Yeast Surface Display of Antheraea pernyi Lysozyme Revealed Î±

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Functional Structure/Activity Relationships

Yeast Surface Display of Antheraea pernyi Lysozyme Revealed #-helical Antibacterial Peptides in Its N-terminal Domain Sai Wen, Tongxin Mao, Dongmei Yao, Tian Li, and Fenghuan Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02489 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 3, 2018

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Journal of Agricultural and Food Chemistry

Yeast Surface Display of Antheraea pernyi Lysozyme Revealed α-helical Antibacterial Peptides in Its N-terminal Domain Sai Wen, Tong-xin Mao, Dong-mei Yao, Tian Li and Feng-huan Wang* Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients, School of Food and Chemical Engineering, Beijing Technology and Business University, Beijing, 100048, China

Corresponding Authors: Prof. Feng-huan Wang, e-mail address: [email protected]

ACS Paragon Plus Environment


1

ABSTRACT:

2

The present study investigated a novel lysozyme ApLyz from the Chinese oak

3

silkmoth, Antheraea pernyi, for its active expression with N- or C-terminus fused to

4

the yeast cell surface, and the antimicrobial activities of the corresponding expressed

5

lysozymes were evaluated. The bactericidal activity of C-terminal fusion of ApLyz

6

surpassed that of the N-terminal fusion, which revealed the implication of N-terminal

7

stretch of ApLyz in the bactericidal fuction based on the structural mobility of this

8

region. Two N-terminal peptides of ApLyz (residues 1-15 and 1-32), which primarily

9

consist of amphiphilic α-helices, exerted similar bactericidal efficacy and had a strong

10

preference for the Gram-negative strains. Further investigation revealed that the

11

N-terminal peptides are membrane-targeting peptides causing cell permeabilization

12

and also possess non-membrane disturbing bactericidal mechanism. Overall, in

13

addition to the key findings of novel bactericidal peptides from silkmoth lysozyme,

14

this work laid the foundation for future improvement of ApLyz by protein

15

engineering.

16 17

KEYWORDS:

18

silkmoth lysozyme, yeast surface display, antimicrobial peptide, membrane

19

permeabilization

20 21 22


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INTRODUCTION

24

Lysozyme (EC.3.2.1.17), coined by Alexander Fleming with the meaning of “an

25

enzyme that actually lyses bacteria”, is normally defined as a 1,4-β-d-N-acetyl

26

muramidase, given its ability to hydrolyze the glycosidic bond between the first

27

carbon of N-acetyl muramic acid and the fourth carbon of N-acetyl glucosamine in

28

peptidoglycans of the cell wall. Recent evidences have also revealed that lysozymes

29

possess some intriguing modes of action, such as perturbation of DNA or RNA

30

synthesis1, self-promoted uptake2, activation of bacterial autolysins3, participation in

31

digestion4, display of β-1,4-N,6-O-diacetyl muramidase activity5 or chitinase activity6.

32

As possibly the best-studied group of bacterial lytic hydrolases7-8, lysozyme has been

33

extensively investigated for food and pharmaceutical applications9-10, including food

34

additives in cheese making11, substitution for sulphite used in wines production12-13,

35

antifouling coatings for food package or implants14-15, preservatives in eye drops16,

36

active ingredient in toothpaste17-18 and treatment for enterotoxigenic E.coli-induced

37

diarrhea in young pigs19, etc.

38

While some lysozymes can be easily produced from egg-white or milk, the

39

construction of a recombinant over-expression system is essential, especially when

40

the lysozyme of interest is rare, or molecular engineering for structural or functional

41

investigations are required. When lysozyme is expressed in Escherichia coli, the

42

target protein remains in cells and is yielded as insoluble inclusion body with an extra

43

methionine residue at N-terminus, which reduces conformational stability and makes

44

the renaturation procedure indispensable20-21. On the other hand, yeast expression



45

system, by taking advantages of soluble secretion, correct folding and comprehensive

46

post-transcriptional modifications of heterologous eukaryotic proteins, has been

47

proved to be a powerful tool for active expression of lysozymes from various sources,

48

such as egg white22-23, human24-25, silkmoth26 and marine invertebrates27. The yeast

49

surface display system is also an emerging technique for protein engineering and

50

screening, which can auto-immobilize proteins on the exterior of yeast cells. This

51

mechanism can endow the recombinant yeast with novel functions, taking lysozyme

52

expression for example, such as whole-cell biocatalyst for antifouling coating, a dual

53

purpose delivery vehicle of lysozyme and yeast for feed, and the screening platform

54

for novel lysozymes.

55

A drawback of natural lysozyme as antimicrobial agent is that the bactericidal activity

56

of lysozyme is directed against Gram-positive bacteria, and to a much less degree

57

against Gram-negative bacteria including foodborne pathogens. Over the past decades,

58

several researches have illustrated that, in addition to N-acetylmuramidase activity,

59

lysozymes can kill bacteria by non-catalytic action. It has been reported that heat

60

denaturation of lysozyme from chicken egg white enhanced bactericidal activity

61

towards Gram-negative bacteria of an enzymatically inactive, more cationic and

62

hydrophobic dimeric form28-30. Further studies on either genetically mutated lysozyme

63

devoid of muramidase activity or partially unfolded lysozyme suggested that the

64

antimicrobial action of lysozyme is due to structural factors rather than enzymatic

65

mechanism31-32. This observation, together with the results of proteolytic digestions

66

and three-dimensional (3D) structural analysis, suggested that lysozyme contains


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cationic and amphiphilic helix or helix hairpin motif that can promote self-uptake,

68

insert into and form pores in negatively charged bacterial membranes33-36. The

69

identified antimicrobial peptides are mostly located in the terminal region of

70

lysozymes in favor of membrane penetration. So far, the nonenzymatic bactericidal

71

properties were found in c-type lysozymes, e.g., chicken-, human- and T4 phage

72

lysozymes37, and in g-type lysozyme from goose egg white38 as well as i-type

73

lysozyems like destabilase-lysozyme (DL)39. This distinct non-enzymatic antibacterial

74

feature of lysozyme is directly related to the killing of Gram-negative bacteria, and

75

thus herald attractive opportunities for developing novel broad-spectrum lysozymes or

76

derived peptides for food or therapeutic applications.

