https://orcid.org/0000-0002-1456-8284
Abstract
Calcium-binding motifs are shared by multiple bacteriophage lysins; however, the influence of calcium on the enzymatic activity and host range of these enzymes is still not understood. To address this, ClyF, a chimeric lysin with a putative calcium-binding motif, was used as a model for in vitro and in vivo investigations.
The concentration of calcium bound to ClyF was determined by atomic absorption spectrometry. The influence of calcium on the structure, activity and host range of ClyF was assessed by circular dichroism and time–kill assays. The bactericidal activity of ClyF was evaluated in various sera and a mouse model of Streptococcus agalactiae bacteraemia.
ClyF has a highly negatively charged surface around the calcium-binding motif that can bind extra calcium, thereby increasing the avidity of ClyF for the negatively charged bacterial cell wall. In line with this, ClyF exhibited significantly enhanced staphylolytic and streptolytic activity in various sera containing physiological calcium, including human serum, heat-inactivated human serum, mouse serum and rabbit serum. In a mouse model of S. agalactiae bacteraemia, intraperitoneal administration of a single dose of 25 μg/mouse ClyF fully protected the mice from lethal infection.
The present data collectively showed that physiological calcium improves the bactericidal activity and host range of ClyF, making it a promising candidate for the treatment of infections caused by multiple staphylococci and streptococci.
Introduction
Antibiotics have played a critical role in combating bacterial infections for about 100 years. However, due to the overuse and misuse of antibiotics, bacteria that are resistant to killing by antibiotics are continuously emerging and spreading, which poses a growing threat to global public health. The lag in the development of new antibiotics further exacerbates this unmet need in antimicrobial resistance, which necessitates the development of novel antibacterial agents with different mechanisms of action from traditional antibiotics.1–3
Bacteriophage-derived lysin, a type of peptidoglycan hydrolase, which is expressed at the late phase of the phage infection cycle, represents a promising alternative to antibiotic therapy.4–7 Lysin usually contains a modular structure, that is, an N-terminal enzymatically active domain (EAD) that is responsible for the cleavage of peptidoglycan bonds and a C-terminal cell-wall binding domain (CBD) that recognizes conserved elements on the surface of the target bacterium.8,9 The robust and rapid bactericidal activity, low risks of resistance, good performance against persisters and biofilms, and high specificity make lysin an ideal alternative to traditional antibiotics.10–15 In fact, many diseases are commonly caused by coinfection with multiple bacteria, which challenges most host-specific lysins and their transmedicinal progress. However, few lysins has been reported to be active against multiple bacterial species. For instance, PlySs2, a Streptococcus suis prophage lysin, is active against Staphylococcus aureus, Streptococcus agalactiae and other closely related streptococci.16 Previously, we reported that a novel chimeolysin, ClyF, consisting of the EAD from the Ply187 lysin, i.e. Pc, and the CBD from the PlySs2 lysin, demonstrated high activity against multiple isolates of staphylococci but not the streptococci tested.17 Interestingly, we observed that ClyF exhibits improved bactericidal activity against MRSA in milk and in human serum (HS);17 however, the mechanisms behind this are still not fully understood. Here, we report that ClyF contains an active calcium-binding motif and unexpectedly gains extended bactericidal activity and host range in the presence of physiological calcium, which was confirmed by time–kill assay, MIC assay and a mouse bacteraemia model of S. agalactiae infection.
Materials and methods
Bacterial strains
All S. aureus strains and the Escherichia coli BL21(DE3) strain (Table S1) used in this study were grown in lysogeny broth (LB) at 37°C with a shaking rate of 200 rpm. All streptococci were cultured in brain heart infusion (BHI) medium.
