aac accepts, published online ahead of print on 4 november...

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A novel chimeric lysin with high antimicrobial activity against 1

methicillin-resistant Staphylococcus aureus in vitro and in vivo 2

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Hang Yang, Yun Zhang, Junping Yu, Yanling Huang, Xian-En Zhang, Hongping Wei* 4

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State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, 6

Wuhan 430071, China. 7

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For correspondence. *E-mail: [email protected]; Tel.: (+86) 27 51319676; Fax: (+86) 27 87199492. 9

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Running title: Elimination of MRSA by chimeric lysin 11

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Key words: Staphylococcus aureus, MRSA, lysin, chimeric lysin, chemotherapy 13

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AAC Accepts, published online ahead of print on 4 November 2013Antimicrob. Agents Chemother. doi:10.1128/AAC.01793-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 23

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The treatment of infections caused by methicillin-resistant Staphylococcus aureus 25

(MRSA) is a challenge worldwide. In our search for novel antimicrobial agents 26

against MRSA, we constructed a chimeric lysin (named as ClyH) by fusing the 27

catalytic domain of Ply187 (Pc) with the non-SH3b-like cell wall binding domain of 28

phiNM3 lysin. Herein, the antimicrobial activity of ClyH against MRSA strains in 29

vitro and in vivo was studied. Results showed that ClyH could kill all the tested 30

clinical isolates of MRSA with higher efficacy than lysostaphin as well as its parental 31

enzyme. The MICs of ClyH against clinical S. aureus strains were found as low as 32

0.05 to 1.61 mg/L. In a mouse model, a single intraperitoneal administration of ClyH 33

protected mice from death caused by MRSA, without obvious harmful effects. The 34

present data suggest that ClyH has potential to be an alternative therapeutic agent for 35

the treatment of infections caused by MRSA. 36

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INTRODUCTION 45

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Staphylococcus aureus is a common pathogen with ability to develop resistance to 47

virtually all classes of antibiotics (1-3). The infections caused by S. aureus, especially, 48

methicillin-resistant S. aureus (MRSA) (4, 5), are becoming a serious problem 49

worldwide, therefore, there is an urgent need to develop effective therapeutic agents 50

against MRSA (6). 51

Among many new antimicrobial agents against S. aureus, bacteriophage lysins have 52

been found promising because of their narrow spectra of activity, rapid antibacterial 53

activity and a low probability for developing resistance (7-11). Currently, a few lysins 54

identified directly from genomes of bacteriophages have been studied for controlling 55

infections caused by MRSA both in vitro and in vivo (10, 12, 13). However, producing 56

a perfect lysin directly from phage genomes remains difficult, because of the poor 57

solubility of the natural lysins when over-expressed in Escherichia coli (14). 58

To circumvent these problems, chimeric lysins have been introduced by shuffling the 59

domains, i.e., the cell wall binding domains (CBDs) and the catalytic domains (CDs) 60

from natural lysins (15-19). Many chimeric lysins have chosen a CBD homologous to 61

SH3b-like domains, similar to that of lysostaphin (Table S1). However, it has been 62

reported that the staphylococcal SH3b domains were not always 63

staphylococcal-specific (20). More importantly, the bacteria might have a chance, 64

although low, to develop potential resistance to the lysins containing SH3b-like 65

domains due to small alternations within the peptide cross-bridges of the bacterial cell 66


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wall as did to lysostaphin (21). While, a few CDs, mainly cysteine and 67

histidine-dependent aminopeptidase/hydrolase (CHAP) (17) and endopeptidase (19, 68

22), have been used as the CDs of chimeric lysins. Among all the CDs, we noted that 69

the CD from lysin Ply187 (Pc, consists of its N-terminal 157 amino acids) was special. 70

It has been reported that the Pc has a much higher amidase activity than the whole 71

lysin (23), and its activity could be further enhanced by adding a known SH3b CBD 72

(24). 73

In the present work, as an effort to find novel chimeric lysins for controlling MRSA, 74