77

Insect lysozymes play a pivotal role in humoral immune response of insects to protect

78

them from a wide range of infectious microbes, and are therefore being increasingly

79

studied. In this study, a novel lepidopteran lysozyme from silkmoth Antheraea pernyi

80

was investigated for active expression and display with its N- or C-terminus tethered

81

to the yeast cell surface. This result of surface displayed A. pernyi lysozyme (ApLyz)

82

to exert antimicrobial potency has guiding significance for application of ApLyz in

83

immobilized form. The study also revealed that the N-terminal domain of ApLyz,

84

which primarily consisted of amphiphilic α-helical peptides, was implicated in the

85

bactericidal property of this lysozyme. In order to explore the structural requirements

86

and peptide-based bactericidal action of ApLyz, two N-terminal stretches of ApLyz

87

were

88

microorganisms, as well as membrane disturbing activity. The potential bactericidal

synthesized

and

tested

for

antimicrobial


efficacy

against

different


89

mechanisms of ApLyz-derived peptides, their functional significance, and feasibility

90

for exploring novel antimicrobial peptides are discussed.

91 92

MATERIALS AND METHODS

93

Construction of recombinant plasmids expressing A. pernyi lysozyme with N- or

94

C-terminus fused to the surface anchor protein

95

The gene encoding mature peptide of A. pernyi lysozyme (aplyz, GenBank:

96

DQ353869.1) was initially identified by cDNA cloning from the hemolymph of the

97

silkworm, A. pernyi, upon Escherichia coli (E. coli) infection of the larvae. The aplyz

98

gene, which is 360 bp in length, and a novel, synthetic gene Raplyz encoding reversed

99

amino acid sequence of ApLyz were both artificially synthesized (Sangon Biotech Co.,

100

Shanghai, China). With regard to yeast surface display of N-terminal fused ApLyz,

101

aplyz gene was amplified by PCR and purified using an E.Z.N.A. Cycle Pure Kit

102

(Omega Bio-Tek, Inc., USA). The insert was subcloned into the yeast surface display

103

vector pYD1(Invitrogen, USA) between the EcoRI (NEB Biolabs, Schwalbach,

104

Germany) and XhoI (NEB) restriction sites to generate vecter pYD1-aplyz. Similarly,

105

as for the display of C-terminal fused ApLyz, Raplyz gene was subcloned into pYD1

106

between EcoRI and XhoI sites to generate vector pYD1-Raplyz. All PCR reactions

107

were performed in 100 µL reactions with high-fidelity DNA polymerase (NEB),

108

following the manufacturer’s protocol; an annealing temperature of 53℃ and

109

extension time of 30 s were used.

110

Yeast surface display of ApLyz with tethered N- or C-terminus


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The recombinant plasmids pYD1-aplyz and pYD1-Raplyz were respectively

112

transformed into S. cerevisiae EBY100 (Invitrogen, USA) following the instruction of

113

the pYD1 Yeast Display Vector Kit (Invitrogen, USA). The Minimal Dextrose

114

tryptophan-free Agar Plates comprising 6.7 g/L YNB without amino acids (BD Difco),

115

20 g/L glucose, 0.1 g/L leucine and 15 g/L agar were used to screen for positive

116

transformants. To induce cell surface protein expression, the positive clones harboring

117

pYD1-aplyz (designated as EBY100/pYD1-aplyz) or pYD1-Raplyz (designated as

118

EBY100/pYD1-Raplyz) were cultured in 10 mL YNB-CAA medium (6.7 g/L YNB, 5

119

g/L casamino acids) containing 20 g/L glucose at 30℃ until OD600 reached between 2

120

and 5. The yeast cells were harvested by centrifugation and resuspended in

121

YNB-CAA medium containing 20 g/L galactose to an OD600 between 0.5 and 1, then

122

cultivated at 20℃ with shaking for 48 hours for optimal expression of lysozyme.

123

Visualization of yeast surface displayed lysozyme and antibacterial activity assay

124

The yeast surface display of lysozyme was visualized through immunostaining using

125

fluorescent antibody and microscopy. The transformants of EBY100/pYD1-aplyz and

126

EBY100/pYD1-Raplyz were respectively harvested by centrifugation, washed with

127

1×sodium phosphate buffe (PBS), and then resuspend in 250 µl of 1×PBS, 1 mg/mL

128

BSA, and 1 µg Anti-Xpress antibody (Invitrogen, USA). The mixtures were chilled on

129

ice for 30 min and washed twice with 1×PBS. After being resuspended in 250 µl of

130

1×PBS, 1 mg/mL BSA and 1µg FITC-labeled Goat anti-mouse IgG (Beyotime

131

Biotech. Co., China), followed by incubation on ice for 30 min in dark with

132

occasional mixing, cells were washed and resuspended again with 1×PBS (W/O BSA),



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133

and visualized using a fluorescence microscope (Imager A1, Zeiss, Germany) with

134

FITC filter.

135

The activity of the ApLyz displayed on the yeast cell surface was assessed against

136

Microcococcus lysodeikticus by cylinder-plate method. The solid medium containing

137

30 mL YPD medium and 1.5 % (w/w) agar was inoculated with 0.25 mL overnight

138

cell culture of M. lysodeikticus before pouring into the Petri dishes. In each plate,

139

three stainless steel cylinders of uniform size were placed on the surface of the solid

140

medium and filled with 200 µL aliquot of cell culture from EBY00/pYD1,

141

EBY00/pYD1-aplyz and EBY00/pYD1-Raplyz, respectively. The plates were

142

incubated at 25℃ aerobically for 24 hours. The growth inhibition zone diameters

143

(mm) were carefully measured with a digital caliper.

144

Generation of Three-Dimensional (3D) Structures and peptide sequence analysis

145

BLAST search in PDB database showed that the amino acid sequence of ApLyz had

146

significant sequence similarity with lysozyme from tasar silkworm, Antheraea mylitta

147

(SMTL ID: 1iiz.1, 84% identity). 3D structures of ApLyz were generated by

148

homology modeling using SWISS-MODEL online service (http://swissmodel. expasy.

149

org), and presented by UCSF Chimera40. Sequence aligment analysis was performed

150

by

151

(http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Computation of the theoretical pI

152

(isoelectric point) and Mw (molecular weight) of the synthetic peptides was

153

performed on an ExPASy server, using Swiss-Prot sequence entries.

154

Antibacterial assay by viable count plating

ClustalX

and

visualized

by

the


online

tool

Espript

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155

In brief, a single colony of bacteria was transferred into 50 mL of trypticase soy broth

156

(TSB) and incubated overnight at 37℃, diluted (1:50) in TBS and regrown at 37℃ to

157

logarithmic phase (1-4×108 cfu/mL). Bacteria were harvested, washed and

158

resuspended (106 cfu/mL) in 1×PBS. A 50 µL aliquot of the bacterial suspension was

159

mixed with 50 µL of water containing the test peptide at defined concentration, then

160

100 µL of 2% TSB in 1×PBS was added. The mixture was incubated at 37℃ for 1

161

hour, serially diluted in 1×PBS, and plated on TSB agar plates. Colony-forming units

162

were obtained after incubation of the plates at 37℃ for 24 hours. Assays were

163

performed in triplicate.