Protein expression and purification
Recombinant proteins were purified according to the method described previously using a Ni-NTA agarose.18 Briefly, E. coli cells were cultured to an OD600 of 0.6–0.8, then induced by 0.2 mM isopropyl-β-D-thiogalactoside and grown for 12 h at 16°C. The bacterial cells were lysed by a high-pressure cell cracker in 20 mM Tris-HCl, pH 8.0. The supernatant was filtered by a 0.45 μm filter and passed through a Ni-NTA column that was pre-equilibrated with 20 mM imidazole. After washing and elution with 40 and 250 mM imidazole, collected proteins were dialysed against PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·H2O, 1.4 mM KH2PO4, pH 7.4), passed through a Detoxi-Gel™ Endotoxin Removing Gel (Thermo Scientific) and finally stored at 4°C after filtration. The residual endotoxin level was confirmed by an EndoLISA kit (ELISA325-based endotoxin detection assay, Hyglos GmbH) to a low level (< 0.5 EU/mL). Protein concentration was determined by bicinchoninic acid assay (Pierce Rockford, IL, USA), using BSA as the standard.
In silico calcium-binding motif analysis and 3D modelling
The calcium-binding motif in ClyF was analysed by Clustal Omega online software (https://www.ebi.ac.uk/Tools/msa/clustalo/) and ESPript (http://espript.ibcp.fr/ESPript/ESPript/index.php) using lysin LysGH15 (PDB ID: 4OLK) as the template. The 3D structure of ClyF was modelled by AlphaFold 2 online software, and the surface contact potential of ClyF was further analysed by PyMOL.
Nano differential scanning fluorimetry (nanoDSF)
The intrinsic emission fluorescence of protein (500 mg/L) at 350 and 330 nm was monitored by a nanoDSF method using a Prometheus NT.48 instrument (NanoTemper Technologies, CA, USA) over a temperature range of 25°C–95°C (increasing step of 1°C/min) in the presence of 0, 0.1, 1, 5 and 10 mM CaCl2, ZnCl2 or MgCl2. The first derivative of the ratio of fluorescence at 350 and 330 nm (first derivative of F350/F330) was calculated automatically by the PR-ThermControl software supplied with the instrumentation. The thermal unfolding transition temperature (Tm) corresponds to peaks of the first derivative of F350/F330. Samples were measured in triplicate.
Circular dichroism
The circular dichroism spectrum of ClyF at 200 mg/L in 20 mM PBS (pH 7.4) was collected by an Applied Photophysics Chirascan Plus circular dichroism spectrometer (Leatherhead, UK) from 190 to 260 nm (0.05 cm path length) at room temperature in the presence of 0, 0.1, 1, 5 and 10 mM CaCl2, ZnCl2 or MgCl2. The spectra of air and PBS (pH 7.4) were recorded as background and baseline, respectively. The secondary structure was calculated by the CDNN V2.1 software supplied by the instrument manufacturer. Samples were measured in triplicate.
Quantification of calcium bound to ClyF
Briefly, 400 mg/L ClyF was incubated with 0, 0.1 and 1 mM CaCl2 for 1 h at room temperature, and then dialysed against 20 mM Tris buffer (pH 7.4) to remove unbound calcium. The concentrations of calcium in resulting protein samples were determined by Agilent 200 Series AA flame atomic absorption spectrometer (Agilent, USA). Standard CaCl2 solution ranging from 0 to 1 mM was measured under the same instrumental conditions. Samples were measured in triplicate.
Electrophoretic mobility shift assay
Purified pET28a(+) plasmid (250 ng) was mixed with 0, 0.1, 1 and 5 mM CaCl2, ZnCl2 or MgCl2 in the presence or absence of 2.5 µM ClyF for 1 h at room temperature, and then underwent electrophoresis against 1% agarose gel. Nucleic acids treated with 5 mM CaCl2, ZnCl2 and MgCl2 were used as controls. Samples were measured in triplicate.
Bacteriolytic analysis
Influence of metal ions on the lytic activities of the EAD of ClyF, i.e. Pc, and the full-length ClyF were determined by turbidity reduction assay as described previously.17 Briefly, an overnight culture of S. aureus N315 was washed and resuspended in PBS to an OD600 of ∼1.0. Cells were then treated with 50 mg/L Pc or ClyF in the presence of 1 mM EDTA, 1 mM EDTA supplemented with 2 mM CaCl2 or MgCl2; the turbidity of each well at 600 nm was monitored by a Synergy H1 microplate reader (BioTek, USA) for 60 min at 37°C. Cells treated with an equal volume of PBS instead of lysin served as controls. All experiments included at least three biological replicates.
