All animal experiments protocols were performed following the Guide for the Care and Use of Laboratory Animals of the Chinese Association for Laboratory Animal Sciences (CALAS) and were approved by the ethics committee of Renji Hospital, School of medicine, Shanghai Jiaotong University, Shanghai, China.

All experiments were performed with S. aureus Newman (NM) or USA300 as the wild-type strains. The bacterial strains and plasmids used in this study are listed in Supplementary Table 1. Escherichia coli and S. aureus were grown in Luria-Bertani (LB) broth and tryptic soy broth (TSB), respectively. However, for transduction of plasmids, heart infusion broth (HIB) supplemented with 5 mM CaCl₂ was used. When necessary, antibiotics were added to the growth media at the following concentrations: ampicillin, 100 μg/ml; erythromycin, 10 μg/ml; and chloramphenicol, 5 μg/ml.

To construct the plasmid for deleting the lytM, sle1, or agr genes in Newman and USA300 strains, we used a ligation independent cloning (LIC) method (Aslanidis and De Jong, 1990). First, vector DNA was PCR-amplified from pKOR1 using the primers P236/237 (Supplementary Table 2). One- kb DNA fragments, upstream and downstream of lytM, sle1, or agr, were amplified by PCR from the chromosomal DNA with the primer pairs P596/597 and P598/599 (for lytM), P632/633, and P634/637 (for sle1) or P19/20 and P21/22 (for agr) (Supplementary Table 2) and PrimSTAR (Takara), a PCR enzyme with high fidelity. The PCR products were treated with T4 DNA polymerase in the presence of dCTP (vector) or dGTP (insert DNA) and mixed together. The DNA mixture was used to transform E. coli DH5α. The resulting plasmids, pKOR1ΔlytM, pKOR1Δsle1, or pKOR1Δagr were electroporated into S. aureus strain RN4220 and subsequently transduced into NM or USA300 with Φ85. The deletion was carried out as described previously (Bae and Schneewind, 2006).

To generate complement plasmids for clpP mutant strain, First, vector DNA was PCR-amplified from pCL55 using the primers P35/80, the clpP gene full length with its own promoter was amplified with the primer pairs P2525/2526 (Supplementary Table 2). The plasmid was constructed with LIC and the resulting plasmid, pclpP, was electroporated into S. aureus strain RN4220 and then into NMΔclpP with Φ85.

To express Sle1 proteins in S. aureus, the gene sle1 was PCR-amplified with the following primer pairs: PL96/97 (Supplementary Table 2). The amplified fragments were digested with either BamHI/SalI. Then they were inserted into the multi-copy plasmid pOS1 under the control of its own promoter, resulting in pOS1-Sle1-his. The plasmids were transformed into E. coli DH5a, and then into S. aureus RN4220. Finally, the plasmids were transduced by Φ85 into wild type and clpP transposon mutants of S. aureus. The test strains in TSB containing chloramphicol (10 μg/ml) were incubated until early stationary growth phase (OD₆₀₀ is around 2). The proteins were detected by Western blot analysis with anti-His-tag antibody.

Unless stated otherwise, all restriction enzymes and DNA modification enzymes were purchased from New England Biolabs. Plasmids and genomic DNA were extracted with plasmid miniprep kit (Omega) according to the manufacturer's instruction. Plasmid DNA was introduced into E. coli by the method of Hanahan (Hanahan, 1983) and into S. aureus RN4220 by electroporation with a gene pulser (Bio-Rad). Subsequent transduction of the plasmids into target strains of S. aureus was carried out with Φ 85.

