Physiological significance of ClpP in Enterococcus faecalis and its regulating proteins were identified by Tandem Mass Tag Mass Spectrometry

ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis is still obscure. Thus a clpP deletion mutant (△clpP) was constructed in E. faecalis OG1RF strain to elucidate a more comprehensive picture of the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with Tandem Mass Tags labeling. The ΔclpP mutant strain showed impaired growth at 20°C or 45°C, at 5% NaCl or 2 mM H2O2. The surviving bacteria of the ΔclpP mutant strain reduced after exposure to the high concentration (50 x MIC) of linezolid or minocycline for 96 h. The ΔclpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 proteins increased, 24 proteins decreased) in the ΔclpP mutant strain. Among those differential abundance proteins, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx (spxA), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the ΔclpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein (rplD), ribosomal protein L7/L12 (rplL2), 50S ribosomal protein L13 (rplM), L18 (rplR), L20 (rplT), 30S ribosomal protein S14 (rpsN2) and S18 (rpsR) all reduced. The abundances of biofilm formation related adapter protein MecA increased, while the abundances of dihydroorotase (pyrC), orotate phosphoribosyltransferase (pyrE) and orotidine-5'-phosphate decarboxylase linezolid or minocycline tolerance of E. faecalis . ClpP participates in the biofilm formation of E. faecalis may by affecting the abundances of adapter protein MecA, orotate phosphoribosyltransferase ( pyrE ), orotidine-5'-phosphate decarboxylase ( pyrF ). Our results also suggest that ClpP may modulate the abundances of FsrA, GelE, and acetyl esterase/lipase participates in the virulence of E. faecalis .

with circular agitation (220 rpm). The diluted suspension was also inoculated into fresh TSB with 5% NaCl pH5.5 or 2 mM H2O2 and incubated at 37°C with circular agitation (220 rpm). OD 600 values for the cultures were determined using an Eppendorf Biospectrometer (Eppendorf, Hamburg, Germany) at one-hour intervals. Three independent experiments were performed.
The sensitivity of the △clpP mutant strain to sodium dodecyl sulphate (SDS) Overnight cultures of the E. faecalis strains were diluted 1:200 in fresh TSB medium and incubated at 37°C for four hours until an OD600 of 1.0 was reached. After 10-fold serial dilution, 5 μL of the aliquot was spotted onto a TSB agar plate containing 0.008% SDS and incubated at 37°C for 24 h. The bacterial colonies on the plates were photographed and counted [28]. Three independent experiments were performed, and the representative results were shown.

Microtiter plate assay of biofilm formation
The biofilm-forming ability of the E. faecalis isolates was detected as previously described with modifications [50]. Overnight cultures were diluted 1:200 in 200 μl of TSBG (TSB with 0.25% glucose) and inoculated into 96 polystyrene microtiter plates. After 12, 24, or 48 h of static incubation at 37°C, the supernatant was discarded, and plates were washed thrice with deionized water to remove unattached cells, stained with 1% crystal violet (CV) for 20 min at room temperature, and rinsed with distilled water. Last, the CV was solubilized in ethanol-acetone (80:20, vol/vol) and optical density at 570 nm (OD 570 ) was determined. Three independent experiments were performed.
Quantification of extracellular DNA Extracellular DNA (eDNA) was quantified, as described previously [51]. Overnight cultures of the E. faecalis strains were diluted to OD600 = 0.001 in AB medium supplemented with 0.5% glucose, 0.05 mM propidium iodide (PI) and 10% TSB. The diluted cultures were transferred to polystyrene microtitre plates (200 μL/well) and incubated for 24 h at 37°C. The cell density was measured at OD 600 using a microtitre plate reader (BioRAD, United States). The fluorescence of PI-bound eDNA was measured by a Varioskan TM LUX multimode microplate reader (Thermo Fisher, United States) with the excitation/emission wavelength at 535/610 nm. Relative amounts of eDNA per OD 600 unit were determined. Three independent experiments were performed.

Determination of MIC and antimicrobials tolerance of strains
The minimal inhibitory concentrations (MICs) of the antimicrobials against the E. faecalis isolates were determined by the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines CLSI-M100-S26, with CLSI-recommended MIC breakpoints. The antimicrobials-tolerance of strains was detected as described previously with modifications [28].
Antimicrobials (at 50 x MIC) were added to the stationary-phase cultures (16 h) of the E. faecalis strains, and then the cultures were incubated at 37°C for 120 h without shaking. At the time points of every 24 h, one-milliliter aliquots were sampled and washed twice with ice-cold saline, ten-fold dilutions were then plated on Muller-Hinton agar, and the numbers of CFU were determined. Three independent experiments were performed.