Lytic assay
The lytic activity of ClyF at 1 h post-treatment was measured by the log killing assay as previously described.15 Briefly, overnight cultures of bacterial cells were resuspended in PBS to a final OD600 of 0.6 to 0.8. The effects of calcium on the killing capacity of ClyF were investigated by exposure of S. aureus N315 to 5 mg/L ClyF for 1 h at 37°C in the presence of 0, 0.1, 0.2, 0.5, 1, 5 and 10 mM CaCl2. When evaluating the effects of metal ions on ClyF activity, S. aureus N315 was treated with 5 mg/L ClyF for 1 h at 37°C in the presence of 3 mM EDTA, 3 mM EDTA supplemented with 4 mM CaCl2, 4 mM ZnCl2 or 4 mM MgCl2. When evaluating the effects of human serum albumin (HSA) on ClyF activity, bacterial cells were treated with 1 or 10 mg/L ClyF for 1 h at 37°C in the presence/absence of 1 mM CaCl2 or 40 mg/mL HSA. The decrease of viable bacterial number (in cfu) after each treatment was determined by plating 10-fold serial dilutions on BHI agar. All experiments included at least three biological replicates.
Time–kill assay
Time–kill curves were performed according to the method described by CLSI with modifications.19 When evaluating the effects of calcium on the bactericidal activity of ClyF against multiple strains, each bacterium was exposed to ClyF in MHB for 24 h in the presence/absence of 0.5 mM CaCl2; viable bacterial number at 0, 0.5, 1, 3, 6 and 24 h were determined by plating serially diluted 10-fold samples to agar plates. The ClyF concentration was 4 mg/L for S. aureus N315, 16 mg/L for S. agalactiae S12, and 20 mg/L for all the other strains tested. When evaluating the effects of HS on the bactericidal activity of ClyF, each type of bacterial cells was exposed to ClyF in MHB containing 25% HS, 25% heat-inactivated HS (iHS), or 1 mM CaCl2 for 24 h, viable bacterial numbers at 0, 0.5, 1, 3, 6 and 24 h were determined by plating serially diluted 10-fold samples onto agar plates. The ClyF concentration was 2 mg/L for S. aureus N315 and 16 mg/L for all the other strains tested. All experiments included at least three biological replicates.
MIC assay
The MICs of ClyF against S. aureus isolates under different conditions were determined by the broth dilution method, as described by CLSI, with modifications.20 HS-MHB and iHS-MHB were prepared by mixing 2 × MHB with an equal volume of PBS 4-fold diluted HS and iHS (the final concentration of HS was 12.5%), respectively. CAMHB was prepared by supplementing MHB with 0.1 mM CaCl2. S. aureus cells with a final concentration of 1 × 104 cfu/well were cocultured with various concentrations of ClyF (0, 1, 2, 4, 8, 16, 32, 64 and 128 mg/L) in MHB, CAMHB, HS-MHB and iHS-MHB for 24 h at 37°C. Resulting cell viability within each well was determined by resazurin assay.21 MIC was defined as the lowest concentration of antimicrobial that completely inhibited the growth of bacteria, that is, the colour was still blue after the treatment of resazurin for 4 h. All experiments included at least three biological replicates.
Mouse experiments
All mouse infection experiments were performed in an ABSL-2 lab and all experimental methods were carried out in accordance with the regulations and guidelines set forth by the Animal Experiments Committee of the Wuhan Institute of Virology, Chinese Academy of Sciences. All experimental protocols were approved by the Animal Experiments Committee of Wuhan Institute of Virology, Chinese Academy of Sciences (WIVA17201602). Animals were housed in individually ventilated cages following a set of animal welfare and ethical criteria during the experiment and euthanized at the end of observation. Female 6–8-week-old BALB/c mice were infected intraperitoneally with 2.9 × 108 cfu/mouse of S. agalactiae S12 as described previously,22 and randomly divided into four groups. After 1 h of infection, mice were administrated intraperitoneally with either a single dose of 6.25 µg/mouse ClyF (n = 7), 12.5 µg/mouse ClyF (n = 7), 25 µg/mouse ClyF (n = 7) or an equal volume of PBS buffer (n = 5). The survival rate for each group was recorded for 10 days and analysed by log-rank (Mantel–Cox) test.