Cells in 3 ml TSB cultures were harvested at the stationary phase (OD₆₀₀ value of 2). Cells were disrupted by shaking with a Mini-Beadbeater (Biospec Products) at maximum speed for 30 s. Tubes were then incubated on ice for 5 min. Then, the suspension was centrifuged. Total RNA isolation from the supernatant was performed according to the manufacturer's instructions (Qiagen). After treatment using a TURBO DNA-freeTM kit (Ambion), ~1 μg of total RNA was reverse-transcribed with a HieffTM first Strand cDNA Synthesis Super Mix for RT-PCR kit (Yeasen Bio). The cDNA was used as a template for real-time PCR using SYBR-green PCR reagents (Roche). Reactions were performed in a MicroAmp Optical 96-well reaction plate using a 7,500 Sequence Detector (Applied Biosystems). Primers used are listed in Supplementary Table 2. All RT-PCR experiments were performed in triplicate, with gyrase B (gyrB) used as an internal control.

Western blot analysis of proteins was carried out as described previously (Sun et al., 2011). The AgrA antibody was generated by GLbiochem, China. The His-tag antibody was bought from Yeasen Bio, China. All other antibodies were generated by GenScript.

Semi-quantitative biofilm assays were performed as described in our previous work (Liu et al., 2011). Briefly, overnight cultures of S. aureus strains were diluted 1:100 into fresh TSB with 0.5% Glucose. The diluted cultures were pipetted into sterile 96-well flat-bottom tissue culture plates and incubated at 37°C for 24 h. Culture supernatants were gently removed, and wells were washed with phosphate-buffered saline (PBS). The adherent organisms at the bottom of the wells were fixed by Bouin fixative over 1 h. Then the fixative was gently removed, wells were washed with PBS and stained with 0.4% (wt/vol) crystal violet. Biofilm formation was measured with a MicroELISA autoreader.

The BioFlux 1000z microfluidic system (Fluxion Biosciences, CA) was used to assess biofilm formation under flow conditions as described (Benoit et al., 2010). To grow biofilms, the microfluidic channels were primed with TSB supplemented with 0.5% glucose at 2 dynes/cm² for 10 min. Each channel of a 48-well plate was coated with 20% platelet-poor human plasma in 50 mM carbonate buffer, PH 9.6 and incubated for 24 h at 4°C before biofilm assays were set-up. The exponential cultures were diluted 1:100 in TSB supplemented with 0.5% glucose. Bacterial suspensions were seeded at 0.2 dyn/cm² for 5 s in all channels. The plate was then incubated at room temperature for 1 h to allow cells to adhere. Excess inoculums were removed and 0.8 ml of TSB supplemented with 0.5% glucose was added to the input wells. Biofilms were grown at 37°C with a flow of fresh media at a constant shear of 0.15 dyn/cm². Images were taken every 10 min for 24 h at 10 × magnification under brightfield.

Cell cultures were grown to exponential phase. Culture supernatants from strain wild-type and its derivatives were filter sterilized, extracted with phenol-chloroform, and ethanol precipitated. Precipitates were suspended in H₂O. The volumes of water were varied to compensate for slight differences in OD₆₀₀ of the initial cultures. The eDNAs were quantified using Nanodrop 2000 (Thermo Scientific) and visualized by agarose gel.

Bacterial cells were grown in TSB containing 1 M NaCl to the exponential growth phase at 37°C with shaking and then washed and suspended in 0.05 M Tris-HCl buffer (pH 7.5) containing 0.1% Triton X-100 and were incubated at 30°C with shaking. The optical density was measured in intervals. Results were normalized to an OD₆₀₀ at time zero.

Cell cultures were grown to exponential phase, and normalized by OD₆₀₀. The normalized supernatants were filtered through 0.22 μM filter and concentrated with a Centricon 3 concentrator (Millipore) at 4°C according to the manufacturer's instructions. Concentrated supernatants were mixed with an equal volume of Laemmli sample buffer, and 20 μl was separated by 12% SDS-PAGE supplemented with 0.2% autoclaved S. aureus RN4220 cell (wt/vol) as a substrate. After electrophoresis, the gels were washed four times with distilled water for 30 min at room temperature, incubated in 25 mM Tris-HCl containing 1.25% Triton X-100 (pH 8.0) at 37°C for 4 h, and then stained with methylene blue in 0.01% KOH(wt/vol), followed by destaining in deionized water.