Pc was fused with a CBD not homologous to SH3b domains, to generate a novel 75

chimeric lysin, named as ClyH. Various tests, including its lytic activity against 76

clinical MRSA isolates in vitro and in vivo, were done to show the antimicrobial 77

efficacy of ClyH. These results supported the potential of ClyH as a novel therapeutic 78

agent for treating multidrug-resistant S. aureus caused infections. 79

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MATERIALS AND METHODS 81

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Bacterial strains 83

Bacterial strains (Table S2) used in this work were routinely grown at 37°C. All the 84

staphylococci strains were grown in trypticase soy broth (TSB) medium. Clinical 85

isolates of S. aureus with different genetic background were collected from various 86

sources in China in order to cover all SCCmec types. MRSA strains were determined 87

by PCR against mecA and femB as described (25), with primers MecA-F 88


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(5’-GTAGAAATGACTGAACGTCCGATAA-3’), MecA-R 89

(5’-CCAATTCCACATTGTTTCGGTCTAA-3’), and FemB-F 90

(5’-TTACAGAGTTAACTGTTACC-3’), FemB-R 91

(5’-ATACAAATCCAGCACGCTCT-3’), respectively. Once confirmed, their SCCmec 92

types were further determined by multiple-PCR as described (26). The lukF/lukS PV 93

was determined by PCR according to the method described (27). 94

Because some lysins (28) were reported to be active against both S. aureus and 95

streptococcal strains, S. thermophilus, S. sobrinus, S. pyogenes and S. suis were tested 96

to evaluate the specificity of ClyH. Other strains used include Lactobacillus 97

acidophilus, Bifidobacterium dentium, Enterococcus faecalis, Enterococcus faecium 98

Enterobacter sakazakii, Salmonella enteritidis, Listeria monocytogenes, Pseudomonas 99

aeruginosa and Xanthomonas oryzae. All these strains were cultured in brain heart 100

infusion (BHI) medium. Bacillus cereus was tested as well but cultivated in 101

Luria-Bertani (LB) medium. Escherichia coli BL21(DE3) was used for cloning and 102

expressing of recombinant proteins. 103

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Construction of expressing plasmids 105

The chimeric lysin ClyH was constructed by fusing the N-terminal 157 amino acids of 106

Ply187 (Pc) with the C-terminal 97 amino acids of phiNM3 lysin. To do this, the DNA 107

fragment encoding the chimeric lysin was chemically synthesized by Songon Biotech 108

(Shanghai, China). The resulted gene corresponding to ClyH was cloned into 109

pBAD24 vector with primers ClyH-F 110


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(5’-AAAAGAATTCATGGCACTGCCTAAAACGGGTAAAC-3’) and ClyH-R 111

(5’-AAACTCGAGTTAAAACACTTCTTTCACAATC-3’) for expressing untagged 112

ClyH, and into pET28a(+) vector with primers pH-F 113

(5’-TTAACCATGGGCATGGCACTGCCTAAAACG-3’) and pH-R 114

(5’-TTAACTCGAGAAACACTTCTTTCACAATCAATC-3’) for expressing 115

his-tagged ClyH (ClyH-his), respectively. To express the his-tagged parental CD 116

(Pc-his), the Pc corresponding gene fragment was cloned into pET28a(+) vector with 117

primers PC-F (5’-AATTCCATGGGCATGGCACTGCCTAAAACG-3’) and PC-R 118

(5’-TTAACTCGAGTGGTGGTGTAGGTTTCGGTTC-3’). After confirmation by 119

sequencing, the correct plasmids were transformed into E. coli BL21(DE3) for 120

expression. 121

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Purification of recombinant proteins 123

The recombinant proteins were expressed by the E. coli BL21(DE3) strain in standard 124

