Adsorption of Insecticidal Crystal Protein Cry11Aa onto Nano-Mg(OH

Oct 11, 2017 - State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops & Key Lab of Biopesticide and Chemical Biology, Ministry of...
0 downloads 5 Views 10MB Size
Subscriber access provided by Southern Cross University Library

Article

Adsorption of Insecticidal Crystal Protein Cry11Aa onto nanoMg(OH)2: Effects on Bioactivity and Anti-Ultraviolet Ability Xiaohong Pan, Zhangyan Xu, Lan Li, Enshi Shao, Saili Chen, Tengzhou Huang, Zhi Chen, Wenhua Rao, Tianpei Huang, Lingling Zhang, Songqing Wu, and Xiong Guan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03410 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 15, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

1

Adsorption of Insecticidal Crystal Protein Cry11Aa onto

2

nano-Mg(OH)2: Effects on Bioactivity and Anti-Ultraviolet Ability a,b

, Zhangyan Xu a, Lan Li a, Enshi Shao

a,b

, Saili Chen a, Tengzhou Huang a, Zhi

3

Xiaohong Pan

4

Chen a, Wenhua Rao a, Tianpei Huang a,b, Lingling Zhang a,b, Songqing Wu a,b, Xiong Guan a,b,*

5

a

6

Biopesticide and Chemical Biology, Ministry of Education & College of Plant Protection &

7

College of Resources and Environmental Sciences & College of Life Sciences & Forestry

8

College, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, P. R. China.

9

b

10

State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops & Key Lab of

Fujian-Taiwan Joint Center for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, P. R.

China.

11 12

*

13

591-83789259.

CORRESPONDING

AUTHOR

E-mail:

[email protected];

14 15

ACS Paragon Plus Environment 1

Tel.

&

Fax:

(+086)

Journal of Agricultural and Food Chemistry

16

ABSTRACT

17

The traditional Bacillus thuringiensis (Bt) formulation for field applications are not resistant

18

to harsh environmental conditions. Hence, the active ingredients of the Bt bioinsecticides could

19

degrade quickly and has low anti-ultraviolet ability in the field, which significantly limits its

20

practical application. In the present study, we developed an efficient and stable delivery system

21

for Bt Cry11Aa toxins. We coated Cry11Aa proteins with Mg(OH)2 nanoparticles (MHNPs), and

22

then assessed the effects of MHNPs on bioactivity and anti-ultraviolet ability of the Cry11Aa

23

proteins. Our results indicated that MHNPs, like “coating clothes”, could effectively protect the

24

Cry protein and enhance the insecticidal bioactivity after UV radiation (the degradation rate was

25

decreased from 64.29% to 16.67%). In addtion, MHNPs could improve the proteolysis of

26

Cry11Aa in the midgut and aggravate the damage of the Cry protein to the gut epithelial cells,

27

leading to increased insecticidal activity against Culex quinquefasciatus. Our results revealed

28

that MHNPs, as an excellent nano-carrier, could substantially improve the insecticidal bioactivity

29

and anti-ultraviolet ability of Cry11Aa.

30

KEY WORDS: Cry11Aa; Nano-Mg(OH)2; Anti-ultraviolet; Bioactivity; Proteolysis

31

ACS Paragon Plus Environment 2

Page 2 of 32

Page 3 of 32

32

Journal of Agricultural and Food Chemistry

1. INTRODUCTION

33

As one of the most widely used bioinsecticides, Bacillus thuringiensis (Bt) produces

34

insecticidal crystal proteins (Cry and Cyt) during the sporulation phase.1-3 Moreover, the Cry

35

toxins are effective against lepidopteran, coleopteran, dipteran insect pests and nematodes.4-6

36

Mosquitoes are responsible for many severe human diseases, such as malaria, dengue fever,

37

yellow fever and so on.7 Bt subsp. israelensis (Bti) is highly active against disease-vector

38

mosquitoes, like Aedes, Culex and Anopheles,4 and the efficacy of Bti is due to the presence of

39

Cry4A, Cry4B, Cry10A, Cry11A, Cyt1A and Cyt2B proteins.8-10 However, there are also some

40

difficulties in using Bti products, such as the degradation of active ingredients, low

41

anti-ultraviolet ability and so on.11 Therefore, how to protect the effective component and

42

increase the anti-ultraviolet capacity of Bti is of great importance from both economical and

43

practical point of view. It has been reported that the Bti products can be adsorbed on clay

44

particles, thus protecting their activity (such as UV radiation and microbial degradation) and