A murine subcutaneous foreign body infection model was performed as described by Pozzi et al. (2012). Briefly, 20 male BALB/c mice were randomly and evenly divided into two groups and infected with wild type or clpP mutant bacteria. One-centimeter long silicon catheters (14 gauge) were implanted subcutaneously on each side at the flank of each mouse. Bacterial cells were grown in TSB to the exponential growth phase and then washed with and suspended in phosphate buffered saline (PBS) to OD₆₀₀ = 1.0. S. aureus (10⁸ CFU) were injected into the catheter bed. After 7 days, the mice were sacrificed. Catheters were aseptically removed and manipulated with the method described previously except with a sonication time of 15 min (Liu et al., 2011). Furthermore, peri-catheter tissue, liver, spleen, kidneys were dissected, weighed, and homogenized in 1 ml PBS. Then serial dilutions of the wash fluid were plated on TSA blood plates, and the recovered S. aureus colonies were counted. Bacteria present in blood were also quantitatively cultured on TSA blood plates for CFU counting.

Statistical analysis was performed using Graph-Pad Prism, version 5. Error bars in all graphs show the standard deviation (± SD).

ClpP has been reported to inhibit biofilm formation in the S. aureus 8325-4 strain (Frees et al., 2004). To examine whether the biofilm inhibitory effect is universal, we analyzed biofilm formation of clpP mutant in the following three strains: S. aureus Newman, a methicillin-sensitive strain (MSSA), USA300, the predominant community-associated methicillin-resistant strain (CA-MRSA) in the USA and ST59 11-775, the most frequent lineage of community-associated infections in China and adjacent Asian countries (Li et al., 2016). In all three strains, clpP mutants showed enhanced biofilm formation (Figure 1), indicating that the biofilm inhibitory effect of ClpP is likely to be universal among S. aureus strains. When biofilm formation was analyzed in the presence of shear stress (0.15 dynes/cm²), which imitates blood flow in veins, the biofilm inhibitory effect of ClpP was still observed (Figure 1B). On the other hand, the mutants of Clp ATPases showed a wild-type level of biofilm formation (Figure 1C), indicating that either the Clp ATPases are not involved in the regulation of the biofilm formation or they have redundant roles.

In the staphylococcal biofilm, the three main components are proteins, extracellular DNA (eDNA) and PIA (Paharik and Horswill, 2016). To determine the main constituents of the biofilm formed by the clpP mutant, we first added Proteinase K or nuclease to the biofilm formed by the Newman clpP mutant. As shown in Figures 2A,B, the biofilm was reduced by either proteinase K or nuclease, indicating proteins and eDNA are the main components of the biofilm formed by the clpP mutant.

The involvement of PIA in the biofilm formation by the clpP mutant was assessed indirectly by comparing the transcriptional levels of icaABC, the genes encoding PIA-synthesis enzymes in wild type and clpP mutant. As shown in Figure 2C, the transcript levels of icaABC were not significantly affected by the clpP mutation, suggesting that PIA probably does not contribute to the increased biofilm formation by the clpP mutant.

In S. aureus, the quorum sensing two component system Agr is known to suppress biofilm formation by up-regulating the production of extracellular proteases (Boles and Horswill, 2008). In the strain 8325-4, clpP mutation is known to reduce the activity of Agr (Frees et al., 2003; Michel et al., 2006). Indeed, the transcript levels of agrA and agrC were greatly reduced in the clpP mutants of both NM and USA300 strains (Figure 3A). The expression level of the response regulator AgrA was also greatly reduced in the clpP mutants (Figure 3B), further confirming the lower Agr activity in the clpP mutant. Finally, we observed that the protease activity was significantly decreased by mutation in either clpP or agr (Figure 3C), indicating that protease activity was reduced due to low Agr activity in clpP mutant strain.