164

CD assay

165

The regular secondary structure content of two synthesized peptides were analyzed

166

using circular dichroism (CD). Ellipticity was scanned over 190~250 nm wavelength

167

on a Bio-Logic spectropolarimeter (Model MOS-500, France) where a concentration

168

of 0.5 mg/mL tested peptide in 0.1 cm path-length cuvette was used. The ellipticity

169

data

170

(http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d3/) to approximate the secondary

171

structure content.

172

Live/dead viability assay

173

25 mL culture of tested strain were cultivated to late log phase as for viable count

174

plating, centrifuged and resuspended in 2 mL of 0.85% NaCl. A 1 mL aliquot of this

175

suspension was added to a centrifuge tube containing 20 mL of 0.85% NaCl and

176

incubated at room temperature for 1 hour, mixing every 15 min. Cells were washed

was

fitted

using

the

web

interface


of

K2D3

program


177

again and resuspended in 10 mL of 0.85% NaCl before staining. A 1:1 mixture of

178

SYTO 9 and propidium iodide (component A and B of the LIVE/DEAD BacLight

179

bacterial viability kit L7012, Molecular Probes, USA) was diluted by adding 3 µL of

180

the dye mixture to each milliliter of cell suspension. The bacterial suspension was

181

divided into three parts of 100 µL each for the addition of synthetic peptide α15 or

182

α32 in 20 µL PBS (200 µg/mL), respectively, or 20 µL PBS only as negative control.

183

2 hours after addition of the peptides or PBS, bacteria were examined separately

184

under a fluorescence microscope with FITC and Rhod bandpass filter set.

185 186

RESULTS

187

Yeast Surface Display of A. pernyi Lysozyme in N- or C-terminal Fusion

188

Constructs

189

Yeast surface display is a novel, cost-effective technique that can exhibit proteins on

190

the surface of yeast cells through a linkage with the surface anchor protein Aga2p, a

191

subunit of yeast mating protein a-agglutinin receptor41. In the most commonly used

192

configurations, the fusion of target protein with Aga2p is constructed at either N- or

193

C-terminus of the target protein42. Whether one side is more favorable for the

194

biological activity of the protein or not has to be evaluated for each fusion construct.

195

With regard to yeast surface display of ApLyz, the gene aplyz (GenBank:

196

DQ353869.1) was cloned into the expression vecter pYD1 in frame with the upstream

197

leader sequence encoding protein Aga2p, a Gly-Ser linker, and the XpressTM epitope.

198

In this way, the N-terminus of expressed ApLyz was fused to the C-terminus of Aga2p,


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199

allowing secretion and display on the cell surface of Saccharomyces cerevisiae

200

EBY100. The configuration of vector pYD1-aplyz was confirmed by double digestion

201

and DNA sequencing. As for the expression of ApLyz with its C-terminus fused to

202

Aga2p, a new DNA sequence encoding the reversed amino acid sequence of ApLyz

203

was synthesized and designated as Raplyz. The corresponding expression vector

204

pYD1-Raplyz was constructed and confirmed analogously.

205

The expression and display of fused ApLyz was induced by culturing recombinant S.

206

cerevisiae EBY100 with 20 g/L galactose as inducer for 48 hours and detected by

207

immunofluorescent staining which used anti-Xpress antibody as primary antibody and

208

FITC-labeled goat anti-mouse IgG (H+L) as secondary antibody. As shown in Figure

209

1A, the fluorescence of negtive control, i.e., EBY100 transformant of empty plasmid

210

pYD1, was barely detectable above background, while intense green fluorescence

211

were both observed on the yeast cells harboring pYD1-aplyz or pYD1-Raplyz,

212

indicating that these two display systems were capable for secretion and surface

213

display of lysozyme. Additionally, fluorescence intensity also illustrated that the N-

214

and C-terminal fusion of ApLyz lysozymes were displayed on the cell surface at a

215

similar level.

216 217

Antibacterial Activity Assay of Yeast Surface Displayed Lysozyme

218

Further analysis of antibacterial activity against Microcococcus lysodeikticus by

219

cylinder-plate method was taken to ascertain the impact of N- and C-terminal fusion

220

modes on the efficacy of displayed lysozymes. A 200 µL aliquot of fermentation broth



221

was added into a cylinder and incubated at 20℃ for 24 hours. Formation of a growth

222

inhibition halo around the well readily indicated antimicrobial action of displayed

223

lysozymes. In contrast to fluorescence intensity, the resulting inhibition zones against

224

M. lysodeikticus showed that EBY100/pYD1-Raplyz exhibited higher antibacterial

225

activity than that of EBY100/pYD1-aplyz (Figure 1B), while EBY100/pYD1

226

presented no obvious activity (data not shown). This result revealed that

227

immobilization of the N-terminus of ApLyz had an adverse effect on the bactericidal

228

activity. Therefore, we speculated that the N-terminal domain of ApLyz was

229

implicated in the bactericidal function based on the structural mobility of this region.

230 231

Amphiphilic Peptides in the N-terminal Domain of ApLyz

232

To explore the N-terminal structure-activity relationship of ApLyz, the 3D structure of

233

ApLyz was generated through homology modeling with lysozyme from tasar

234

silkworm, Antheraea mylitta (SMTL ID: 1iiz.1, 100% coverage, 84% identity) as

235

template. As shown in Figure 2A, mature ApLyz contains four α-helices, designated

236

as H1 (residues Lys5-Gln15), H2 (residues Arg23-Glu32), H3 (residues Thr85-Arg97), and

237

H4 (residues Tyr104-Asn108), respectively, and two short double-stranded β-sheet

238

motifs. Catalytic residues of Glu32 and Asp50 are highly conserved in c-type

239

lysozymes and located between α-helices H2 and H3. Four pairs of disulfide bonds

240

are present between Cys6 and Cys120, Cys27 and Cys110, Cys62 and Cys76, and Cys72

241

and Cys90.

242

In the N-terminal domain of ApLyz, the remote H1 helix is rich in basic amino acids


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243

and exhibited +3 net positive charge at physiological pH. Corresponding helical wheel

244

projection and 3-D structure model showed that the hydrophobic residues were

245

mainly on one side of H1 helix, and polar residues were on the other side (Figure

246

2B-a).