45

enhancing the persistence of the product.12

46

Nanoparticles (NPs) are widely used in industrial, medical, personal and military applications

47

due to their small size (one or more dimensions in the order of 100 nm or less), high specific

48

surface area, strong adsorption capacity, as well as unique optical and electrical properties.13, 14

49

At present, several studies have focused on NPs application in pesticides, and their results have

50

indicated that the nano-loading of NPs (such as TiO2, ZnO, SiO2 and CaCO3) can improve the

51

utilization rates and other properties.15-20 However, such application in biopesticides also

52

requires

53

agro-biotechnological applications. As a low cost and nontoxic material, Mg(OH)2 NPs (MHNPs)

54

have already been applied in the removal of acid, dye and heavy metals from wastewater, and

relatively

high

cost

and

direct

environmental

ACS Paragon Plus Environment 3

exposure,

hindering

the

Journal of Agricultural and Food Chemistry

55

previous studies have confirmed their low cell toxicity.21, 22 Therefore, MHNPs are regarded as

56

environmentally friendly nanomaterials.23-27 However, till now, only few studies have focused on

57

the application of MHNPs in biopesticides.

58

In this study, we aimed to develop an efficient and stable delivery system for Cry protein by

59

loading MHNPs in biocontrol applications. Cry11Aa toxins was loaded on MHNPs carriers, the

60

bioactivity and anti-ultraviolet radiation ability were investigated, the proteolysis of Cry11Aa by

61

different treatments was carried out, and the midgut tissues of Culex quinquefasciatus were

62

examined. The results indicated that MHNPs could effectively protect the Cry protein after UV

63

radiation, enhance the insecticidal bioactivity, improve the proteolysis of Cry11Aa in the midgut

64

and aggravate the damage of the Cry protein to the gut epithelial cells. Our findings offered

65

valuable information for the practical application of MHNPs in biopesticides.

66

2. MATERIALS AND METHODS

67

2.1 Preparation and characterization of MHNPs and Cry11Aa toxin

68

MHNPs were prepared by co-precipitation of magnesium chloride hexahydrate and sodium

69

hydroxide in double distilled water (ddH2O) at room temperature. The resulting suspensions

70

were washed with ddH2O for three times and then centrifuged at 10,000 g for 10 min. The pH

71

value of the synthesized Mg(OH)2 slurry (1 g/L) was around 10.3. The morphology and size of

72

the synthesized nano-Mg(OH)2 samples were characterized on a JSM-6700F scanning electron

73

microscope (SEM) (JEOL Ltd., Japan) equipped with an Oxford-INCA energy-dispersive X-ray

74

spectroscopy (EDS). Meanwhile, the size distribution of MHNPs was determined by a dynamic

75

light scattering (DLS) instrument produced by Malvern Instruments Corporation, MHNPs were

76

sonicated before DLS determination, and an average Z-Average value was obtained.

ACS Paragon Plus Environment 4

Page 4 of 32

Page 5 of 32

Journal of Agricultural and Food Chemistry

77

The Bt strain was grown in 1/2 LB medium until 80% of the crystal was released. After

78

centrifugation at 8,000 g for 10 min at 4 ◦C, the mixture of crystals, spores and debris was

79

collected and washed with 1 M NaCl, followed by a wash with distilled water. The mixture of

80

crystals, spores and debris was directly resuspended in a solubilization buffer (50 mM Na2CO3,

81

pH 9.5) and then centrifuged at 13,000 g for 20 min to remove the insoluble debris. The

82

supernatant was collected, its pH was adjusted to 4.5 with HAc, and then the supernatant kept at

83

4 ◦C for at least 4 h. The pellet was collected by centrifugation at 10,000 g for 15 min, washed

84

with distilled water for three times, and dissolved in 50 mM Na2CO3 (pH 10.5). The final

85

product was Cry11Aa protein. Cry11Aa protein and the insoluble debris (from centrifugation at

86

13,000 g for 20 min) were separately collected and analyzed by SDS-PAGE. All chemicals were

87

analytical grade, and the treatment was repeated twice.

88

2.2 The stable property analysis of MHNPs

89

The pH tolerance experiment: Briefly, 1 mL MES (C6H13NO4S) medium (30 mM, pH=5.6,

90

simulated natural environment) was added into MHNPs in 2-mL centrifuge tube at room

91

temperature with gentle agitation, and the supernatant was collected every 12 h and replaced

92

with fresh MES medium for seven times. The pH value was monitored, and the initial and final

93

weights of MHNPs were measured.