Virulence of E. faecalis in Galleria mellonella
Infection of G. mellonella larvae with E. faecalis strains was performed as described previously for other pathogens [52]. G. mellonella larvae in groups of 40 were infected in the left posterior proleg with 20 μl inocula of E. faecalis strains containing 5 × 10 6 CFU/mL. Survival of G. mellonella larvae was recorded at 12 h intervals for 72 h p.i.. Every trial included a group of 20 G. mellonella larvae injected with saline as a method control. Experiments were performed in at least three independent tests, and the representative results were shown.

Protein extraction and detected by a mass spectrometer with Tandem Mass Tags (TMT) labeling
The E. faecalis strains OG1RF and the ΔclpP mutant were inoculated into TSB and cultured at 37°C for 4 h to logarithmic phase or for 12 h to stationary phase. The cells were harvested at 4°C centrifugation and minced individually with liquid nitrogen and lysed in lysis buffer, followed by 5 min of ultrasonication on ice. Protein concentration was determined again by the Bradford protein assay.
The supernatant from each sample, containing precisely 0.1 mg of protein, was digested with Trypsin Gold (Promega) at 1:50 enzyme-to-substrate ratio. After 16 h of digestion at 37°C, peptides were desalted with C18 cartridge to remove the high urea, and desalted peptides were dried by vacuum centrifugation. Desalted peptides were labeled with TMT6/10-plex reagents (TMT6/10plex™ Isobaric Label Reagent Set, Thermo Fisher), following the manufacturer's instructions. For 0.1 mg of the peptide, 1 unit of labeling reagent was used. Peptides were dissolved in 100 μL of 0.1 M TEAB, and the labeling reagent was dissolved in 41 μL of acetonitrile. After incubation for 1 h, the reaction was stopped with ammonium hydroxide. Differently labeled peptides were mixed equally and then desalted by peptide desalting spin columns (Thermo Fisher, 89852). TMT-labeled peptide mix was fractionated using a C18 column (Waters BEH C18 4.6×250 mm, 5 μm) on a Rigol L3000 HPLC operating at 1 mL/min, and the column oven was set at 50°C. Shotgun proteomics analyses were performed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) operated in the data-dependent acquisition (DDA) mode. Q Exactive HF-X mass spectrometer was operated in positive polarity mode with a spray voltage of 2.3 kV and capillary temperature of 320°C. Two independent experiments were performed.

Global protein abundance analysis
The resulting spectra from each fraction were searched separately against NCBI E. faecalis strains OG1RF (CP002621.1) database (https://www.ncbi.nlm.nih.gov/nuccore/CP002621.1) by the search engines: Proteome Discoverer 2.2 (PD 2.2, Thermo). The searched parameters as follows: A mass tolerance of 10 ppm for precursor ion scans and a mass tolerance of 0.02 Da for the production scans were used. Carbamidomethyl was specified in PD 2.2 as fixed modifications. Oxidation of methionine, acetylation of the N-terminus and TMT of lysine were specified in PD 2.2 as variable modifications. A maximum of 2 miscleavage sites was allowed. For protein identification, a protein with at least one unique peptide was identified at FDR less than 1.0% on peptide and protein level, respectively.
Proteins containing similar peptides and could not be distinguished based on MS/MS analysis were grouped separately as protein groups. Reporter Quantification (TMT) was used for TMT quantification.
The protein quantitation results were statistically analyzed by Mann-Whitney Test, the significant ratios, defined as p < 0.05 and ratio > 1.2 or ratio < 0.83 [fold change, FC], were used to screen the differential abundance proteins (DAPs). Gene Ontology (GO) and InterPro (IPR) analysis were conducted using the interproscan-5 program against the non-redundant protein database (including Pfam, PRINTS, ProDom, SMART, ProSiteProfiles, PANTHER), and the databases COG (Clusters of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used to analyze the protein family and pathway. The probable interacting partners were predicted using the STRINGdb server (http://string.embl.de/) based on the related species. STRING is a database of both known and predicted protein-protein interactions. The enrichment pipeline was used to perform the enrichment analysis of GO, IPR, and KEGG, respectively.

RNA isolation and RT-qPCR
The RNA isolation of E. faecalis strains were performed as described previously with some modifications [28]. The E. faecalis strains OG1RF and the ΔclpP mutant were inoculated into TSB and cultured at 37°C for 4 h to logarithmic phase or for 12 h to stationary phase, the following operations were performed at 4°C centrifugation or on ice: bacterial cultures were centrifuged at 5,000 x g for 5 min, and then the pellets were washed twice with 0.9% saline; the culture was homogenized 5 times using 0.1-mm zirconia-silica beads in a mini-BeadBeater (Biospec, Bartlesville, OK) at 5,000 rpm for 60 s at 1-min intervals; the samples were centrifuged at 15,000 rpm and the bacterial RNA in the supernatant was purified using an RNeasy minikit (Qiagen, Hilden, Germany) and quantified using an  Table S2.