LB medium and purified following procedures described previously (24, 29), with 125

minor modifications. ClyH was induced overnight in BL21(DE3) cells with 126

L-arabinose in a final concentration of 0.2% at 20°C. Briefly, cells were harvested by 127

centrifugation and resuspended in 20 mM phosphate buffer (pH 7.4). After sonication, 128

the supernatant was collected by centrifugation at 10,000 g for 30 min at 4°C. The 129

supernatant was applied to a HiTrap Q Sepharose FF column (GE Healthcare), then 130

bound to a HiTrap SP Sepharose FF column (GE Healthcare) and eluted in a linear 131

gradient from 0.02 M to 1 M NaCl solution. For the purification of ClyH-his as well 132


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as Pc-his, protein was expressed by inducing the bacteria with 1 mM isopropyl 133

ȕ-D-thiogalactoside (IPTG) when an optical density of 0.6-0.8 was reached. After 134

induction, the bacteria were incubated overnight at 16°C to allow expression. 135

Purification was achieved through His6 tag using a nickel nitrilotriacetic acid column, 136

washing and eluting with 60 and 265 mM imidazole solutions, respectively. Active 137

fractions were pooled and dialyzed against 1×PBS (137 mM NaCl, 2.7 mM KCl, 4.3 138

mM Na2HPO4·H2O, 1.4 mM KH2PO4, pH 7.4). After quantitation by the Bradford 139

assay, the purified proteins were stored at -80°C until use. 140

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Quantification of ClyH activity 142

Lytic activity was measured as previously described (8), with some modifications. 143

Briefly, S. aureus strain CCTCC AB91118 (also called AB918 for short) was grown to 144

an optical density of 0.2 to 0.3 at OD600, centrifuged, and resuspended in PBS (pH 7.4) 145

to a final OD600 of 1.0. Then, 100 ȝl of the purified ClyH in twofold serial dilutions 146

were mixed with 100 ȝl of the bacterial suspension in 96-well plates (Perkin-Elmer, 147

USA), respectively. The drop of OD600 was monitored by a microplate reader 148

(Synergy H1, BioTek, USA) for 60 min at 37°C. A unit of ClyH activity was defined 149

as the highest dilution that decreased the absorbance by 50% within 15 min (8). The 150

lytic activities of ClyH at different pH values were also measured using a universal 151

buffer described before (30). The buffer was prepared by mixing equal parts of 20 152

mM boric acid and 20 mM phosphoric acid, followed by titration with sodium 153

hydroxide from pH 2 to 12. 154


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After mixing 1 U of ClyH with the S. aureus strain AB918 suspension, the decrease in 155

viable cells corresponding to the loss of turbidity was also tested by plating the 156

aliquots from the lytic assay at various time points (5, 15, 30 and 60 min) to TSB agar 157

for counting CFU. The action of ClyH on the cell wall was monitored by thin-section 158

transmission electron microscopy (Tecnai G2 20 TWIN, FEI, USA). The bacterial 159

suspensions were incubated with 1 U of ClyH at 37°C for 3, 5 and 10 min, 160

respectively. Then the reaction was terminated by adding 2.5% glutaraldehyde before 161

the TEM analysis. 162

To compare the activity of ClyH with that of lysostaphin and Pc-his, mid-log-phase 163

cultures of randomly selected S. aureus strains were pelleted and resuspended in PBS 164

to a final OD600 of 1.0, respectively. One hundred microliters of ClyH or lysostaphin 165

under the same concentration (0.16 ȝM) was added into the bacterial suspension (100 166

ȝl), respectively. The decrease in OD600 was monitored by the spectrophotometer. To 167

minimize the effect of his-tag on the enzymatic activity, ClyH-his (1.2 ȝM) was used 168

to compare with Pc-his under the same concentration. 169

To determine the specificity of ClyH, the lytic activities of ClyH to various bacterial 170

strains were measured as the drop in milli-OD600 per minute (-mOD600/min) in the 171

first 15 min as described elsewhere (31). 172

All the above experiments were performed in triplicate and bacteria cells treated with 173