94

Thermal gravity analysis (TGA): The TGA of MHNPs was recorded by the simultaneous

95

thermal analyzer (STA449C, Netzsch Co.) in argon atmosphere within the temperature range of

96

27~1000 ◦C and with the heating rate of 10 ◦C/min.

97

Ultraviolet radiation resistance capacity: MHNPs were placed at a straight distance of 30

98

cm from the front surface of UV lamp and radiated for 18 h (4.5-h UV radiation per time, four

99

times). After radiation, the sample was analyzed by X-ray diffraction (XRD) and Fourier

ACS Paragon Plus Environment 5

Journal of Agricultural and Food Chemistry

100

transforming infrared spectrum (FT-IR) to determine the possible structural change of MHNPs.

101

XRD patterns were identified using a PANalytical X’Pert PRO diffractometer with Cu Kα

102

radiation (40 kV, 40 mA) in a continuous scanning mode. The 2θ scanning ranged from 5° to 85°

103

in steps of 0.017° with a collection time of 20 s per step. The average crystallite size was

104

determined from the peak broadening according to the Scherrer equation. FT-IR spectra were

105

recorded by the Thermo Nicolet iS50 spectrometer within the range of 400~4,000 cm-1 by the

106

KBr pellet method.

107

2.3 Cry11Aa protein loading

108

MHNPs (3.0 mg) were suspended in 300 µL double distilled water. An aliquot of Cry11Aa in

109

Na2CO3 was added to the suspension and ultrasonicated at 4 ◦C for 30 min. The particles were

110

collected by centrifugation. After loading, the supernatant and precipitate were extracted by

111

centrifugation (10,000 g for 10 min), the supernatant was residual Cry11Aa protein, and the

112

precipitate was Cry11Aa-loaded MHNPs (designated as Cry11Aa-MHNPs). The amount of

113

Cry11Aa loaded onto MHNPs was calculated by subtracting the amount of residual Cry11Aa

114

from the total amount of Cry11Aa added to the sample. The protein concentration was

115

determined at a wavelength of 595 nm by Bradford method using bovine serum albumin as the

116

standard.

117

2.4 The anti-ultraviolet and bioactivity assays

118

Briefly, the untreated Cry11Aa protein was set as the control, and both the Cry protein and

119

Cry11Aa-MHNPs were placed at a straight distance of 30 cm from the front surface of UV lamp

120

and radiated for 4.5 h. After radiation, all samples were analyzed by SDS-PAGE, and the

121

concentrations of Cry protein before and after UV radiation were determined as described in

122

Section 2.3.

ACS Paragon Plus Environment 6

Page 6 of 32

Page 7 of 32

Journal of Agricultural and Food Chemistry

123

Meanwhile, the insecticidal activity of the prepared samples was examined against C.

124

quinquefasciatus. The C. quinquefasciatus, previously provided by the Jiangsu Center for

125

Disease Prevention and Control (Jiangsu Province, China), was reared in an environmentally

126

controlled room (T=25±3◦C, RH=80%, L:D=14:10). Mosquitocidal bioassays of different

127

samples were assayed against 30 third-instar larvae in 10 mL of dechlorinated water, and the

128

samples included Cry11Aa protein, Cry11Aa-MHNPs, Cry11Aa protein-UV 4.5 h and

129

Cry11Aa-MHNPs-UV 4.5 h. The initial concentration of Cry11Aa was 0.057 g/L, and the

130

volume of protein was set as 50, 100, 150, 200, 250 and 300 µL. Each treatment was replicated

131

for three times, and the mortality was recorded at 12 h and 36 h. Moreover, the mean 50% lethal

132

concentration (LC50) and 95% confidence limits (CL) was estimated by SPSS analysis (version

133

17.0) using statistical parameters.

134

2.5 Extraction of C. quinquefasciatus gut proteases and in vitro proteolysis of Cry11Aa

135

The extraction of the guts was conducted as previously described.28,

29

Briefly, 200 C.

136

quinquefasciatus larvae (third-instar) were excised by dissection, placed in 200 µL PBS buffer

137

(pH 7.3) and stored at -80 ◦C. Gut tissues in individual tubes were homogenized and then

138

centrifuged at 12,000 g for 20 min at 4 ◦C. The supernatants (containing the stomach and midgut

139

fractions) were collected, placed into fresh tubes and marked as lumen fractions. In addition, the

140

residual pellets were resuspended in 200 µL PBS buffer (pH 7.3), homogenized, labelled as

141

membrane fractions and stored at -80 ◦C prior to further analysis.