Next we decided to determine the origin of the eDNA in the biofilm of the clpP mutant (Figure 2B). Since the release of eDNA is likely due to autolysis of S. aureus cells, we focused on staphylococcal cell wall hydrolases. Among either proven or putative cell wall hydrolases in S. aureus, LytM and Sle1 are known to be affected by ClpP. The transcription of lytM was up-regulated in the clpP mutant of S. aureus 8325 strain (Michel et al., 2006), whereas, in the S. aureus strains 8325-4 and Newman, Sle1 was shown to bind to ClpPtrap, the protease-deficient mutant of ClpP (Feng et al., 2013). Therefore, we examined whether either LytM or Sle1 play a role in the biofilm formation by the clpP mutant. As can be seen, while the deletion of lytM in the clpP mutant did not decrease the biofilm formation (clpP/lytM in Figure 4A), the sle1 deletion did (clpP/sle1 in Figure 4A), indicating that sle1 is required for the biofilm formation by the clpP mutant. Indeed, a higher level of Sle1 was detected in the clpP mutant as compared with the wild type (Figure 4B). However, the transcription of sle1 was either not significantly affected or reduced in the clpP mutants (Figure 4C). These results are consistent with the idea that the precursor of Sle1 is a ClpP substrate (Feng et al., 2013). Finally, to investigate whether the increased level of Sle1 leads to the release of more eDNA, we compared the amount of eDNA in culture supernatants of the test strains. Indeed, the culture supernatant of the clpP mutant contained a higher level of eDNA, as compared with that of wild type strain (clpP in Figure 4D). However, the level of eDNA was restored to that of wild type by further deletion of sle1 (clpP/sle1 in Figure 4D), strongly suggesting that the increased level of Sle1 is responsible for the heightened release of eDNA, leading to more robust biofilm formation of the clpP mutant.

Sle1 is a peptidoglycan hydrolase, preferentially cleaving the N-acetylmuramyl-L-Ala bonds in dimeric cross-bridges that interlink the two murein strands in the peptidoglycan (Kajimura et al., 2005). Therefore, it is likely that the increased amount of eDNA in the culture supernatant of clpP mutant is due to the heightened cell lysis. To examine this notion, we measured autolysis activity of the test strains in the presence of Triton X-100. Indeed, the clpP mutation increased the autolysis activity of S. aureus Newman and USA300 (clpP in Figure 5A). Although the further deletion of lytM did not affect the autolysis activity of the clpP mutant (clpP vs. clpP/lytM in Figure 5A), the deletion of sle1 greatly reduced the autolysis activity of the clpP mutant (clpP vs. clpP/sle1 in Figure 5A). Zymography analysis showed that, in the test conditions, the cell wall hydrolase activities are mostly provided by the major autolysin Atl and Sle1, while the clpP mutant produced a higher level of Atl compared to WT only in USA300 strain. However, the clpP mutation specifically increases the overall activity of Sle1 (clpP vs. clpP/sle1 in Figure 5B).

ClpP is required for pathogenesis in a systemic staphylococcal infection as well as skin abscess infection (Frees et al., 2003; Farrand et al., 2013), indicating that ClpP plays important roles in S. aureus pathogenesis. Although the clpP mutant showed a slight growth defect especially during early exponential phase, the final cell density was similar to that of wild-type at lag phase (Supplementary Figure 1). Because clpP mutant showed an increased biofilm formation, we asked whether the increased biofilm formation can compensate the important virulence contributions of ClpP. A murine model of subcutaneous foreign body infection was used to investigate the impact of clpP on biofilm-associated infection in vivo. Progression of disease was measured by determining bacterial loads on implanted catheters while dissemination of the bacteria was examined by detecting bacterial loads on peri-tissues and organs. Wild type and the clpP mutant showed similar bacterial loads in the infected animals (Figure 6A). However, significantly higher number of bacteria were found in peri-catheter tissue as well as blood, liver, spleen, and kidneys in the animals infected by the wild-type strain, as compared with the animals infected by the clpP mutant strain (Figures 6B–F), indicating that ClpP plays a critical role in the dissemination of S. aureus, and that the increased biofilm formation capability cannot compensate the critical role played by ClpP.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.