247

(http://www.bioinformatics.nl/emboss-explorer/),

248

amphiphilic. Given that cationic and amphiphilic property is a common trait shared

249

amongst α-helical antimicrobial peptides (AMPs), we speculated that the stretch of

250

residues Lys1-Gln15 containing H1 helix might be similarly effective at conferring

251

antibacterial function.

252

Although the adjacent H2 helix was demonstrated to be an amphiphilic, but anionic

253

peptide (-2 net negative charge) (Figure 2B-b), we noted that H1 and H2 helices

254

together formed a helix-loop-helix motif (HLH, Lys1-Glu32) within the N-terminal

255

region of ApLyz. It is known that HLH structural motif is generally found in

256

DNA-binding proteins43 and membrane-active pore-forming proteins44-46. AMPs with

257

such a structure have been proved to be capable of forming channels through bacterial

258

membrane. For example, Cecropins B, the antimicrobial peptide present in the

259

silkmoth lymph after a bacterial infection, consists of two α-helices joined by a small

260

loop with membrane permeabilization action. The residues of N-terminal helix of

261

Cecropins B are cationic and hydrophobic, while the C-terminal helical residues are

262

primarily hydrophobic47-48. It is inferred that the N-terminal HLH motif of ApLyz

263

might also serve as a domain with antimicrobial function.

The

calculated

hydrophobic

moment

of

H1

helix

proving

this

peptide

264


is

0.48 to

be


265

Antimicrobial Activity Assay of Amphipathic Peptides

266

To test the structural requirements and hypothesized antimicrobial action of

267

N-terminal stretch of ApLyz, peptide α15 (residues 1-15, KWFTKCGLVHELRRQ)

268

and α32 (residues 1-32, KWFTKCGLVHELRRQGFDESLMRDWVCLVENE) were

269

synthesized and tested for bactericidal activity against two Gram-negative (E. coli and

270

K. pmeumonieae) and two Gram-positive (S. aureus and M. lysodeikticus) bacteria.

271

As shown in Figure 3, peptide α15 and α32 were both effective against all tested

272

strains, whereas their bactericidal activity against S. aureus are much weaker than that

273

against three other strains. Bactericidal activity curves of peptide α15 and α32

274

presented analogous trends of dose-dependent activity suggesting they have similar

275

bactericidal potency, except for peptide α15 being more efficient than α32 in killing of

276

M. lysodeikticus. Overall, the Gram-negtive strains, E. coli and K. pmeumonieae, are

277

more sensitive to the bactericidal action of ApLyz-derived peptides than

278

Gram-positive ones, S. aureus and M. lysodeikticus. In contrast, the full lysozyme

279

ApLyz showed an extremely low activity against E. coli according to agar diffusion

280

assay (data not shown), while it exerted superior activity against M. lysodeikticus.

281

Furthermore, to examine whether the tested ApLyz-derived peptides possess

282

native-like α-helical structures, circular dichroism spectroscopy (CD) was used to

283

investigate the respective secondary structure of peptide α15 and α32 in aqueous

284

solution (Figure 4). Secondary structure analysis, which was performed using an

285

online secondary structure estimation program K2D349, indicated that both peptides

286

were composed primarily of α-helices: an estimate of 80% and 95% of α-helical


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287

content in α15 and α32, respectively, with the rest mostly random coils. In addition,

288

the unstructured sequences of peptides may well form helical structure upon

289

membrane-interaction, given that lipid membranes manifest a diverse array of surface

290

forces which can orient and fold an approaching antimicrobial peptide50. In summary,

291

the tested peptides exhibit basically the same secondary structure in the N-terminus of

292

ApLyz.

293 294

Membrane disturbing activity assay of Amphipathic Peptides

295

In light of the known bactericidal fuction of AMPs which primarily correlates to

296

membrane binding and pore-forming, the N-terminal peptides α15 and α32 were used

297

in LIVE/DEAD Baclight assays for a direct proof of membrane disturbing action. In

298

this system, the green-fluorescent nucleic acid stain SYTO 9 alone labels all bacteria

299

regardless of cell membrane integrity, while the red-fluorescent nucleic acid stain

300

propidium iodide (PI) penetrates only bacteria with damaged membranes, causing a

301

reduction in the SYTO 9 stain fluorescence when both dyes are present.

302

Late log phase cultures of E. coli, K. pmeumonieae, M. lysodeikticus and S. aureus

303

were treated with α15 or α32 peptides at a concertration of 200 µg/mL, respectively.

304

After 2 hours of incubation, the dye mixture was added for staining live/dead cells,

305

where bacteria with intact cell membranes stain fluorescent green and damaged

306

membranes stain fluorescent red. Although some cells were already dead before

307

staining, remarkable increases in the population of cells staining red after peptide

308

treatment were readily observed, indicating reduced viability of cells due to



309

peptide-induced membrane permeabilization (Figure 5). In the cases of E. coli, M.

310

lysodeikticus and S. aureus, fluorescent results from LIVE/DEAD Baclight assay

311

paralleled the bactericidal activity tests of α15 and α32 peptides, whereas the

312

membrane damage induced by two peptides in Gram-negative K. pmeumonieae was

313

marginal, which is not aligned with their bactericidal potencies against this strain

314

(Figure 3). A possible explanation for this phenomenon is that ApLyz-derived

315

N-terminal peptides kill K. pmeumonieae either by forming metastable and restorable

316

pores in cell membrane51-52, followed by the exertion of intracellular bactericidal

317

effects53-54, or through distinct mechanisms like the blockage of proton-motive force

318

(respiration) by dissipating the transmembrane electrical potential gradient33. Overall,

319

the data presented bring forth a conclusion that the antimicrobial function of

320

lysozyme-derived peptide is a concerted process involving multiple killing

321

mechenisms.

322 323

DISCUSSION

324

Antimicrobial enzymes, especially lysozyme, has emerged as favorable alternative to

325

create bacteria-resistant surfaces of both food equipment and packaging materials that

326

are proposed to prevent cross-contamination, inhibit biofilm formation, and increase

327

commodity shelf life55-57. However, the prerequisite for developing lysozyme-based

328

antimicrobial material is to immobilize the protein on the packing matrix without

329

compromising its activity. Therefore, it is important to ascertain the effect of

330

immobilization pattern on the functionality of lysozyme that correlates primarily to


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331

the structure-activity relationship.

