Purification and Characterization of a Secretory Alkaline

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Bioactive Constituents, Metabolites, and Functions

Purification and characterization of a secretory alkaline metalloprotease with highly potent antiviral activity from Serratia marcescens strain S3 Yuanxia Qin, Jie Wang, Fenglong Wang, Lili Shen, Haixiang Zhou, Hangjun Sun, Kaiqiang Hao, Liyun Song, Zhicheng Zhou, Chaoqun Zhang, Yuanhua Wu, and Jinguang Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06909 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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Journal of Agricultural and Food Chemistry

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Purification and characterization of a secretory alkaline metalloprotease

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with highly potent antiviral activity from Serratia marcescens strain S3

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Yuanxia Qin,1,2 Jie Wang,2 Fenglong Wang,2 Lili Shen,2 Haixiang Zhou,2

4

Hangjun Sun,2 Kaiqiang Hao,2 Liyun Song,1,2 Zhicheng Zhou,3 Chaoqun

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Zhang,4 Yuanhua Wu,1* Jinguang Yang2*

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1. College of Plant Protection, Shenyang Agricultural University, Shenyang,

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110866, China

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2. Key Laboratory of Tobacco Pest Monitoring Controlling & Integrated

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Management, Tobacco Research Institute of Chinese Academy of Agricultural

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Sciences, Qingdao, 266101, China.

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3. Hunan Tobacco Science Institute, Changsha, 410004, China.

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4. Jiangxi Institute of Tobacco Leaf Science, Nanchang, 330025, China1 *Corresponding authors. E-mail address: [email protected] (W. Y.); [email protected] (Y. J.).

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ACS Paragon Plus Environment


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Abstract

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In this study we report a secretory protein that was purified from Serratia

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marcescens strain S3 isolated from soil from the tobacco rhizosphere.

16

Subsequent mass spectrometry and annotation characterized the protein as

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secretory alkaline metalloprotease (SAMP). SAMP plays a crucial role in

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inhibiting Tobacco mosaic virus (TMV). Transmission electron microscopy

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(TEM), dynamic light scattering (DLS), confocal microscopy, and microscale

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thermophoresis (MST) were employed to investigate the anti-TMV mechanism

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of SAMP. Our results demonstrated that SAMP, as a hydrolytic metal protease,

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combined and hydrolyzed TMV coat proteins to destroy the virus particles.

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This study is the first to investigate the antiviral effects of a S. marcescens

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metalloprotease, and our finding suggests that S. marcescens-S3 may be

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agronomically useful as a disease-controlling factor active against Tobacco

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mosaic virus.

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Key words: Serratia marcescens, secretory alkaline metalloprotease,

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Tobacco mosaic virus, antiviral activity

2


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Introduction

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Tobacco mosaic virus (TMV), could infect at least 125 species from nine

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plant families distributed throughout the world. Together with the high

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frequency of occurrence and destructiveness, TMV often caused serious yield

34

losses every year.1, 2 Once the plant infected with phytopathogenic viruses, it

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hardly to suppress efficiently by chemical pesticides. In consequence, resistant

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crop species (including trans-genesis crops), vector controlling and

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cross-protection are frequently used for plant virus management.1-3 Scientists

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continue to screen antiviral compounds for putative usefulness in virus

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management; these compounds have generally been sourced from plants,

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beneficial microorganisms or other natural sources. In other cases, candidate

41

compounds can also be studied to develop novel biogenic pesticides. To date,

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multitudes of studies have been conducted to isolate and identify candidate

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compounds from Pseudomomas spp,1,2 Bacillus spp,4,5 Actinomycesspp,

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Acinetobacter spp6 and fungal strains such as Trichoderma albolutescens.7

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Serratia marcescens is a Gram-negative enteric bacterium with ubiquitous

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habitation.8 In soil, it may be among the plant growth promoting rhizobacteria

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(PGPR). For example, S. marcescens strain 90-166 induces systematic

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resistance via a quorum sensing-dependent mechanism, and can stimulate

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plant resistance to several plant pathogens including Cucumber mosaic virus.9

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In addition, S. marcescens has been proven to produce various extracellular

