably PIA, which was visualized by wheat germ agglutinin (WGA) lectin staining (39). After 24 h of growth, dead cells and eDNA were observed in biofilms grown in TSB but not biofilms grown in IMDM. In addition, after 24 h, accumulation of N-acetylglucosamine was abundant in the IMDM-cultured biofilms but was not present in the TSB-cultured biofilms. This might be due to the activity of the SaeRS locus (40). The SaeRS locus, which regulates nuc transcription in vitro (40), may be activated differently in these two growth media. In mammalian cell culture medium that contains plasma, the Sae locus is required for biofilm formation, while in TSB, the Sae locus is dispensable (41). SaeRS can be activated by limited or no free iron in medium, such as in IMDM (41), and may stimulate both the production of PIA and thermonuclease (42–44). This could also explain why biofilms grown in IMDM produce more thermonuclease. Because thermonuclease breaks down DNA, it has been described to be produced by biofilms to regulate biofilm mass (26, 35). This would indeed be the case for biofilms grown in TSB, as eDNA is abundantly present. However, when grown in IMDM, the early and robust production of thermonuclease might break down the eDNA in these biofilms. The lack of eDNA in the tested S. aureus biofilms may be the reason why we were unable to demonstrate the activation of human TLR9 by S. aureus biofilms since it is known that TLR9 is expressed at the surface of human polymorphonuclear leukocytes (PMNs) (45) and can be activated by unmethylated CpG motifs of bacterial DNA (25).

Neutrophils are the first immune cells to respond to injury, and one of their defense responses is the formation of NETs (46). NETs have been described as an immune response against pathogens (46) and can be induced by bacteria (47, 48), fungi (49), and bacterial products such as lipopolysaccharides (LPS) (50). S. aureus is a very strong inducer of NETosis (47, 48, 51, 52; T. Hoppenbrouwers, A. R. Sultan, T. E. Abraham, N. Lemmens-den Toom et al., submitted for publication), the process of NET formation in which neutrophils excrete their DNA, histones, and antimicrobial proteins into the ECM (27). NETs trap the bacteria and limit their spreading (46). When we incubated these early-stage biofilms with neutrophils for 90 min, only very limited amounts of NETs were present in biofilms of the wild-type and the nuc complementation strains. In contrast, NETs were visible after 90 min of incubation with neutrophils in biofilms of the nuc mutant strain. Planktonic S. aureus has been described to break down NETs with thermonuclease (27, 28). We confirmed that early-stage biofilms are strong inducers of NETosis, but we also showed that these early biofilms are able to produce thermonuclease to degrade NETs as well as planktonic S. aureus. Therefore, this study provides the first step in understanding the interaction between the early stages of S. aureus biofilms and NETosis. Future studies need to be conducted to further elucidate how the balance between the parallel induction and degradation of NETs by biofilms is regulated. Also of interest is how this balance affects the role of NETosis in the pathophysiology of biofilm, particularly during the early stages of biofilm formation.

Finally, we did not observe ROS production of neutrophils in our biofilm experiments. Neutrophils typically have three different antimicrobial mechanisms as follows: (i) phagocytosis, where pathogens are engulfed into a phagosome and destroyed by NADPH-dependent mechanisms (ROS production) or antimicrobial proteins; (ii) degranulation, where pathogens are killed by ROS and antimicrobial proteins that are released into the extracellular matrix by PMN; and (iii) NETosis (53). NETosis can be induced by both ROS-dependent and ROS-independent pathways (54). In our biofilm experiments, we saw ROS-independent NETosis, indicated by NET formation without visible ROS activation. This is in line with previous literature on planktonic S. aureus (48) and Leishmania (55).

In this biofilm study, we were able to confirm that the protective role of thermonuclease towards NETs during the early stages of biofilm formation is similar to what has been described previously for planktonic cells (27, 28). The mechanism by which biofilm can avoid recognition and activation of hTLR9 and ROS still needs to be addressed properly. Since there are indications that thermonuclease does not act alone in the modulation of early host innate responses (52, 56), other immune modulators present during the early stage of biofilm formation should be explored. Investigation into the role of other SaeRS-related immune modulators such as Eap (map) (57) and sasH (AdsA) should be prioritized, since the role of these immune modulators in host immune evasion related to thermonuclease has already been described elegantly in planktonic S. aureus studies (52, 56, 58–60) but not for biofilms grown in mammalian cell culture medium.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The S. aureus strains used in this study are listed in Table 2. The wild-type and nuc mutant strains were plated on Trypticase soy agar (TSA) with 5% sheep blood for overnight incubation at 37°C (Becton Dickinson, Breda, The Netherlands). The nuc complementation strain was plated on chloramphenicol-containing TSA-blood agar.