Results
ClyF contains an active calcium-binding motif
Sequence alignment analysis revealed that the EAD of ClyF, i.e. Pc, shares a homologous calcium-binding motif with LysGH15,23 with one residual replacement (Figure 1a). To identify the function of the putative calcium-binding motif in ClyF, we purified Pc and examined the effects of calcium on its activity. Results showed that the bacteriolytic activity of Pc could be completely abolished by EDTA and restored by supplementing with CaCl2 (Figure 1b), suggesting that Pc contains an active calcium-binding motif. In line with these observations, the lytic activity of the full-length chimera, ClyF, is extremely sensitive to EDTA, and could be restored by calcium, but not other metal ions (Figure 1c and d).
ClyF contains an active calcium-binding motif. (a) Alignment of the calcium-binding motif of ClyF with that of LysGH15. Alignment was generated by Clustal Omega and visualized by ESPript. The calcium-binding residues (conserved positions 1, 3, 5, 7 and 12) are indicated by asterisks. (b and c) Bacteriolytic activity of Pc and ClyF (50 mg/L) against S. aureus N315 under various conditions. 1 mM EDTA, 2 mM CaCl2 and MgCl2 were used. (d) The effects of different ions on ClyF activity. ClyF was inactivated by 3 mM EDTA and dialysed against PBS; residual enzymatic activity was then tested against S. aureus N315 in the presence of 4 mM CaCl2, ZnCl2 or MgCl2. ***, P < 0.001. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
Calcium improves the overall positive charge of ClyF with minor effects on its secondary structure
It is reported that the equilibrium dissociation constant of the LysGH15 CHAP domain for calcium is approximately 27 µM.23 Interestingly, calcium under millimolar concentrations still showed a dose-dependent enhancement of the killing capacity of ClyF (Figure 2a), indicating that excess calcium promotes ClyF activity via another as yet unknown mechanism of action. The protein contact potential analysis by PyMOL showed that ClyF contains multiple negative charges around the calcium-binding motif (Figure 2b and Figure S1), which could serve as extra hosts for positively charged calcium ions. Supporting this, calcium bound to ClyF increased linearly with the increased concentration of CaCl2 in dialysis buffer (Figure 2c). Specifically, the original concentration of calcium was 18.16 μM in freshly purified 400 mg/L ClyF (14 μM), corresponding to a molar ratio of ∼1:1 between calcium and ClyF (Figure 2c), which is consistent with the model of binding between calcium and the calcium-binding motif revealed by crystallography. 23 However, the concentration of bound calcium increased to 35.97 and 177.39 μM when ClyF was dialysed against 100 and 1000 μM CaCl2, respectively (Figure 2c). As shown in Figure 2d, increased binding with positively charged calcium enhanced the non-specific binding of ClyF to negatively charged nucleic acids, resulting in a reduced free band on the gel corresponding to the size of the nucleic acids. Circular dichroism analysis further showed that a minor difference is observed in the adsorption profiles of ClyF in the presence of various concentrations of CaCl2 (Figure 2g), suggesting that calcium only has a minor influence on the secondary structure of ClyF (Table S2). In contrast, other divalent metal ions, such as magnesium and zinc, showed minor effects on the shift of nucleic acids in combination with ClyF (Figure 2e and f), suggesting that magnesium and zinc probably cannot conjugate to the surface of ClyF stably. In addition, ClyF was conformationally unstable in the presence of ZnCl2 (Figure 2h); specifically, the content of the α-helix was significantly decreased, and the content of parallel/antiparallel and random coil was significantly increased (Table S3), which partially explains the abolished bactericidal activity of ClyF in excess concentrations of zinc (Figure 1d). In contrast, minor structural change of ClyF was observed in the presence of MgCl2 (Figure 2i). To further understand the effects of calcium on the structure of ClyF, we investigated the thermostability of ClyF by nanoDSF in the presence of varied concentrations of CaCl2. The thermodynamics analysis showed that the unfolding curve of ClyF shifts rightwards, along with the increased concentration of calcium (Figure S2a). In other words, calcium enhanced the Tm of ClyF in a dose-dependent manner; specifically, the unfolding temperature of ClyF increased from 50.7°C to 56.5°C in the presence of 10 mM CaCl2 (Figure S2b), suggesting that calcium-binding motif-mediated binding of calcium enhances the conformational stability of the entire complex.