332

In

333

surface-immobilization of silkmoth lysozyme ApLyz on the exterior of S. cerevisiae

334

cells. S. cerevisiae has long been an efficient cell factory for the expression of

335

eukaryotic proteins and can perform complex post-translational modifications, such as

336

glycosylation, disulfide formation, phosphorylation and acylation. For glycoproteins,

337

however, S. cerevisiae typically produces high-mannose type glycan stretches, which

338

can be antigenic for recombinant mammalian proteins58. Nonetheless, the

339

glycosylation pathway in yeast is similar with that of the insect, by which

340

glycoproteins are modified with heterogeneous mannose-type N-glycan structures59.

341

In particular, the yeast and insect systems have identical steps of glycosylation in the

342

endoplasmic reticulum (ER), which are proved to be intimately coupled with the

343

quality control of protein folding60. In this context, yeast expression system can

344

potentially yield correctly folded insect lysozyme ApLyz and minimize the negative

345

impact of yeast glycosylation on its bioactivity. By using yeast display system, the

346

impact of N- or C-terminal fusion mode on the bactericidal activity of ApLyz was

347

explored. It was shown that the N- and C-terminal fusions of lysozyme both exerted

348

potent antibacterial activities against the tested strain; this is particularly advantageous

349

because it holds promise for ApLyz as active antimicrobial agent for immobilization

350

on the surfaces of food packaging materials or implants.

351

Furthermore, we found that the C-terminal tethered ApLyz exhibited higher

352

bactericidal activity than that of the N-terminal tethered ApLyz. A rational explanation

this

research,

we

investigated

the

heterologous


expression

and


353

is that the N-terminal domain is directly implicated in the bactericidal function of

354

ApLyz based on the structural mobility of this region. In light of the simulated

355

structure of ApLyz, its N-terminal domain primarily consists of two α-helices (i.e., H1

356

and H2), which are joined to form a typical helix-loop-helix structure (HLH). In

357

particular, the H1 α-helix is rich of basic and hydrophobic residues, and adopts an

358

amphiphilic secondary structure resembling the typical cationic antimicrobial peptides

359

(AMPs). Accumulating findings indicated that the cationic host-defense AMPs can

360

fold into amphiphilic structure by interaction with membranes and subsequently

361

induce permeabilization61. We speculated that the innate cationic and amphiphilic

362

peptides present at the terminal stretch of lysozyme, as well as surface-exposed, are in

363

favor of membrane binding and penetration. This is in line with previous reports of

364

the identified antimicrobial peptides mostly resided in the terminal region of

365

lysozymes. Besides antimicrobial peptides found in N-terminus, the C-terminal

366

peptide stretches in human (residues 87-115) and chicken lysozyme (residues 87-114)

367

have also been identified to possess in vitro bactericidal activity62-63. Given the

368

structural similarity of these c-type lysozymes, it can be inferred that the C-terminal

369

stretch of ApLyz, which mainly consist of α-helices, might also serve as an

370

antimicrobial domain. Nonetheless, as shown in our experiment, the effect of

371

immobilization of C-terminus of ApLyz on the bactericidal activity of ApLyz was

372

inferior to that of the N-terminal fusion, and therefore, the functional significance of

373

C-terminal peptides awaits further investigation.

374

In order to unravel the functional significance of N-terminal α-helical peptide motifs


Page 18 of 35

Page 19 of 35


375

of ApLyz, peptide α15 and α32 were synthesized and tested for antimicrobial activity

376

against Gram-negative and Gram-positive strains. As expected, the viable cell counts

377

showed that α15 and α32 peptides were both effective, to different extents, in the

378

killing of tested bacteria and displayed highest antibacterial activity toward

379

Gram-negative bacteria, which compensates for the weakness of parent lysozyme

380

ApLyz in this aspect. Furthermore, CD spectra of these two peptides in aqueous

381

solution manifested that the predominant structures of them are α-helices, which is

382

essentially the same with the N-terminal domain in ApLyz. Given the inconsistency

383

between the pI values of peptide α15 ( pI=10.05 ) and α32 ( pI=5.62 ), we inferred

384

that the structural factor, rather than cationicity, is mostly responsible for

385

antimicrobial function of the N-terminal domain of ApLyz.

386

The hypothesized membrane permeabilization mechanism of ApLyz-derived peptides

387

was strongly reinforced by the results of fluorescence staining assays which clearly

388

demonstrated a membrane perturbing activity of both α15 and α32 peptides. However,

389

a lack of correlation between membrane permeabilization and antibacterial activity of

390

these peptides against K. pmeumonieae was observed, similar to the results of buforin

391

II64 and indolicidin65. These findings suggested that multiple bactericidal mechanisms

392

of membrane- and non-membrane permeabilizing modes are adopted by antimicrobial

393

peptides, of which the predominant mechanism is target strain-specific and might

394

prove an important area for indepth research.

395

Comparing with intensively studied human- and chicken lysozyme, the amino acid

396

sequence identity between ApLyz and these two c-type lysozymes are relatively low



397

(37%, 39%) (Figure 6), and the N-terminal α-helical peptides derived from respective

398

lysozymes differ in their bactericidal performance33, 35, 62. The sequence diversity of

399

homologous lysozymes contribute to the diverse antimicrobial actions. For instance,

400

some lysozyme-like proteins from silkmoth, having 50-60% protein sequence

401

homology with the silkmoth lysozyme, were recently identified to possess remarkable

402

bacteriostatic rather than bactericidal activity against both Gram-negative and

403

Gram-positive bacteria, but lack of peptidoglycan hydrolysis ability66. Additionally, it

404

was reported that even single amino acid substitution either in hinge or α-helical

405

region of natural or lysozyme-derived antimicrobial peptide can affect the

406

antimicrobial mechanism dramaticlly63, 67. Therefore, lysozyme-derived peptides from

407

different origins furnish a promising source of natural antimicrobial peptides for food,

408

fodder and pharmaceutical applications.

409

In summary, this study demonstrated the presence of a new class of potent bactericidal

410

peptides found in the N-terminal domain of silkmoth lysozyme ApLyz and enabled

411

the selection of novel antimicrobial peptides to complement conventional antibiotics

412

for enhanced food safety and hygiene. Moreover, the discovery of non-catalytic

413

antimicrobial motif of ApLyz, together with the established yeast surface display

414

system, opened up an opportunity for future improvement of lysozyme in bactericidal

415

efficacy, especially against Gram-negtive strains, by high-throughput screening of

416

corresponding mutant library.

417 418


Page 20 of 35

Page 21 of 35


419

AUTHOR INFORMATION

420

Conflict of interest

421

The authors declare no conflict of interest.

422

Funding

423

This work was supported by the National Natural Science Foundation of China (No.