PBS were used as the blank controls. 174

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MIC determinations 176


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MICs of antibiotics (penicillin, gentamicin, vancomycin and oxacillin) and ClyH were 177

determined by microtiter broth dilution as described by the Clinical and Laboratory 178

Standards Institute (CLSI) (32). MIC was defined as the lowest concentration of 179

antibiotic producing inhibition of visible growth. 180

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Immunological neutralization test 182

The neutralization effect of ClyH-specific antibodies to the activity of ClyH was 183

tested using a standard immunological protocol as described (10). ClyH (200 U) 184

solutions were injected into the peritoneal cavities of mice for 3 times at a 10-day 185

interval. Mice sera were sampled 15 days after the last injection and the serum titers 186

were checked by ELISA using horseradish peroxidase-conjugated goat anti-mouse 187

IgG. The detailed procedure of ELISA was performed following the instructions of the 188

manufacturer of a commercial ELISA kit (QF-Bio, Shanghai, China). Before the 189

neutralization test, ClyH (about 0.5 U) was reacted with 80 µl of the ClyH immunized 190

mouse serum at 37°C for 15 min, using non-immunized mouse serum and PBS as the 191

controls. Then, the neutralization effect was performed immediately by testing the 192

lytic activity of each mixture to S. aureus strain AM025 using the same procedure as 193

the lytic activity assay described above. 194

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Mouse protection experiments 196

All mouse experiments were conducted with the approval of the Animal Experiments 197

Committee of Wuhan Institute of Virology, Chinese Academy of Sciences 198


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(WIVA17201203). Female BALB/c mice (6-8 weeks old) were injected 199

intraperitoneally with different concentrations of MRSA strain AM025 to determine 200

the minimal lethal dose (MLD) that caused 100% mortality within two days. In the 201

mouse protection assay, mice were inoculated intraperitoneally with 2×MLD of 202

AM025 cells, and then divided into 3 groups randomly. Three hours after challenging, 203

two groups (6 each) received 180 U and 360 U (900 µg) of ClyH intraperitoneally, 204

respectively, and the other group (n=8) was injected with PBS buffer. Another group 205

(n=6) without MRSA infection received 540 U of ClyH only. The survival rates of all 206

the groups were observed for 10 days after the infection. To check the toxicity of 207

ClyH, 5 mice without injecting the bacteria were given the ClyH solution for seven 208

days (200 U/injection, one injection/day, total dose of 1400 U), and the survival rate, 209

their body weights and activities were observed for 10 days after the last injection. 210

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RESULTS 212

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Characteristics of ClyH 214

The purified ClyH, ClyH-his as well as Pc-his displayed high purities (>90%) in 12% 215

SDS-PAGE gels (Fig. 1A and 1B). As shown in Fig. 1C, after addition of ClyH, the 216

OD600 of S. aureus AB918 suspension decreased rapidly with reaction time, while the 217

OD600 of the S. aureus suspension without ClyH had small changes. Fig. 1C also 218

showed that the loss of turbidity correlated with the decrease in viable cells. 219

The influence of pH, temperature and ionic strength on the activity of ClyH was also 220


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studied. As shown in Fig. 1D, ClyH remained high activity against AB918 cells in a 221

broad pH range from pH 5 to 10, and reached its maximum activity at pH 6. The 222

temperature had a significant effect on the lytic activity of ClyH. High lytic activities 223

were observed at temperatures between 35°C and 45°C (Figure S1A). The ionic 224

strength ranging from 137 mM to 500 mM NaCl had no significant effect on ClyH 225

activity (Figure S1B). 226

We also tested the stability of ClyH at 4°C (Figure S2), and found that ClyH retained 227

63.7% and 21.2% lytic activity in terms of the initial lytic activity after storing for 4 228

and 8 weeks, respectively (Figure S2B). 229

The TEM analysis showed that AB918 cells exposed to ClyH suffered a process from 230

deformation to extrusion and then disruption in cell wall at single or multiple sites, 231

which was quite consistent with the typical phenomenon of lysin-mediated cell lysis. 232