Calcium improves the overall positive charge of ClyF with minor effects on its secondary structure. (a) Dose-dependent enhancement of calcium on the bactericidal activity of ClyF against S. aureus N315. (b) Surface contact potential analysis of ClyF by PyMOL. The calcium-binding motif is marked with dashed oval. Negatively charged regions are labelled in red, while positively charge regions are in blue. (c) Concentrations of calcium bound to ClyF under different conditions. ClyF was mixed with 0, 0.1 and 1 mM CaCl2 for 1 h at room temperature and dialysed against 20 mM Tris. Concentrations of calcium in resulting samples were determined by colorimetric assay. (d–f) Effects of calcium, magnesium and zinc on the non-specific binding of ClyF to nucleic acids. Purified pET28a(+) plasmid was mixed with 0, 0.1, 1 and 5 mM CaCl2 (d), ZnCl2 (e) and MgCl2 (f) in the presence or absence of 2.5 µM ClyF for 1 h at room temperature, and then underwent electrophoresis on 1% agarose gel. The first lane on the left is the standard DNA ladder. The retention band and size of pET28a(+) are marked with triangles. (g–i) Circular dichroism spectra of ClyF under different concentrations of CaCl2, ZnCl2 and MgCl2. Spectra of ClyF in PBS (pH 7.4) containing 0.1, 1, 5 and 10 mM CaCl2 (g), ZnCl2 (h) and MgCl2 (i) were obtained by scanning with a circular dichroism spectrometer from 190 to 260 nm at room temperature and data from 200 to 260 nm are shown. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
Calcium modulates the activity and host range of ClyF
Since ClyF showed enhanced activity against S. aureus in the presence of calcium (Figure 1d), we wonder whether this phenomenon could be extended to other bacteria. To answer this question, we examined the growth of multiple Gram-positive bacteria exposed to ClyF for 24 h in the presence of 0.5 mM CaCl2. Results showed that calcium significantly improved the killing activity of ClyF against all strains tested during the entire incubation process (Figure 3). Specifically, obvious delayed regrowth was observed for S. aureus (Figure 3a), Streptococcus dysgalactiae (Figure 3c), Streptococcus pyogenes (Figure 3h) and Streptococcus pneumoniae (Figure 3e) in the presence of calcium. Notably, increased killing of S. pneumoniae, Enterococcus faecalis and Streptoccus mutans by ClyF was observed in the presence of calcium (Figure 3e–g), indicating an extended spectrum of activity.
Calcium extends the bactericidal activity and host range of ClyF. Overnight cultures of S. aureus N315 (a), S. agalactiae S12 (b), S. dysgalactiae ATCC 35666 (c), S. suis SC84 (d), S. pneumoniae NS26 (e), E. faecalis 493 (f), S. mutans UA159 (g) and S. pyogenes ATCC 12344 (h) were exposed to ClyF in MHB for 24 h at 37°C in the presence or absence of 0.5 mM CaCl2; viable bacterial number at 0, 0.5, 1, 3, 6 and 24 h was determined by plating serial dilutions onto LB or BHI agar. The ClyF concentration was 4 mg/L for S. aureus, 16 mg/L for S. agalactiae, and 20 mg/L for all the other strains. The PBS-treated groups were used as controls. Data are shown as mean ± SD (n = 3). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
ClyF gains enhanced bactericidal activity in various sera
Since the above observations showed that ClyF gains enhanced activity at physiological calcium concentration (1–2 mM), we wondered whether ClyF shows a similar improved bactericidal activity in serum. To this end, we first ascertained the bactericidal activity of ClyF in human serum, taking four bacteria, i.e. S. aureus, S. agalactiae, S. dysgalactiae and S. pyogenes, which are commonly recognized in bloodstream infections, as examples. As shown in Figure 4, improved killing by ClyF was observed in all strains tested under conditions of 1 mM CaCl2 or HS. Notably, ClyF showed improved killing capacity against S. aureus in HS in comparison with that of its activity in 1 mM calcium-supplemented PBS (Figure 4a), indicating that possibly other as yet unrevealed factors in HS facilitate the killing of S. aureus by ClyF. To exclude the effects of certain proteins in human serum, we incubated HS for 20 min at 75°C, filtered it and examined the activity of ClyF in iHS. Results showed that similar regrowth profiles are observed for S. agalactiae, S. dysgalactiae and S. pyogenes in the presence of HS, iHS and calcium (Figure 4b–d), suggesting that HS, either heat-treated or not, shows a comparable enhancement effect on ClyF activity to that of calcium. In addition, ClyF gained improved killing capacity in both mouse serum (MS) and rabbit serum (RS), as it did in the presence of 1 mM CaCl2 (Figure 4e).