TABLE 2.

Strains of S. aureus that were used in this study

Strain	Genetic background	Description	Reference
Newman	ST8	Laboratory strain	71
RN6390	ST8	Laboratory strain	72
M116	ST8, ST239	Clinical isolate from a patient with an osteomyelitis infection in Indonesia	15
RWW50	ST8, USA300	Clinical isolate from the collection of MMIZ Erasmus MC, The Netherlands	15
UAMS-1	ST30	Wild-type (WT) clinical isolate from a patient with an osteomyelitis infection in Arkansas, USA	73
UAMS-1471	ST30	Δnuc, thermonuclease mutant strain of UAMS-1	74
UAMS-1552	ST30	nuc+, complementation of the nuc mutant strain with a plasmid-borne version of the nuc gene	74

Bacterial growth and biofilm assessment.

Biofilm formation was assessed in vitro using a dynamic microtiter plate biofilm formation assay as previously described (61–64) with slight modifications (15). S. aureus bacteria cultured overnight on TSA were suspended in 4 ml of 0.9% NaCl to reach an optical density at 660 nm (OD₆₆₀) of 2.0 (±0.2). One microliter of this suspension was added to 199 μl Iscove’s modified Dulbecco’s medium (IMDM) (Gibco, Bleiswijk, The Netherlands), without the presence of human serum, in separate wells of a sterile flat-bottom 96-well polystyrene tissue culture plate (Cellstar; Greiner Bio-One, Alphen aan den Rijn, The Netherlands) (64). Plates were incubated for 1, 2, 4, and 24 h under 150 rpm orbital shaking at 37°C. At these time points, the formed biofilms were washed once with 200 μl sterile water, air dried for 30 min at room temperature, and stained for 2 min with 50 μl of 1% crystal violet (Sigma-Aldrich, Zwijndrecht, The Netherlands). Excess crystal violet was removed by washing the plates five times with 200 μl of sterile water. The biofilms were dissolved in 200 μl extraction solution consisting of 50% distilled water (dH₂O), 40% ethanol (Sigma-Aldrich), and 10% acetic acid (Sigma-Aldrich). Lastly, to determine the biofilm mass, the absorbance of crystal violet was measured at OD₄₉₀ in a microplate reader (Epoch 2 microplate reader; BioTek Instruments, Inc., Winooski, VT, USA).

Determination of nuc presence.

The presence of nuc was determined by a PCR protocol as described previously (65).

Collecting biofilm supernatants.

Biofilm supernatants were collected according to protocol described previously (15). Briefly, S. aureus RN6390 (8325-4), Newman, and clinical strains M82 (ST20), M116 (ST239), and RWW50 (USA300) were cultured as described above. We cultured the biofilm for 1, 3, or 23 h. At these time points, we discarded the supernatant to remove all nonattached or planktonic bacteria, washed the biofilm with 200 μl IMDM once, added 200 μl of fresh IMDM, and proceeded for 1 h of incubation at the conditions described above. Subsequently, we collected the supernatants and labeled them as the 2nd, 4th, and 24th hour biofilm supernatants. All individual supernatants from all time points and all different strains were filtered through a 0.2-μm sterile filter (Whatman; GE Healthcare Life Sciences, Little Chalfont, UK) and stored at −20°C until further usage. The supernatant collected after 1 h of biofilm formation served as the negative reference control.

Quantification of thermonuclease Nuc1 production with competitive Luminex assay.