The weakening and rupture of the cell wall resulted in the loss of cytoplasmic 233

contents partly or totally (Fig. 1E and 1F), and formation of cell ghost (Fig. 1G). 234

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The specificity of ClyH 236

As shown in Fig. 2, ClyH had an effective lytic activity against staphylococci strains, 237

including methicillin-sensitive S. aureus (MSSA) and MRSA strains tested (Table S2), 238

but not other species tested except S. sobrinus. This observation was quite consistent 239

with an early report indicating that the CBD of phiNM3 was highly specific to 240

staphylococci (19). Moreover, the lytic velocities were quite fast for all the clinical 241

isolated MRSA strains, regardless of their SCCmec types. 242


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Comparison of lytic activity of ClyH with that of other antimicrobials 244

To compare the activity of ClyH with that of other antimicrobial agents against S. 245

aureus, the antimicrobial activity of ClyH were tested together with lysostaphin, 246

Pc-his and several antibiotics. As shown in Fig. 3A, ClyH displayed a higher activity 247

than lysostaphin. Furthermore, ClyH could even lyse two strains (AM016 and AM045) 248

that lysostaphin could not lyse. We also observed an obvious strain-to-strain variation 249

of ClyH activity, similar to those observed for other lysins (19, 28, 31, 33). It has been 250

reported that the cell wall thickening is associated with adaptive resistance to 251

antibiotics in MRSA clinical isolates (34), which may contribute to the observed 252

variability of ClyH activity. To minimize the effect of his-tag on the enzymatic 253

activity, we expressed a his-tagged ClyH (ClyH-his) (Fig. 1B) and compared its lytic 254

activity with that of Pc-his. Results displayed that the lytic activity of ClyH-his was 255

quite close to that of ClyH (Figure S3), and higher than that of Pc-his, improved 3.7 to 256

13.6 folds for the strains tested (Fig. 3B). 257

MIC tests (Table 1) showed that all the isolates tested were highly resistant to 258

penicillin with minimum inhibition concentration (MIC) values higher than 319.4 259

mg/L, except for strain AM058. MRSA strains displayed a relatively higher resistance 260

to gentamicin than MSSA strains. However, all the strains were highly sensitive to 261

vancomycin and ClyH, with MIC values ranging from 0.53 to 1.99 mg/L, and 0.05 to 262

1.61 mg/L, respectively. 263

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Elimination of MRSA by ClyH in a mouse model 265

The in vivo protective efficacy of ClyH was tested in a mouse model. As shown in Fig. 266

4A, administration of 180 U of ClyH at 3 h after challenging 4×109 CFU 267

AM025/mouse protected 66.7% mice against lethality in the 10-day course of 268

experiments. The protective efficacy was improved to 100% when the dose of ClyH 269

increased to 360 U. While in the group receiving no injection of ClyH, all mice were 270

dead within 24 h after the challenge. Further tests showed that a single administration 271

of higher doses (540 U) and the repeated administration (total dose of 1400 U in 7 272

days) of ClyH alone neither influenced the survival rate nor produced adverse effects 273

to the mice in terms of body weight and activity. However, as shown in Fig. 4B, the 274

ELISA experiments demonstrated that repeated injection of ClyH (200 U) could 275

induce immune response in the mice (the antibody titers were over 4×105). 276

Fortunately, the immunized serum showed no obvious neutralization effect on the 277

lytic activity of ClyH (Fig. 4C). 278

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DISCUSSIONS 280

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The modular structure of lysin makes it possible to swap different catalytic domains 282

and binding domains to create novel chimeric lysins, which may not only retain the 283

binding specificity and/or lytic activity of the original lysins (35, 36), but also have 284

better antimicrobial properties. As shown in Table S1, besides ClyH, several other 285

chimeric lysins have been reported previously against S. aureus (17, 19, 22, 24). The 286