Statistical analysis
Experimental data were analyzed with SPSS software (version 16.0; SPSS, Chicago, IL, USA) and compared using the Student's t test, one-way analysis of variance, Mann-Whitney test, or the log-rank test. Differences with a P value of <0.05 were considered statistically significant.

Construction of clpP deletion mutant and the complemented strain
To explore the role of ClpP in E. faecalis, we constructed the clpP deletion mutant in the E. faecalis OG1RF strain using the temperature-sensitive plasmid pJRS233. The deletion mutant strain was verified by PCR and direct sequencing and was referred to as the OG1RF ΔclpP mutant strain. The complemented ΔclpP strain was constructed using shuttle vector pIB166, which named OG1RF ΔclpP/pIB166::clpP and also verified by PCR and direct sequencing. The ΔclpP strain containing the empty vector pIB166 was designated as OG1RF ΔclpP/pIB166. The RNA levels of the clpP gene of all the above four E. faecalis OG1RF strains were determined by RT-qPCR and shown in Fig. S1.
ΔclpP mutant strain showed impaired growth at 20℃, 45℃, 5%NaCl or 2 mM H2O2 Previous research indicated that ClpP participated in the stress of low or high temperature and the oxidative stress response in S. aureus [23], however, these issues are still unknown in E. faecalis.
Thus we first investigated the effects of clpP deletion on the growth of E. faecalis under the stress of low or high temperature, hyperosmotic pressure, low pH, and oxidative stress. At 37℃, there were no significant growth differences between the E. faecalis OG1RF parent strain and its ΔclpP mutant.
However, under the 20℃ or 45℃, the ΔclpP mutant strain showed a lower OD 600 than was observed for the wild-type after entering logarithmic phase growth (Fig.1). As Fig.2 indicated, the growth of ΔclpP mutant strain was also impaired under 5%NaCl (logarithmic phase) or 2 mM H2O2 (later logarithmic phase or stationary phase).

Deletion of clpP leads to decreased biofilm formation
The polystyrene microtiter plate assay was performed to evaluate the role of clpP in the biofilm formation of E. faecalis under static conditions. The biofilm formation of E. faecalis OG1RF parent strain and its ΔclpP mutant was monitored at 12, 24, and 48 h on microtiter plates stained with crystal violet, and the OD 570 were determined. The biofilms of the ΔclpP mutant strain (OD 570 , 0.835 ± labeling. The protein quantitation results were statistically analyzed by Mann-Whitney Test, the significant ratios, defined as p < 0.05 and ratio > 1.2 or ratio < 0.83 [fold change, FC], were used to screen the differential abundance proteins (DAPs).The protein quantitation results were given as the means from two independent experiments, the repeatability of the two independent experiments were evaluated by the coefficient of variation (CV), and as Fig. S2 indicated that the CV of the two independent experiments was very low. AlltheDAPsweresummarized in Table 1, the abundances of 135 proteins changed in the ΔclpP mutant strain, of which 111 proteins increased, while 24 proteins decreased.

Gene Ontology (GO) and KEGG analysis of DAPs
All the DAPs between the △clpP mutant and the parent strain were analyzed by the Gene Ontology (GO) and KEGG analysis. As Fig.6 GOanalysisshowed, the increased DAPs in the △clpP mutant strain (logarithmic phase) were mainly concentrated in the following molecular functions: Nacetyltransferase activity, coenzyme binding, cofactor binding, ATPase activity, nucleosidetriphosphatase activity, hydrolase activity, ATP binding, kinase activity, nucleotide binding, organic cyclic compound binding, heterocyclic compound binding, DNA binding and nucleic acid binding.
However, the decreased DAPs were mainly included in the following molecular functions: structural constituent of ribosome, rRNA binding, orotidine-5'-phosphate decarboxylase activity, hydrolase activity, organic cyclic compound binding, heterocyclic compound binding, and nucleic acid binding.
The KEGG analysis demonstrated that functions of most of these DAPs in the △clpP mutant (logarithmic phase) belonged to the ribosome, fructose and mannose metabolism, pyrimidine metabolism, purine metabolism, pentose phosphate pathway, glycolysis/gluconeogenesis, ABC transporters (Fig.7). The functions of DAPs in the stationary phase of △clpP mutant strain were similar to that in the logarithmic phase (Fig.S3).