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enzymes including serralysin, chitinase, metalloprotease, bacteriocins, lipase,

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thiol proteases, and nucleases.10-13 In this study we isolated PGPR strain S3

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from a tobacco field in Shanxi province, and found that this strain strongly

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suppresses TMV in tobacco. In this study, this S3 strain was confirmed as S. 3



55

marcescens, and a protein was purified via ion-exchange and size exclusion

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chromatography based on the bioassay-guided method. The isolated protein

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was identified as a secreted alkaline metalloprotease (SAMP) which was

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revealed to contain 471 amino acids by mass spectrometry (MS). We obtained

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pure SAMP through recombinant expression and studied the mechanism of its

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anti-TMV effect. Here we discovered that SAMP can interact with TMV coat

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protein (TMV CP) and subsequently hydrolyze TMV CP after combining, which

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could destroy TMV particles then reduce viral invasion.

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Materials and methods

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Identification of S. marcescens-S3

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S. marcescens-S3 was identified by biological plate and16S rRNA gene

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sequence analysis. The 16S rRNA gene was cloned according to previously

67

described protocols with the following primers 27f/1492r14 and sequenced by

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Sangon Biotech (Shanghai) Co. Ltd. We performed a BLAST search on sibling

69

species via NCBI and downloaded these sequences from GenBank to

70

determine the phylogeny of the genus. We used Clustal W15 to compare the

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multiple sequence alignments produced by MEGA v6.0, and used this data to

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construct a phylogenetic tree.

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Extraction of crude protein

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To obtain samples of the crude protein, S. marcescens-S3 was cultured in

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a 3-L volume of Luria Bertani (LB) medium for centrifugation (8000 ×g, 15 min).

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After centrifugation, the gathered supernatant was pump-filtered using a 0.5

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μm micro-membrane (Millipore, Germany) to remove residual bacteria.1 Solid

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ammonium sulfate was then added into the supernatant until it reached 80%

79

(wt/vol) relative saturation. After resting overnight at 4 °C, the sediment was 4


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separated from the supernatant by centrifugation (10,000 ×g, 15 min), and 100

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mL of buffer A (PBS buffer, pH = 7.2, 20 mmol·L-1) was added to the resulting

82

solution. The crude protein solution was desalted by dialysis as per the method

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described by Wu8 and Nam.16 After desalting, the protein solution was freeze

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dried by a vacuum freeze dryer; the resulting solid powder was then harvested

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and re-dissolved in buffer A.

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Anti-TMV protein isolation and identification

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The crude protein solution was filtered through a 0.22 μm micro-membrane

88

(Millipore, Germany) and was then loaded onto an anion-exchange

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chromatography Q Separose Fast Flow column pre-equilibrated with elution

90

buffer B (Tris-HCl, pH = 5.0, 20 mmol·L-1). Next, we eluted the bound proteins

91

using an elution buffer containing an increasing gradient of NaCl at 1 mL·min-1.

92

Fractions harvested were desalted with AMICON Ultra 15mL-3kDa centrifugal

93

filter (Merck Millipore Ltd., Ireland) for anti-TMV testing. All effective fractions

94

were loaded for size-exclusion chromatographic analysis using a Sephacryl

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S100 HR column and buffer A for elution. Each elution section was collected

96

for further anti-TMV testing. The active fraction was then centrifuged and

97

concentrated (4,000×g, 45 min) using the AMICON centrifugal filter described

98

above. Proteins were separated using SDS-PAGE, and then recovered for

99

later analyses using mass spectrometry (MS).

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Construction of recombinant plasmids

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We obtained the genomic DNA of S. marcescens-S3 by extracting total

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DNA using an Easy Pure® Genomic DNA Kit. Using this genomic DNA as a

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template, we then amplified the coding region of SAMP by PCR using primers

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SAMP-Fw/SAMP-Re. The retrieved SAMP fragment was then cloned into 5



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pET-28a(+) and created the recombinant expression vector pET-28a(+)-SAMP,

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and transformed into E. coli DE3 chemically competent cells.17 For expression

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in plant cells, the coding sequence of SAMP was cloned into the FU46-RFP

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entry vector and then recombined into the destination vector pEarleygate100.