424

21406005); the Scientific Research Starting Foundation for Young Talents in Beijing

425

Technology and Business University (No. QNJJ2014-28).

426 427

ACKNOWLEDGEMENTS

428

We want to acknowledge Prof. Fan, Li-hai for providing yeast strain and plasmid in

429

the experiment of yeast surface display.

430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449

REFERENCES (1) Pellegrini, A.; Thomas, U.; Wild, P.; Schraner, E.; Von Fellenberg, R., Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli. Microbiological Research 2000, 155 (2), 69-77. (2)

Masschalck, B.; Van Houdt, R.; Van Haver, E. G. R.; Michiels, C. W., Inactivation of

Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure. Applied and Environmental Microbiology 2001, 67 (1), 339-344. (3)

Iacono, V. J.; Zove, S. M.; Grossbard, B. L.; Pollock, J. J.; Fine, D. H.; Greene, L. S.,

Lysozyme-mediated aggregation and lysis of the periodontal microorganism Capnocytophaga gingivalis 2010. Infection and Immunity 1985, 47 (2), 457-64. (4)

Sahoo, N. R.; Kumar, P.; Bhusan, B.; Bhattacharya, T. K.; Dayal, S.; Sahoo, M., Lysozyme

in livestock: a guide to selection for disease resistance: a review. Journal of Animal Science Advances 2012, 2 (4), 347-360. (5)

Rau, A.; Hogg, T.; Marquardt, R.; Hilgenfeld, R., A new lysozyme fold. Crystal structure of

the muramidase from Streptomyces coelicolor at 1.65 A resolution. Journal of Biological Chemistry 2001, 276 (34), 31994-9. (6)

Ghasemi, S.; Ahmadian, G.; Sadeghi, M.; Zeigler, D. R.; Rahimian, H.; Ghandili, S.;

Naghibzadeh, N.; Dehestani, A., First report of a bifunctional chitinase/lysozyme produced by Bacillus pumilus SG2. Enzyme and Microbial Technology 2011, 48 (3), 225-31.



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Callewaert, L.; Van Herreweghe, J. M.; Vanderkelen, L.; Leysen, S.; Voet, A.; Michiels, C.

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Salazar, O.; Asenjo, J. A., Enzymatic lysis of microbial cells. Biotechnology Letter 2007, 29

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Hughey, V. L.; Johnson, E. A., Antimicrobial activity of lysozyme against bacteria involved

in food spoilage and food-borne disease. Applied and Environmental Microbiology 1987, 53 (9), 2165-70. (10) Parisien, A.; Allain, B.; Zhang, J.; Mandeville, R.; Lan, C. Q., Novel alternatives to antibiotics: bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides. Journal of Applied Microbiology 2008, 104 (1), 1-13. (11) Losso, J. N.; Nakai, S.; Charter, E. A., Lysozyme. CRC Press: New York, 2002. (12) Zeuthen, P.; Bùgh-Sùrensen, L., Food preservation techniques. CRC Press: New York, 2003. (13) Masschalck, B.; Michiels, C. W., Antimicrobial properties of lysozyme in relation to foodborne vegetative bacteria. Critical Reviews in Microbiology 2003, 29 (3), 191-214. (14) Zhou, B.; Li, Y.; Deng, H.; Hu, Y.; Li, B., Antibacterial multilayer films fabricated by layer-by-layer immobilizing lysozyme and gold nanoparticles on nanofibers. Colloids and Surfaces B: Biointerfaces 2014, 116, 432-8. (15) Eby, D. M.; Luckarift, H. R.; Johnson, G. R., Hybrid antimicrobial enzyme and silver nanoparticle coatings for medical instruments. ACS Applied Materials & Interfaces 2009, 1 (7), 1553-60. (16) Shigeyasu, C.; Yamada, M.; Akune, Y., Influence of Ophthalmic Solutions on Tear Components. Cornea 2016, 35 (1), S71-S77. (17) Shao, Y.; Xue, J.; Peng, C.; Li, C.; Zhang, S.; Zhou, H., Clinical evaluation of a toothpaste containing lysozyme for removal of extrinsic stains on the tooth surface: An 8-week, double-blind, randomized study. American Journal of Dentistry 2017, 30 (3), 147-150. (18) Hannig, C.; Spitzmuller, B.; Lux, H. C.; Altenburger, M.; Al-Ahmad, A.; Hannig, M., Efficacy of enzymatic toothpastes for immobilisation of protective enzymes in the in situ pellicle. Archives of Oral Biology 2010, 55 (7), 463-9. (19) Cooper, C. A.; Garas Klobas, L. C.; Maga, E. A.; Murray, J. D., Consuming Transgenic Goats' Milk Containing the Antimicrobial Protein Lysozyme Helps Resolve Diarrhea in Young Pigs. PLOS ONE 2013, 8 (3), e58409. (20) Časaitė, V.; Bružytė, S.; Bukauskas, V.; Šetkus, A.; Morozova-Roche, L. A.; Meškys, R., Expression and purification of active recombinant equine lysozyme in Escherichia coli. Protein Engineering, Design and Selection 2009, 22 (11), 649-654. (21) Mine, S.; Ueda, T.; Hashimoto, Y.; Imoto, T., Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser. Protein Engineering, Design and Selection 1997, 10 (11), 1333-1338. (22) Masuda, T.; Ueno, Y.; Kitabatake, N., High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris. Protein Expression and Purification 2005, 39 (1), 35-42. (23) Kawamura, S.; Fukamizo, T.; Araki, T.; Torikata, T., Expression of a Synthetic Gene Coding for Ostrich Egg-White Lysozyme in Pichia Pastoris and Its Enzymatic Activity. The Journal of Biochemistry 2003, 133 (1), 123-131. (24) Goda, S.; Takano, K.; Yamagata, Y.; Katakura, Y.; Yutani, K., Effect of extra N-terminal