Thermonuclease Nuc1 production during biofilm formation was quantified using a multiplex competitive Luminex assay. With the competitive Luminex assay, we can assign semiquantitatively the amount of an antigen present by determining the ratio of bound and unbound antibodies to the antigen after preexposing serum antibodies to a liquid containing an unknown number of free antigens. The thermonuclease Nuc1 production was measured in the biofilm supernatant of the 1st, 2nd, 4th, and 24th hours of biofilm formation, and the assay was performed according to previously described protocols (15, 29, 61). Briefly, 30 μl of the diluted biofilm-derived supernatants was incubated separately with 30 μl of 1/200 diluted human pooled serum (HPS) (29, 61). The mixture was incubated at 22°C in a U shape 96-well microplate (Greiner Bio-One) placed within Thermostat plus (Eppendorf, Nijmegen, The Netherlands) for 35 min and continuously shaken at 800 rpm. Next, 50 μl of this mixture was directly assayed for the semiquantification of the noncaptured IgGs specific for S. aureus biofilm-derived thermonuclease Nuc1 using Luminex bead-based flow cytometry (Luminex Corporation, Austin, TX, USA). The thermonuclease Nuc1 (N3755; Sigma-Aldrich, Zwijndrecht, The Netherlands) used in this assay was coupled to MagPlex beads (Luminex Corporation) according to the manufacturer’s instructions. The antigen concentration used for coupling was 2 μg per antigen per 10⁶ MagPlex beads as optimized by Hansenová Maňásková et al. (29). The beads were diluted to a final concentration of 1,500 beads/μl. Negative control beads (beads to which no antigen was added during coupling) were included in all assays. The Luminex assay was performed as described previously (66) with one modification; that is, we measured 50 beads per region (29). All samples were analyzed on the Luminex Bio-Plex 200 system using Bio-Plex Manager software version 6.1 (Bio-Rad Laboratories, Inc., USA). All data are based on three separate experiments. The median fluorescence intensity (MFI) values of these triplicates were averaged if the coefficient of variation (CV) value was lower than 25%, and the standard error of the mean (SEM) was calculated. For each sample, we calculated the proportion (%) of uncaptured IgG specific for the thermonuclease Nuc1 using the following formula: Y = (A/B) × 100, in which A is the unbound IgG specific for thermonuclease Nuc1 present in the HPS-biofilm supernatant mix, and B is the level of thermonuclease Nuc1-specific IgG present in untreated HPS. Y can be considered a semiquantitative measurement of the antigen-specific antibody absorption from HPS by biofilm supernatant, thus indirectly reflecting the immune modulators present in the biofilm supernatant (67, 68) and subtracting this percentage from 100%. Statistical differences between the time points were calculated by comparing the percent proportions of the 2nd, 4th, and 24th hour time points and using the 1st hour as the reference control.

Identification of the thermonuclease by mass spectrometry.

Protocol was previously described (15). Briefly, 100 μl of supernatants derived from the 4th hour during biofilm formation of the RWW50 (USA300) and Newman strains was precipitated at 1:10 (vol/vol) with ice-cold acetone and centrifuged for 10 min at 18,407 × g at 4°C. Subsequently, pellets were washed twice with 50 μl of cold acetone. Then, pellets were air dried for 30 min and dissolved in 10 μl of 50 mM NH₄HCO₃. Solution digestion was performed according to an adjusted protocol as described by Dekker et al. (69). Ten microliters of 0.2% RapiGest (Waters Corporation, Milford, MA, USA), which was previously diluted in 50 mM NH₄HCO₃, was added to the 10-μl sample. This mixture was then reduced with 5 mM dithiothreitol (DTT) (Thermo Fisher Scientific, Waltham, MA, USA) at 60°C and 450 rpm for 30 min. After cooling down to room temperature, this mixture was alkylated in the dark with 15 mM iodoacetamide (Thermo Fisher Scientific) at ambient temperature for 30 min and digested overnight with 1:100 (wt/wt) trypsin (Promega Corporation, Fitchburg, WI, USA) at 37°C and 450 rpm. Five percent trifluoroacetic acid was added to obtain a final concentration of 0.5% trifluoroacetic acid (pH < 2). After 45 min of incubation at 37°C and 450 rpm, the samples were centrifuged at 13,000 × g for 10 min, and the supernatant was used for liquid chromatography-mass spectrometry (LC-MS). Mass spectrometry and data analyses were performed as described elsewhere (69). A database search was performed against the uniprot_sprot_v151112 database (selected for Bacteria, 332,280 entries).

Quantification of thermonuclease activity with FRET-based assay.