142

The obtained lumen and membrane fractions were incubated with Cry11Aa and

143

Cry11Aa-MHNPs, respectively. The Cry11Aa was incubated at 37 ◦C for trypsin activation as

144

the positive control. Certain volumes of lumen and membrane fractions were added to 40 µL

145

sample (Cry11Aa, Cry11Aa-MHNPs) with a ratio of 10:1 (Cry11Aa: gut samples, w/w). The

ACS Paragon Plus Environment 7

Journal of Agricultural and Food Chemistry

146

mixtures were incubated at 37 ◦C for different time intervals for the proteolysis of Cry protoxin.

147

The reaction was terminated by addition of SDS-PAGE sample buffer (5×loading buffer) and

148

immediately heated at 100 ◦C for 5 min. Finally, the proteolysis fragments of Cry11Aa were

149

analyzed by SDS-PAGE.

150

2.6 Sample preparation for transmission electron microscopy (TEM)

151

The third-instar of C. quinquefasciatus larvae were either fed different samples as mentioned

152

in Section 2.4 or water only (control). A total of 25 larvae from all replicates were dissected to

153

isolate their midguts in 24 h, the peritrophic membranes and malpighian tubules were removed

154

under a stereoscopic microscope, and then the isolated midgut tissues were rinsed with PBS for

155

three times.30, 31 Subsequently, the midguts were immediately fixed with 2.5% glutaraldehyde in

156

phosphate buffer (0.1 M, pH 7.0) for more than 4 h, then post-fixed with 1% OsO4 in phosphate

157

buffer (0.1 M, pH 7.0) for 2 h and dehydrated in a graded series of ethanol (30%, 50%, 70%,

158

80%, 90%, 95% and 100%) for 5 min at each step. Subsequently, the specimens were embedded

159

in 618-resin and sectioned in LEICA EM UC7 ultratome. Then the sections were sequentially

160

stained by uranyl acetate and alkaline lead citrate for 5 to 10 min. Transversally sectioned gut

161

samples were observed by Hitachi Model H-7650 TEM.

162

3. RESULTS AND DISCUSSION

163

3.1 Characterization of MHNPs and Cry protein

164

The morphology of MHNPs and Cry11Aa-MHNPs was characterized by SEM. We found that

165

the synthesized MHNPs contained a lot of small flakes around tens of nanometers (Figure 1a).

166

Meanwhile, the size distribution was determined in order to monitor the agglomeration state of

167

MHNPs in water. Figure 1d illustrates that the size of MHNPs was well distributed, and the

ACS Paragon Plus Environment 8

Page 8 of 32

Page 9 of 32

Journal of Agricultural and Food Chemistry

168

average particle size (Z-Average value) was 287.3 nm, indicating that the synthesized MHNPs

169

maintained a relatively stable state at nanometer level and had a little agglomeration in water.

170

As to Cry11Aa-MHNPs, the MHNPs were densely aggregated but still with structure of

171

nano-flakes (Figure 1b), and the EDS analysis revealed that the compound contained C, N and S

172

elements (insert picture of Figure 1b), confirming that the Cry11Aa protein was successfully

173

loaded onto MHNPs. Additionally, the Cry protein was analyzed by SDS-PAGE, and the

174

molecular mass of 68 kDa belonged to Cry11Aa (Figure 1c, Line 2). Moreover, the molecular

175

mass of Cry11Aa protein barely changed after loaded onto MHNPs (Figure 1c, Line 1), and the

176

protein band at 68 kDa changed shallow, this result also confirming that Cry11Aa was loaded

177

onto MHNPs. Furthermore, the adsorption experiments indicated that the adsorption capacity of

178

protein by MHNPs could achieve as high as 136 mg/g, suggesting that the MHNPs could be used

179

as a good nano-carrier with high adsorption ability of Cry protein.

ACS Paragon Plus Environment 9

Journal of Agricultural and Food Chemistry

Page 10 of 32

180 181

Figure 1. Characterization of MHNPs and Cry11Aa loading on MHNPs. a) SEM images of

182

MHNPs. b) SEM images of Cry11Aa-MHNPs, the insert picture was EDS analysis (note the C,

183

N and S elements). c) SDS-PAGE analysis of Cry11Aa before and after loading with Cry11Aa

184

(M: Prestained Markers, which was purchased from Thermo; Line 1: Cry11Aa-MHNPs; Line 2:

185

Cry11Aa protein). d) size distribution of MHNPs, the dispersant was water.