ClyF gains enhanced bactericidal activity in various sera. (a–d) Regrowth of bacteria exposed to ClyF in HS. S. aureus N315 (a) was exposed to 2 mg/L ClyF and the strains of S. agalactiae S12 (b), S. dysgalactiae ATCC 35666 (c) and S. pyogenes ATCC 12344 (d) were exposed to 16 mg/L ClyF in MHB for 24 h in the presence of calcium, HS and iHS; viable bacterial number at 0, 0.5, 1, 3, 6 and 24 h was determined by plating serial dilutions onto LB or BHI agar. (e) Bactericidal activity of ClyF in MS and RS. S. aureus N315 was treated with 1 mg/L ClyF for 1 h at 37°C. The number of viable bacteria in each treatment was determined by the serial dilution method. Data are shown as mean ± SD. **, P < 0.01. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
To further confirm the modulation effects of calcium on the activity of ClyF, we determined the MIC of ClyF against multiple S. aureus strains and representative isolates of S. agalactiae, S. dysgalactiae and S. pyogenes in MHB with or without CaCl2 and HS. As shown in Table 1, the MIC value of ClyF in MHB is higher than its MIC values tested under the other conditions, suggesting that calcium, supplemented exogenously or in HS, could equivalently improve the antibacterial activity of ClyF. Notably, minor differences in the MIC values of ClyF were observed among different S. aureus strains under conditions of uninactivated HS and iHS. This may be due to the heterogeneity of different strains, or related to the antibacterial effects of HS itself.24
MICs of ClyF under different conditions
| Strain | MIC (mg/L) | |||
|---|---|---|---|---|
| MHB | CAMHB | HS-MHB | iHS-MHB | |
| S. aureus N315 | 8 | 8 | 4 | 4 |
| S. aureus WHS11032 | 16 | 8 | 8 | 4 |
| S. aureus RN4220 | 16 | 8 | 8 | 8 |
| S. aureus WHS11011 | 16 | 8 | 8 | 8 |
| S. aureus WHS11018 | 16 | 8 | 4 | 8 |
| S. aureus CCTCC AB91118 | 8 | 8 | 8 | 4 |
| S. dysgalactiae ATCC 35666 | 32 | 16 | 16 | 16 |
| S. pyogenes ATCC 12344 | 32 | 16 | 16 | 16 |
| S. agalactiae S12 | 64 | 32 | 32 | 32 |
| Strain | MIC (mg/L) | |||
|---|---|---|---|---|
| MHB | CAMHB | HS-MHB | iHS-MHB | |
| S. aureus N315 | 8 | 8 | 4 | 4 |
| S. aureus WHS11032 | 16 | 8 | 8 | 4 |
| S. aureus RN4220 | 16 | 8 | 8 | 8 |
| S. aureus WHS11011 | 16 | 8 | 8 | 8 |
| S. aureus WHS11018 | 16 | 8 | 4 | 8 |
| S. aureus CCTCC AB91118 | 8 | 8 | 8 | 4 |
| S. dysgalactiae ATCC 35666 | 32 | 16 | 16 | 16 |
| S. pyogenes ATCC 12344 | 32 | 16 | 16 | 16 |
| S. agalactiae S12 | 64 | 32 | 32 | 32 |
MICs of ClyF under different conditions
| Strain | MIC (mg/L) | |||
|---|---|---|---|---|
| MHB | CAMHB | HS-MHB | iHS-MHB | |
| S. aureus N315 | 8 | 8 | 4 | 4 |
| S. aureus WHS11032 | 16 | 8 | 8 | 4 |
| S. aureus RN4220 | 16 | 8 | 8 | 8 |
| S. aureus WHS11011 | 16 | 8 | 8 | 8 |