We quantified the thermonuclease activity using a previously described fluorescence resonance energy transfer (FRET)-based assay (37). The FRET substrate was purchased from Eurogentec (Maastricht, The Netherlands) (35). The FRET substrate was diluted to 2 μM in Tris buffer (20 mM Tris, pH 8.0, and 10 mM CaCl₂) (35, 37). To measure the activity of the biofilm-associated nuclease of each strain, biofilms were grown in a flat-bottom 96-well black polystyrene tissue culture plate (Costar; Corning Incorporated, NY, USA) at 37°C with continuous 150-rpm agitation. After incubation for 3 h, biofilms were washed once with IMDM or TSB. Twenty-five microliters of new IMDM or TSB (with or without tested compounds) and 25 μl diluted FRET substrate were added to the biofilms. The biofilms were then incubated in a Fluostar Optima microplate fluorescence reader (BMG Lab Technologies) at 30°C with 150-rpm periodic rotational shaking for 2 h. The accumulation of fluorescence during these 2 h, used as a measurement for thermonuclease activity, was determined automatically every 5 min (excitation, 552 nm; emission, 580 nm; gain, 800). Results are reported as fluorescence units. The mean fluorescence intensity (MFI) of three separate experiments was calculated.

LIVE/DEAD-WGA staining for biofilm.

Biofilms were stained with a LIVE/DEAD BacLight bacterial viability kit (Thermo Fisher Scientific, Bleiswijk, The Netherlands) and wheat germ agglutinin (WGA)-Alexa Fluor 350 conjugate (Invitrogen BV, Breda, The Netherlands) according to the manufacturer’s protocol with slight modification. This staining method used propidium iodide (PI) and SYTO 9 to stain the DNA. The affinity of SYTO 9 to DNA is stronger than that of PI, and contrary to PI, SYTO 9 can penetrate live or intact cells. Therefore, in this double staining, SYTO 9 will bind the DNA of live cells while PI will bind to the dead ones or to extracellular DNA (eDNA). As an addition, wheat germ agglutinin (WGA) lectin staining is used to detect the presence of N-acetylglucosamine, which is, for instance, the primary building block of PIA/PNAG (poly-N-acetylglucosamine) (70). In this experiment, 4-h-old biofilms were washed once with 200 μl of IMDM. The biofilms were then incubated with 50 μl of IMDM, 50 μl of 15 μM propidium iodide (PI), 50 μl of 2.5 μM SYTO 9, and 0.5 μl of 1 mg/ml WGA-Alexa Fluor 350 conjugate on each well. The plate was incubated at 22°C on an orbital shaker (300 rpm) in the dark for 35 min. The biofilms were imaged using an Olympus IX51 fluorescence microscope (Olympus Nederland B.V., Zoeterwoude, The Netherlands) with ×20 and ×40 magnification. For relative quantification, the same staining procedure was followed, only the stains were applied individually to separate wells in phosphate-buffered saline (PBS) containing S. aureus biofilms. After staining, the PBS in the wells was replaced with new PBS, and the plate was measured in a Fluostar Optima microplate fluorescence reader (BMG Lab Technologies) at 37°C at an excitation/emission for SYTO of 485 nm/520 nm (gain, 1,280), for PI of 544 nm/612 nm (gain, 2,560), and for WGA-Alexa Fluor 350 conjugate of 340 nm/360 nm (gain, 1,280). Results for both PI (eDNA) and WGA (PIA) are reported as the mean ratio (of three separate experiments) of the SYTO 9 signal (%).

NETosis assay.

Human neutrophils were isolated as described previously (47, 51) and stained with Hoechst 34580 (1:10,000; Life Technologies, Landsmeer, The Netherlands) and PI (1:400; Sigma-Aldrich, Zwijndrecht, The Netherlands). One hundred microliters of 10⁷/ml neutrophils was added to a 3-h-old biofilm formed by Newman, RWW50, UAMS-1, UAMS-1471, and UAMS-1552 in the presence or absence of 5 μg/ml exogenous thermonuclease Nuc1 (N3755; Sigma-Aldrich) in a flat-bottom 96-well polystyrene tissue culture plate (Cellstar; Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Biofilm and neutrophil interactions were imaged with a confocal microscope (Leica SP5 AOBS) after 20 min of incubation at 37°C. Hoechst and PI were excited by 405-nm and 561-nm lasers. NETs were visible as PI-positive elongated structures and were quantified. Furthermore, using the same experimental setup, the presence or absence of NETs was quantified by measuring the amount of fluorescence in a Fluostar Optima microplate fluorescence reader (BMG Lab Technologies) at an excitation and emission of 485 nm and 612 nm, respectively (gain, 1280). One exception to this assay was that the neutrophils were only stained with PI (1:400) before addition to the biofilms. The mean fluorescence intensities (MFIs) of three separate experiments were calculated.