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residues on the stability and folding of human lysozyme expressed in Pichia pastoris. Protein Engineering, Design and Selection 2000, 13 (4), 299-307. (25) Taniyama, Y.; Yamamoto, Y.; Nakao, M.; Kikuchi, M.; Ikehara, M., Role of disulfide bonds in folding and secretion of human lysozyme in Saccharomyces cerevisiae. Biochemical and Biophysical Research Communications 1988, 152 (3), 962-967. (26) Koganesawa, N.; Aizawa, T.; Masaki, K.; Matsuura, A.; Nimori, T.; Bando, H.; Kawano, K.; Nitta, K., Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system. Protein Engineering, Design and Selection 2001, 14 (9), 705-710. (27) Wang, T.; Xu, Y.; Liu, W.; Sun, Y.; Jin, L., Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris. Protein Expression and Purification 2011, 77 (1), 20-25. (28) Pellegrini, A.; Thomas, U.; von Fellenberg, R.; Wild, P., Bactericidal activities of lysozyme and aprotinin against gram-negative and gram-positive bacteria related to their basic character. Journal of Applied Microbiology 1992, 72 (3), 180-7. (29) Ibrahim, H. R.; Higashiguchi, S.; Juneja, L. R.; Kim, M.; Yamamoto, T., A Structural Phase of Heat-Denatured Lysozyme with Novel Antimicrobial Action. Journal of Agricultural and Food Chemistry 1996, 44 (6), 1416-1423. (30) Derde, M.; Guerin-Dubiard, C.; Lechevalier, V.; Cochet, M. F.; Jan, S.; Baron, F.; Gautier, M.; Vie, V.; Nau, F., Dry-heating of lysozyme increases its activity against Escherichia coli membranes. Journal of Agricultural and Food Chemistry 2014, 62 (7), 1692-700. (31) Ibrahim, H. R.; Higashiguchi, S.; Koketsu, M.; Juneja, L. R.; Kim, M.; Yamamoto, T.; Sugimoto, Y.; Aoki, T., Partially Unfolded Lysozyme at Neutral pH Agglutinates and Kills Gram-Negative and Gram-Positive Bacteria through Membrane Damage Mechanism. Journal of Agricultural and Food Chemistry 1996, 44 (12), 3799-3806. (32) Ibrahim, H. R.; Higashiguchi, S.; Sugimoto, Y.; Aoki, T., Role of Divalent Cations in the Novel Bactericidal Activity of the Partially Unfolded Lysozyme. Journal of Agricultural and Food Chemistry 1997, 45 (1), 89-94. (33) Ibrahim, H. R.; Imazato, K.; Ono, H., Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration. Journal of Agricultural and Food Chemistry 2011, 59 (18), 10336-45. (34) Mine, Y.; Ma, F.; Lauriau, S., Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme. Journal of Agricultural and Food Chemistry 2004, 52 (5), 1088-94. (35) Ibrahim, H. R.; Inazaki, D.; Abdou, A.; Aoki, T.; Kim, M., Processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach. Biochimica et Biophysica Acta 2005, 30 (1), 102-14. (36) Hunter, H. N.; Jing, W.; Schibli, D. J.; Trinh, T.; Park, I. Y.; Kim, S. C.; Vogel, H. J., The interactions of antimicrobial peptides derived from lysozyme with model membrane systems. Biochimica et Biophysica Acta 2005, 1 (2), 175-89. (37) During, K.; Porsch, P.; Mahn, A.; Brinkmann, O.; Gieffers, W., The non-enzymatic microbicidal activity of lysozymes. FEBS Lett 1999, 449 (2-3), 93-100. (38) Thammasirirak, S.; Pukcothanung, Y.; Preecharram, S.; Daduang, S.; Patramanon, R.; Fukamizo, T.; Araki, T., Antimicrobial peptides derived from goose egg white lysozyme. Comparative Biochemistry and Physiology - Part C: Toxicology and Pharmacology 2010, 151 (1), 84-91. (39) Zavalova, L. L.; Yudina, T. G.; Artamonova, II; Baskova, I. P., Antibacterial non-glycosidase



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activity of invertebrate destabilase-lysozyme and of its helical amphipathic peptides. Chemotherapy 2006, 52 (3), 158-60. (40) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E., UCSF Chimera--a visualization system for exploratory research and analysis. Journal of Computational Chemistry 2004, 25 (13), 1605-12. (41) Liu, Z.; Ho, S. H.; Hasunuma, T.; Chang, J. S.; Ren, N. Q.; Kondo, A., Recent advances in yeast cell-surface display technologies for waste biorefineries. Bioresource Technology 2016, 215, 324-333. (42) Cruz-Teran, C. A.; Tiruthani, K.; Mischler, A.; Rao, B. M., Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae. ACS Synthetic Biology 2017, 6 (11), 2096-2107. (43) Massari, M. E.; Murre, C., Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Molecular and Cellular Biology 2000, 20 (2), 429-40. (44) Agirre, A.; Barco, A.; Carrasco, L.; Nieva, J. L., Viroporin-mediated membrane permeabilization. Pore formation by nonstructural poliovirus 2B protein. Journal of Biological Chemistry 2002, 277 (43), 40434-41. (45) Sanchez-Martinez, S.; Madan, V.; Carrasco, L.; Nieva, J. L., Membrane-active peptides derived from picornavirus 2B viroporin. Current Protein & Peptide Science 2012, 13 (7), 632-43. (46) Srisailam, S.; Arunkumar, A. I.; Wang, W.; Yu, C.; Chen, H. M., Conformational study of a custom antibacterial peptide cecropin B1: implications of the lytic activity. Biochimica et Biophysica Acta 2000, 15, 1-2. (47) Chakraborty, S.; Phu, M.; de Morais, T. P.; Nascimento, R.; Goulart, L. R.; Rao, B. J.; Asgeirsson, B.; Dandekar, A. M., The PDB database is a rich source of alpha-helical anti-microbial peptides to combat disease causing pathogens. F1000Res 2014, 3 (295). (48) Boman, H. G.; Faye, I.; Gudmundsson, G. H.; Lee, J. Y.; Lidholm, D. A., Cell-free immunity in Cecropia. A model system for antibacterial proteins. European Journal of Biochemistry 1991, 201 (1), 23-31. (49) Caroline, L.-J.; A., A.-N. M.; Carol, P.-I., Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins: Structure, Function, and Bioinformatics 2012, 80 (2), 374-381. (50) Silvestro, L.; Axelsen, P. H., Membrane-induced folding of Cecropin A. Biophys J 2000, 79 (3), 1465-77. (51) Bechinger, B.; Gorr, S. U., Antimicrobial Peptides: Mechanisms of Action and Resistance. Journal of Dental Research 2017, 96 (3), 254-260. (52) Tosatto, L.; Andrighetti, A. O.; Plotegher, N.; Antonini, V.; Tessari, I.; Ricci, L.; Bubacco, L.; Dalla Serra, M., Alpha-synuclein pore forming activity upon membrane association. Biochimica et Biophysica Acta 2012, 11 (83), 20. (53) Le, C. F.; Fang, C. M.; Sekaran, S. D., Intracellular Targeting Mechanisms by Antimicrobial Peptides. Antimicrobial Agents and Chemotherapy 2017, 61 (4), 02340-16. (54) Chongsiriwatana, N. P.; Lin, J. S.; Kapoor, R.; Wetzler, M.; Rea, J. A. C.; Didwania, M. K.; Contag, C. H.; Barron, A. E., Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids. Scientific Report 2017, 7 (1), 017-16180. (55) Sebaa, S.; Hizette, N.; Boucherit-Otmani, Z.; Courtois, P., Dosedependent effect of lysozyme