| S. aureus WHS11018 | 16 | 8 | 4 | 8 |
| S. aureus CCTCC AB91118 | 8 | 8 | 8 | 4 |
| S. dysgalactiae ATCC 35666 | 32 | 16 | 16 | 16 |
| S. pyogenes ATCC 12344 | 32 | 16 | 16 | 16 |
| S. agalactiae S12 | 64 | 32 | 32 | 32 |
| Strain | MIC (mg/L) | |||
|---|---|---|---|---|
| MHB | CAMHB | HS-MHB | iHS-MHB | |
| S. aureus N315 | 8 | 8 | 4 | 4 |
| S. aureus WHS11032 | 16 | 8 | 8 | 4 |
| S. aureus RN4220 | 16 | 8 | 8 | 8 |
| S. aureus WHS11011 | 16 | 8 | 8 | 8 |
| S. aureus WHS11018 | 16 | 8 | 4 | 8 |
| S. aureus CCTCC AB91118 | 8 | 8 | 8 | 4 |
| S. dysgalactiae ATCC 35666 | 32 | 16 | 16 | 16 |
| S. pyogenes ATCC 12344 | 32 | 16 | 16 | 16 |
| S. agalactiae S12 | 64 | 32 | 32 | 32 |
HSA promotes the bactericidal activity of ClyF
Previous studies showed that HS is rich in calcium, of which approximately 50% exists in the form of free ions and the rest is bound to proteins, mainly to HSA.25,26 Therefore, we evaluated the influence of HSA on ClyF activity to understand the contribution of the key human blood factor. Unexpectedly, HSA also improved the bactericidal activity of ClyF against S. aureus in a dose-dependent manner (Figure S3). Consistent with this observation, HSA improved the activity of ClyF against other bacteria tested, although the enhancement was slightly weaker than that of calcium at 1 h after treatment (Figure 5). These observations suggested that HSA also plays an as yet not fully understood role in the improved killing activity of ClyF.
HSA promotes ClyF activity. Overnight cultures of S. aureus N315 (a), S. agalactiae S12 (b), S. dysgalactiae ATCC 35666 (c) and S. pyogenes ATCC 12344 (d) were incubated with ClyF in PBS in the presence of 1 mM CaCl2, or 40 mg/mL HSA for 1 h at 37°C. The number of viable bacteria was determined by plating serial dilutions onto LB or BHI agar. The final concentration of ClyF was 1 mg/L for S. aureus and 10 mg/L for the streptococci tested. Data are shown as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
ClyF protects mice from S. agalactiae infection
Further, we took the S. agalactiae-infected mouse model of bacteraemia as an example to evaluate the efficacy of ClyF in vivo. To this end, female BALB/c mice were infected intraperitoneally with 2.9 × 108 cfu/mouse S. agalactiae S12, an optimized dose that ensured migration and dissemination of bacteria from the site of infection to blood and organs within 1 h in our previous work,22 and then intraperitoneally administrated different doses of ClyF at 1 h post infection. In line with the in vitro observations, a single dose of 25 μg/mouse ClyF fully protected mice from lethal S. agalactiae infection (Figure 6), indicating that ClyF has extended bactericidal activity against S. agalactiae in vivo.