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difference of ClyH to other chimeric lysins is its unique fusion of the CD of Ply187 287

lysin with the CBD of phage phiNM3 lysin. Upon exposure of S. aureus AB918 cells 288

to ClyH, the rapid loss of turbidity and the cell wall damage (Fig. 1) indicated that 289

ClyH was highly active against S. aureus. The specific activity of ClyH was about 400 290

U/mg, which is about 2-fold higher than that of its closely related chimer ClyS (19). 291

Furthermore, unlike most lysins which are usually active only in pH range from 5 to 8 292

(28, 37), ClyH retained a high lytic activity (above 30% of the maximum) under pH 5 293

to 10. Besides pH, ionic strength also had a minor effect on the activity of ClyH. 294

These properties make ClyH suitable to work under some environmental conditions 295

that render other lysins inactive. 296

In vitro tests showed that ClyH was a highly potent agent to kill S. aureus. Its 297

capability to lyse all the tested clinical MRSA isolates (Fig. 2), regardless of their 298

SCCmec types, indicated that ClyH might be used to control all kinds of MRSA in 299

vitro. The greatly improved lytic activity of ClyH (ranging from 3.7 to 13.6 folds) 300

than that of Pc-his indicated that the non-SH3b CBD could add activity to the whole 301

lysin, which is similar to that found in the chimer Ply187AN-KSH3b (where a 10-fold 302

higher lytic activity than Pc-his was found after adding a SH3b CBD) (24). Since Pc 303

has been reported having a higher lytic activity than the whole lysin Ply187 (23), it is 304

easy to conclude that ClyH has a significantly improved lytic activity compared to 305

Ply187. Moreover, ClyH displayed not only higher lytic activity than lysostaphin, but 306

also a broader lytic spectrum to the two clinical MRSA isolates (AM016 and AM045), 307

which were resistant to lysostaphin (Fig. 3A). This may be due to the non-SH3b 308


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binding domain of ClyH, which is much more difficult to evoke resistance than the 309

SH3b domain of lysostaphin (21). 310

The low MIC values of ClyH suggested that ClyH has the potential to be used as an 311

antimicrobial agent for the treatment of infections caused by MRSA in vivo. Our 312

initial study demonstrated that a single intraperitoneal administration of a low dose of 313

ClyH could greatly improve the survival rate of mice infected by a lethal dose of 314

MRSA (Fig. 4). Importantly, an accumulated excessive dose of ClyH (up to 1400 U) 315

showed no adverse effects on the body weights and the activities of the mice, which 316

indicated that ClyH did not have obvious toxicity. As a protein, ClyH could induce a 317

humoral immune response, which might block its usage for treating repeated 318

infections. Fortunately, our neutralization test showed that although repeated 319

administration of ClyH did evoke obvious immune response, the antibodies induced 320

did not influence the activity of ClyH. All these results suggested that ClyH might be 321

systematically administrated with safety to combat the increasing infections caused by 322

multidrug-resistant S. aureus. 323

In conclusion, the novel chimeric lysin ClyH showed good antimicrobial activities 324

against all clinical MRSA isolates tested and some improved properties over other 325

lysins. Although more tests are needed, the present data strongly supported that ClyH 326

offers a great potential to be used as a novel agent for the treatment of infections 327

caused by MRSA. 328

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ACKNOWLEDGEMENTS 330


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331

This work was supported by the Basic Research Program of the Ministry of Science 332

and Technology of China (2012CB721102 to HP Wei and JP Yu), the National Natural 333

Science Foundation of China (21075131), the Post-Graduate Scientific and 334

Technological Innovation Project of Chinese Academy of Sciences (Y204081YZ1) 335

and the Key Laboratory on Emerging Infectious Diseases and Biosafety in Wuhan. 336

We thank Prof. Xiancai Rao from Third Military Medical University for providing the 337