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upon Candida albicans biofilm. Molecular Medicine Reports 2017, 15 (3), 1135-1142. (56) Muriel-Galet, V.; Talbert, J. N.; Hernandez-Munoz, P.; Gavara, R.; Goddard, J. M., Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications. Journal of Agricultural and Food Chemistry 2013, 61 (27), 6720-7. (57) Appendini, P.; Hotchkiss, J. H., Immobilization of Lysozyme on Food Contact Polymers as Potential Antimicrobial Films. Packaging Technology and Science 1997, 10, 271-279. (58) Nakamura, S.; Takasaki, H.; Kobayashi, K.; Kato, A., Hyperglycosylation of hen egg white lysozyme in yeast. The Journal of Biological Chemistry 1993, 268, 12706-12712. (59) Rendi, D.; Wilson, I. B. H.; Paschinger, K., The Glycosylation Capacity of Insect Cells. Croatica Chemica Acta 2008, 81 (1), 7-21. (60) Trombetta, E. S., The contribution of N-glycans and their processing in the endoplasmic reticulum to glycoprotein biosynthesis. Glycobiology 2003, 13 (9), 77R-91R. (61) Powers, J. P.; Hancock, R. E., The relationship between peptide structure and antibacterial activity. Peptides 2003, 24 (11), 1681-91. (62) Ibrahim, H. R.; Thomas, U.; Pellegrini, A., A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action. Journal of Biological Chemistry 2001, 276 (47), 43767-74. (63) Pellegrini, A.; Thomas, U.; Bramaz, N.; Klauser, S.; Hunziker, P., Identification and isolation of a bactericidal domain in chicken egg white lysozyme. Journal of Applied Microbiology 1997, 82 (3), 372-378. (64) Park, C. B.; Kim, H. S.; Kim, S. C., Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochemical and Biophysical Research Communications 1998, 244 (1), 253-7. (65) Hale, J. D.; Hancock, R. E., Alternative mechanisms of action of cationic antimicrobial peptides on bacteria. Expert Review of Anti-infective Therapy 2007, 5 (6), 951-9. (66) Gandhe, A. S.; Janardhan, G.; Nagaraju, J., Immune upregulation of novel antibacterial proteins from silkmoths (Lepidoptera) that resemble lysozymes but lack muramidase activity. Insect Biochemistry and Molecular Biology 2007, 37 (7), 655-66. (67) Park, C. B.; Yi, K.-S.; Matsuzaki, K.; Kim, M. S.; Kim, S. C., Structure–activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II. Proceedings of the National Academy of Sciences 2000, 97 (15), 8245-8250.

615



Page 26 of 35

616

Figure captions

617

Figure 1. (A) Bright field (upper panel) and immunofluorescence (bottom panel)

618

microscopic images of EBY100 (a-b), EBY100/pYD1-LYZ (c-d) and

619

EBY100/pYD1-RPLYZ (e-f) staining with anti-Xpress-FITC antibodies.

620

Antimicrobial activity of yeast surface displayed lysozymes LYZ and RPLYZ against

621

M. lysodeikticus using the cylinder-plate method (inhibition zone). Experiments were

622

carried out in triplicate, and values were shown as means ± S.D. “**”(p< 0.01)

623

indicates significant difference obtained from the Student’s t test.

(B)

624 625

Figure 2. (A) Schematic representation of 3-D structure of ApLyz based on homology

626

modeling. The HLH α-helical motif (boxed, dashed line) is located at the upper side

627

of the active site cleft. The two catalytic residues (Glu32 and Asp50) and four

628

disulfide bonds are shown. (B) Helical wheel projections of H1(a) and H2(b)

629

α-helices (http://rzlab.ucr.edu/scripts/wheel). By default the output presents the

630

hydrophilic residues as circles, hydrophobic residues as diamonds, potentially

631

negatively charged as triangles, and potentially positively charged as pentagons.

632

Hydrophobicity is color coded as well: the most hydrophobic residue is green, and the

633

amount of green is decreasing proportionally to the hydrophobicity, with zero

634

hydrophobicity coded as yellow. Hydrophilic residues are coded red with pure red

635

being the most hydrophilic (uncharged) residue, and the amount of red decreasing

636

proportionally to the hydrophilicity. The potentially charged residues are light blue.

637


Page 27 of 35


638

Figure 3 Bactericidal activity of synthesized α15 and α32 peptides against

639

Gram-negtive strains, E. coli and K. pmeumonieae, and Gram-positive strains, M.

640

lysodeikticus and S. aureus. The colony forming unit (CFU) was counted to determine

641

the number of viable cells after incubation with different concentrations (50-250

642

µg/mL) of α15 or α32 peptides for 1 h. Values represent the means ± S.D. of three

643

independent experiments.

644

Circular dichroism spectra of α15 and α32 peptide. Mean residue

645

Figure 4

646

ellipticity has units of deg.cm2dmol-1.

647

Membrane disturbing activity of synthesized α15 and α32 peptide.

648

Figure 5

649

LIVE/DEAD staining of cells followed the incubation with α15 or α32 at a final

650

concentration of 200 µg/mL for 120 min. For four types of bacterial cells, left: PBS

651

control, center: α15 peptide, right: α32 peptide. Bacterial cells with damaged

652

membranes fluoresce red; bacteria with intact membranes fluoresce green.

653

Multiple sequence alignment of lysozymes from human (PDB ID: 1lz1),

654

Figure 6

655

hen egg white (PDB ID: 1dpx) and A. perni (GenBank: ABC73705.1). Gaps are

656

indicated by “.”. Secondary structure elements are shown at the top. Sequence

657

alignment was carried out by clustalW and visualized using ESPript3.0 .

658



659 660 661

Figure 1


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662 663 664 665

Figure 2



666 667

Figure 3

668


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669 670

Figure 4

671



672

673 674 675

Figure 5


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676 677 678

Figure 6



679 680

TOC Graphic


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TOC graphic 48x26mm (300 x 300 DPI)


Yeast Surface Display of Antheraea pernyi Lysozyme Revealed Î±

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