ClyF protects mice from lethal S. agalactiae infection. Female BALB/c mice (6–8 weeks old) were injected intraperitoneally with 2.9 × 108 cfu/mouse S. agalactiae S12 and divided randomly into four groups. One-hour post-infection, groups (n = 7 each) received a single intraperitoneal dose of 6.25, 12.5 or 25 μg/mouse ClyF, or an equal volume of PBS (n = 5). The survival rates for all groups were recorded for 10 days. Survival curves were analysed by the log-rank (Mantel–Cox) and Gehan–Breslow–Wilcoxon tests. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
Discussion
Abundant evidence demonstrating the high bactericidal efficiency of lysins in vitro and in animal infection models has been established.6,10,27 However, lysins that have a spectrum of action across multiple bacterial species are still not adequately addressed. In the present study, we report that a chimeric lysin, ClyF, with prime activity against staphylococci, gains extended bactericidal activity against multiple streptococci and E. faecalis in the presence of calcium, in serum, and in a mouse model, suggesting that ClyF is a calcium-dependent broad-spectrum lysin.
Our previous study also showed that ClyC, a chimeric lysin consisting of Pc, and the CBD from the LysSA97 lysin, gains improved staphylolytic activity owing to the putative calcium-binding motif of Pc.28 In the present study, we further confirmed that Pc contains a calcium-dependent staphylolytic activity domain, and moreover, we showed that calcium is not only essential for the lytic activity of ClyF, but also improves the bactericidal activity and host range of ClyF, i.e. calcium expands the spectrum of action of ClyF against E. faecalis and multiple streptococci, including S. agalactiae, S. dysgalactiae, S. suis, S. pneumoniae, S. mutans and S. pyogenes. In addition, the improved bactericidal activity and host range of ClyF were also observed in HS, either heat-inactivated or not, as well as a mouse model of S. agalactiae bacteraemia. Although the role played by the other factors in serum could not be fully excluded, our present data collectively indicate that physiological calcium is probably the major causative agent responsible for the extended spectrum of ClyF. Interestingly, a previous study showed that PlySs2 lysin gains enhanced bactericidal activity in HS by binding to HSA.16 HSA contains a high dose of calcium, which could serve as a calcium pool for ClyF activity; however, the detailed mechanism underlying the promoting effect of HSA on ClyF activity still needs further study.
Since the cell wall of Gram-positive bacteria contains teichoic acid and other negatively charged components,29 the bactericidal activity of lysins could be improved by increasing their positive charge;30 therefore, one possible reason is that excess calcium increases the positive charge on the surface of ClyF, facilitating electrostatic interactions between the lysin and the bacterial surface, thereby enhancing the lytic efficiency of ClyF. Supporting this hypothesis, calcium enhanced the block efficacy of ClyF on the mobility of negatively charged nucleic acids. Currently, we have no idea whether calcium-extended bactericidal activity observed in ClyF is shared by other lysins that contain a conserved calcium-binding motif. However, a recent study showed that CHAPk, another calcium-binding motif-containing domain from the staphylococcal-specific LysK lysin, also displays improved antimicrobial activity against S. agalactiae in TSB medium and milk that is rich in calcium ions.31
Altogether, our present data showed that physiological calcium improves the bactericidal activity and host range of ClyF, making it a promising candidate for the treatment of infections caused by multiple staphylococci and streptococci.
Acknowledgements
We thank Mrs Pei Zhang, Dr Jia Wu and Dr Yanfeng Yao from the Core Facility and Technical Support, Wuhan Institute of Virology for their assistance in microscopy analysis and animal experiments.
Funding
This work was financially supported by the National Natural Science Foundation of China (Nos. 32070187, 32161133003 and 31770192) and the Youth Innovation Promotion Association CAS.
Transparency declarations
None to declare.
Author contributions
Conceived the study: H.Y.; collection of data: M.J., W.Z., D.L., H.X., X.Z. and Z.Z.; data analysis: F.H., M.Z. and X.L., supervised by H.Y., H.W. and J.H.; initial drafting of manuscript: M.J. and H.Y. Review and editing of manuscript: H.Y; correction and approval of the manuscript: all authors.
Supplementary data
Figures S1 to S3 and Tables S1 to S3 are available as Supplementary data at JAC Online.
References
Author notes
Hongping Wei and Hang Yang contributed equally and are co-senior authors of this work.