All data were corrected for the background signal (neutrophils without bacteria).

Cellular ROS activation assay.

NETosis can be induced by both ROS-dependent and ROS-independent pathways (54). Therefore, to determine which pathway of NET formation is induced during the early stages of S. aureus biofilm formation, we quantified ROS activation of neutrophils during coincubation with S. aureus biofilm. Three-hour-old biofilms grown in IMDM were washed and refreshed with 100 μl of new IMDM (in the presence or absence of 5 μg/ml exogenous thermonuclease Nuc1 [N3755; Sigma-Aldrich]). One hundred microliters of 10⁷/ml neutrophils was then added to the biofilms. The biofilms were then prepared for the cellular ROS activation assay (GeneCopoeia, Nivelles, Belgium) according to the manufacturer’s protocol. The kinetics of the first 145 min of ROS activation was measured in a Fluostar Optima microplate fluorescence reader (excitation, 552 nm; emission, 580 nm; gain, 800). Results were reported as fluorescence units. The MFIs of three independent experiments were calculated.

Human TLR9 activation assay.

To study the effect of bacterial DNA accumulation in biofilm toward host immune responses, we measured the activity of human Toll-like receptor 9 (TLR9), which is known to be able to bind to bacterial DNA (30, 31). For this experiment, 3-h-old biofilms prepared in IMDM were washed once with 200 μl endotoxin-free water. Two hundred microliters of HEK-Blue detection medium (InvivoGen, Toulouse, France) containing 5 × 10⁴ human embryonic kidney 293 cells expressing human TLR9 (HEK-Blue hTLR9) (InvivoGen, Toulouse, France) was added to the biofilms (in the presence or absence of 5 μg/ml exogenous thermonuclease Nuc1 [N3755; Sigma-Aldrich]). As a positive control, HEK-Blue hTLR9 cells were pretreated with class B CpG oligonucleotides (ODN 2006; InvivoGen). After overnight incubation at 37°C in a 5% CO₂ incubator, the OD₆₂₀ was measured in a microplate reader (Epoch 2 microplate reader; BioTek Instruments, Inc., Winooski, VT, USA).

Biofilm survival assay with microcalorimeter.

Using isothermal microcalorimetry, we observed the effects of neutrophils on the metabolic status of biofilm-associated S. aureus in real time. This metabolic activity directly reflects the fitness of the biofilm-associated bacterial cells and the consequence of coincubation with neutrophils. Wild-type, nuc mutant, and nuc complemented S. aureus UAMS strains (Table 2) cultured overnight on TSA were suspended in 5 ml of 0.9% NaCl to reach an OD₆₀₀ of 0.5 (±0.05). Ten microliters of this suspension was added to 9,990 μl 0.9% NaCl in a sterile 15-ml tube (Greiner Bio-One, Alphen aan de Rijn, The Netherlands). Then, 10 μl of this bacterial solution was added to 190 μl IMDM (Gibco, Bleiswijk, The Netherlands) in a sterile flat-bottom calWell insert (CalScreener; Symcel Sverige AB, Kista, Sweden). After 3 h of incubation under 150-rpm orbital shaking at 37°C, the biofilms within each individual calWell insert were washed and refreshed with 100 μl of new IMDM and 100 μl of medium containing 10⁷ human neutrophils per milliliter. These individual calWell inserts that contain biofilms and neutrophils were inserted into a titanium tube set (SymCel Sverige AB, Kista, Sweden) and incubated at 37°C overnight in the isothermal microcalorimeter (CalScreener; SymCel Sverige AB, Kista, Sweden) to measure the metabolic activity of the bacteria within the biofilms.

Statistical analysis.

Statistical analysis was performed using the Prism 5.0 package (Graph Pad Software, San Diego, CA, USA) and Microsoft Excel 2010. We used the unpaired t test or one-way analysis of variance (ANOVA) for data analysis, where a P value of ≤0.05 was considered statistically significant. All experiments were independently repeated three times and depicted with standard error of the mean (SEM).