DAPs associated with the stress response or virulence, biofilm formation of E. faecalis
Based on the literatures, we selected the DAPs which may associate with stress response or virulence, biofilm formation of E. faecalis for further analyzed. The abundance of DAPs which associated with the stress response or virulence of E. faecalis, including the FsrA response regulator and gelatinase GelE, ATPase/chaperone ClpC, chaperonin GroEL, acetyl esterase/lipase, and transcriptional regulator proteins, HrcA, CtsR, and Spx increased in the △clpP mutant strain (Table 2). However, the abundances of ribosomal proteins L4/L1, L7/L12, L13, L18, L20, S14, and S18 decreased in the △clpP mutant strain. The abundance of the biofilm formation of E. faecalis associated DAPs, adapter protein MecA increased in the △clpP mutant strain, while the abundances of orotate phosphoribosyltransferase, orotidine-5'-phosphate decarboxylase and dihydroorotase reduced ( Table   2).The RNA levels of all the above DAPs which may associate with stress response, virulence or biofilm formation of E. faecalis, were verified by RT-qPCR and were consistent with protein abundance changes in the △clpP mutant strain.

Discussion
ClpP as a protease of Hsp100/Clp family is very important for bacterial growth and plays an irreplaceable role in cellular protein quality control systems by refolding or degrading damaged proteins in stressed cells [14]. Up to now, ClpP has been found participating in many essential activities of bacteria, such as stress response, which including abnormal temperature stress, hyperosmotic pressure, low pH, and oxidative stress response, virulence, biofilm formation and so on. Among those DAPs, we also found the transcriptional regulators CtsR and Spx, the ClpC adaptor proteins MecA and FtsZ-interacting cell division protein YlmF, which were similar to that in S. aureus strains, and interestingly we also found other new proteins, such as Acetyl esterase/lipase, ribosomal protein, orotidine-5'-phosphate decarboxylase and so on.
The ClpP has been shown to participate in stress tolerance by refolding or degrading damaged proteins during the growth of bacteria, and several studies have indicated that the ΔclpP mutant strain showed a growth defect over a broad range of temperatures, which included high (  Previous studies have found that ClpP can significantly affect the biofilm formation of bacteria, but the effects of clpP on the biofilm formation in different genera of bacteria were obviously different, or even opposite [15,16,18,19,21]. This study first found that biofilm formation decreased when the This study also found that the sensitivities to linezolid or minocycline of the ΔclpP mutant strain increased. As we knew, linezolid as an inhibitor of bacterial protein synthesis which acts on the 50S ribosome subunit of gram-positive bacteria, and minocycline is a synthetic tetracycline derivative which acts on the 30S ribosome subunit of gram-positive or gram-negative bacteria [45,46]. The present study indicated that the abundance of 50S ribosomal proteins L13, L18, L20, the 30S ribosomal proteins S14, S18, all reduced in the △clpP mutant strain, thus might lead to the increased sensitivities of the △clpP mutant strain to linezolid or minocycline.
In the B. subtilis, Spx plays a significant role in the protection system against oxidative stresses [47].
Recently Rojas-Tapias DF and Helmann JD found that the Spx was itself a regulator of the ctsR operon, and the ctsR operon was found regulated the expression of clpC and clpP [48]. The present study indicated that when the clpP was deleted in E. faecalis OG1RF, the abundance of ClpC, CtsR, and Spx all increased, this was similar with that observed in S. aureus [30]. In S. aureus, the RNA levels of the clpC operon (ctsR-mcsA-mcsB-clpC), groE, and dnaK were induced in response to an accumulation of misfolded proteins, which supported the notion that ClpP proteases served to degrade misfolded proteins [30]. Our study found the abundances of ClpC, GroEL, and the DnaB, but not the DnaK, increased in the △clpP mutant strain, may also due to the accumulation of misfolded proteins.
It is easy to understand that ClpP, as a protease, has a significant effect on the abundance of proteins, but not on the RNA level of genes. In the present study, the abundance of many transcription regulation related proteins changed in the △clpP mutant strain, such as regulatory protein Spx (spxA), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR and so on, and this issue was also observed in other studies [29, 30]. As we knew, the transcriptional regulators usually control the transcription and RNA levels of their functional genes. So the ClpP may through affecting the abundance of transcriptional regulators to alter the RNA levels of other genes, and the RNA levels of many genes changed in the ΔclpP mutant strain in this study, and the similar results were also observed in other studies [23, 30]. Since ClpP was a protease which involved in protein degradation, thus its absence should provoke the accumulation of proteins, and this was consistent with our result that the abundance of most of the DAPs increased in the △clpP mutant strain. However, the abundance of some proteins and their corresponding RNA levels of genes decreased in the △clpP mutant strain in the present study, and the similar results were also found in another study [30]. The reason for this issue may be that as mentioned above, ClpP reduced the transcription and expression of those genes by regulating the abundance of transcriptional regulators.

Conclusions
The present study indicates that ClpP may through affecting the abundance of ribosomal proteins L4/L1, L7/L12, L13, L18, L20, S14, and S18 participates in the stress response, and the linezolid or     Survival of the ΔclpP mutant and the parent strain with antimicrobials exposure over time.
Three independent experiments were performed and the data represent as means ± SD.
The dashed line indicates the detection limit of this assay