186

3.2 The stable property analysis of MHNPs in the delivery system

187

It has been reported that Mg(OH)2 is an environmentally friendly material owing to its special

188

physicochemical properties, such as large availability, high energy storage density, non-toxicity

189

and safety.32 In order to evaluate the stable property of synthesized MHNPs in this delivery

190

system, we assessed the pH tolerance, thermostability and ultraviolet radiation resistance

191

capacity of MHNPs. Our data showed that pH had no obvious changes during the seven cycles

ACS Paragon Plus Environment 10

Page 11 of 32

Journal of Agricultural and Food Chemistry

192

(Figure 2a), and the weight of MHNPs was slightly decreased (Figure 2b, from 0.01 g to 0.0078

193

g). The result indicated that MHNPs could tolerate the pH alteration in natural environment,

194

suggesting that MHNPs could maintain the persistent period. Meanwhile, the pH value of

195

Cry11Aa-MHNPs in MES medium (Figure 2a) was slight changed during the seven cycles (from

196

10.22 to 8.93), and the desorption rate of Cry11Aa in the medium was only 31.5% at the seven

197

cycles (data not shown). This result also implied that Cry11Aa could effectively load on MHNPs,

198

and the MHNPs would be a stable nano-carrier.

199

Figure 2c shows the TGA analysis of MHNPs. The result indicated that the weight loss of

200

MHNPs at the temperature of 300 ◦C might be attributed to the H2O loss from the MHNPs.

201

Moreover, the weight loss of MHNPs within the temperature range of 300~411 ◦C was about

202

27.83%, which might be caused by the decomposition of Mg(OH)2 and subsequent

203

transformation into MgO.33 Moreover, we assessed the ultraviolet radiation resistance capacity of

204

MHNPs by the XRD and FT-IR. The result of XRD analysis (Figure 2d) indicated that the sizes

205

of MHNPs at the (101) direction had slightly changed (from 12.0 ± 0.5 nm to 14.7 ± 0.8 nm)

206

after 18 h UV radiation, but all diffraction peaks had no obvious shifted. Meanwhile, FT-IR

207

patterns (Figure 2e) of MHNPs had no obvious changes before and after UV radiation. Our data

208

suggested that MHNPs possessed super ultraviolet radiation resistance capacity.

209

Additionally, previous studies have indicated that the pure Mg(OH)2 material has a relatively

210

high heat storage capacity (690 kJ/kg),32 and CO2 can slightly react with MgO and Mg(OH)2

211

during heat storage and release processes.34 Their results also suggested that MHNPs exhibit the

212

good storage stability. Overall, MHNPs would be a perfect nano-carrier for efficient and stable

213

delivery of insecticidal crystal proteins.

ACS Paragon Plus Environment 11

Journal of Agricultural and Food Chemistry

Page 12 of 32

214 215

Figure 2. The stable property analysis of MHNPs. a) The pH value of MHNPs treated with

216

MES medium and H2O, the pH value of Cry11Aa-MHNPs in MES medium was also monitored;

217

b) the weight of MHNPs before and after treatment of MES medium and H2O; c) TGA analysis

218

of MHNPs; d) XRD patterns of MHNPs before and after UV radiation, the vertical lines

219

represented the reference database for Mg(OH)2 (JCPDF044-1482). e) FT-IR patterns of MHNPs

220

before and after UV radiation.

221

3.3 The effects on anti-ultraviolet and insecticidal bioactivity

222

Ultraviolet exposure caused decreased or lost insecticidal activity of Cry protein. Therefore,

223

we evaluated the anti-ultraviolet activity of Cry11Aa before and after loading on MHNPs in

224

order to verify the possible protective effects of MHNPs. The toxicities of Cry11Aa against

225

third-instar C. quinquefasciatus larvae were compared after different treatments. Figure 3a shows

226

that the Cry11Aa protein was partially degraded (Lane 3) under UV radiation compared with the

227

control group (Lane 2), while protein bands of Cry11Aa-MHNPs were still clear after UV

228

radiation (Lane 5). Meanwhile, we also compared the degree of protein degradation in different

ACS Paragon Plus Environment 12

Page 13 of 32

Journal of Agricultural and Food Chemistry

229

treatment groups measuring by Bradford method. Figure 3b exhibits that the protein

230

concentration of Cry11Aa-MHNPs was decreased after 4.5-h UV radiation (from 0.36 to 0.30

231

mg/mL, p