S. aureus strains and Dr. Yingle Liu from Wuhan University for his kind help in 338

collecting the S. aureus strains from hospitals in Wuhan. 339

340

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467

TABLES AND FIGURE LEGENDS 468

469

Table 1. The MIC values of S. aureus isolates. 470

Strain

Isolator

Penicillin

mg/L

Gentamicin

mg/L

Oxacillin

mg/L

Vancomycin

mg/L

ClyH

mg/L

MSSA

AB918 >319.42 160.00 1.00 0.53 0.19

AM058 >159.71 80.00 0.50 1.99 0.09

AM061 >319.42 20.00 0.50 1.99 0.09

AM062 >319.42 10.00 0.50 1.99 0.19

AM065 >319.42 20.00 0.50 1.99 0.09

AM066 >319.42 10.00 0.50 1.99 0.09

AM067 >319.42 20.00 0.50 1.99 0.19

AM068 >319.42 5.00 1.00 1.99 0.09

MRSA

AM054 >319.42 160.00 80.01 1.99 0.09

AM044 >319.42 >319.98 >320.31 1.99 0.38


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AM052 >319.42 80.00 >320.31 1.00 0.20

AM022 >319.42 80.00 >320.31 1.99 0.20

AM032 >319.42 319.98 >320.31 1.99 1.61

AM031 >319.42 40.00 >320.31 1.00 1.61

AM041 >319.42 160.00 320.31 1.99 0.05

AM012 >319.42 40.00 >320.31 1.00 0.20

AM036 >319.42 40.00 >320.31 1.99 1.61

AM001 >319.42 319.98 4.01 1.00 0.20

AM016 >319.42 40.00 >320.31 1.00 0.05

AM008 >319.42 10.00 8.01 1.00 0.82

471

FIG 1 Characteristics of ClyH activity. (A) SDS-PAGE of Purified ClyH and Pc-his. (B) 472

SDS-PAGE of Purified ClyH-his and Pc-his. M: protein molecular weight markers. ClyH-his: 473

his-tagged ClyH. Pc-his: the catalytic domain of lysin Ply187 fused with a his-tag. (C) The lytic 474

activity against S. aureus AB918 in vitro. The decrease in OD600 was monitored after addition of 475

ClyH (solid square) with PBS as a control (open circle). Viability of treated cells measured as 476

LogCFU/ml was determined by serially diluting and plating to TSB agar plates (star). (D) The 477

relative activities of ClyH against AB918 cells in buffers at different pHs. (E)-(G) The TEM 478

images of AB918 cells exposed to ClyH. ClyH causes cell wall deformation (E), extrusion and 479

loss of cytoplasmic contents partly or totally (F) and ultimately formation of cell ghost (G). Bar 480

sizes: 200 nm. 481

482


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FIG 2 Lytic activity of ClyH (0.5 U) against different strains in vitro. The activity of lysis is 483

defined as the initial velocity of the decrease in OD600 over time. Error bars show the standard 484

error of three independent assays. 485

486

FIG 3 Comparison of the activity of ClyH/ClyH-his with that of lysostaphin and Pc-his, 487

respectively. (A) The lytic activity of ClyH (0.16 ȝM) in comparison with that of lysostaphin 488

under the same concentration. (B) The lytic activity of ClyH-his (1.2 ȝM) in comparison with 489

that of Pc-his under the same concentration. Error bars represent three independent assays. 490

491

FIG 4 The protective effect of ClyH on mice from death caused by MRSA. (A) Curative effects in 492

a mouse model of systemic MRSA infection. Three hours after infection, one group of mice was 493

given 180 U of ClyH, the second group was given 360 U of ClyH, and the third group was given 494

PBS buffer. Meanwhile, another group of mice without MRSA infection were given 540 U of 495

ClyH to test its toxicity. (B) The titers of anti-ClyH antibody induced by repeated injection of 496

ClyH. The control serum is non-immunized mouse serum. (C) Effect of ClyH-immunized serum 497

on the lytic activity of ClyH against AM025. 498

499

SUPPLEMENTAL MATERIAL 500

501

Additional supporting information may be found in the online version of this article. 502


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