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Recombinant plasmid of TMV coat proteins (TR-His-TMV-CP19) was by

Li,

X.,18

110

provided

and

we

constructed

another

plasmid

the

111

TR-His-TMV-CP19-YFP by inserting the gene fragment of yellow fluorescent

112

protein (YFP) into XhoI site of TR-His-TMV-CP19. All primers used in

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recombinant plasmids construction were listed in supplement Table S1.

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Heterologous expression and purification of recombinant protein

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E. coli DE3 including pET-28a(+)-SAMP vector were cultured (at 37 °C and

116

160 rpm) overnight in 10 mL LB medium with 50 μmol·L-1 kanamycin, and then

117

in 2-L LB medium with 50 μmol·L-1 kanamycin until the OD600 reached 0.6. We

118

then added 0.5 mmol·L-1 β-D-1-thiogalactopyranoside (IPTG) and continued to

119

incubate the cultures for 6 h. After centrifugation (8,000×g, 15 min), collected

120

and resuspended the cells in 100 mL of buffer C (100 mmol·L-1 NaCl, 50

121

mmol·L-1 phosphate buffer and 10 mmol·L-1 β-mercaptoethanol, pH=7.2). The

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cells were then fully disrupted via ultrasonic disruption on ice, and inclusion

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bodies in the precipitate were harvested and dissolved in buffer D (50 mmol·L-1

124

sodium phosphate, 8 mol·L-1 urea, 300 mmol·L-1 NaCl, and 20 mmol·L-1

125

imidazole; pH=7.2). After filtering with a 0.22 μm micro-membrane, the crude

126

protein solution onto a His60 Ni2+ Superflow Resin & Gravity Column (Clontech

127

Laboratories, Inc.), and eluted bound protein with an increasing gradient of

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imidazole in buffer E (50 mmol·L-1 Na3PO4, 8 mol·L-1 urea, 300 mmol·L-1 NaCl;

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pH=7.4) at 2 mL·min-1. All fractions were desalted and analyzed using 6


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SDS-PAGE to determine the optimal concentration of imidazole. Next, the

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fraction of the optimal imidazole concentration was collected, renatured, and

132

saved for future analyses.19,20

133

TR-His-TMV-CP19 and TR-His-TMV-CP19-YFP were expressed in E. coli

134

DE3 as described above, and their expression were induced with 0.5 mmol·L-1

135

IPTG at 16 °C for 16 h. The cells were resuspended in 40 mL buffer C after

136

centrifugation, then fully disrupted by ultrasonic disruption on ice, and

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centrifuged (13,400×g, 30 min, 4 °C) to collect the supernatant. The

138

supernatant was forced through a 0.22 μm syringe filter and was then loaded

139

onto a His60 Ni2+ Superflow Resin & Gravity Column. Next, the column was

140

washed with 5 column volumes of 40 mmol·L-1 imidazole and the protein was

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eluted with 400 mmol·L-1 imidazole. We then concentrated the elution using a

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10 kDa MW filter, and loaded it onto a Hi Load 16/60 Superdex G 100 pg

143

column pre-equilibrated with a dialysis solution (10 mmol·L-1 phosphate buffer

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and 100 mmol·L-1 NaCl solution, pH=7.2).18

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Characterization of SAMP

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The enzymatic activity of the purified protease was assayed by the Folin

147

method.16 With casein as a substrate, the amount of enzyme that produced

148

1µg Folin-stained amino acids or peptides in 1 min was defined as 1 unit of

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enzyme activity. We also measured enzyme characters over a pH range from

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4.0 to 12.0, and the stability of the protease was determined over the same

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range. The buffers used for pH tests were citrate–phosphate buffer (50

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mmol·L-1, pH range 4.0–6.0), Na3PO4 buffer (50 mmol·L-1, pH range 7.0–8.0),

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and carbonate–bicarbonate buffer (50 mmol·L-1, pH range 9.0–12.0).21 To

154

determine pH stability, we incubated the recombinant protein at 40°C for 1 h in 7



155

different buffers, with residual protease activity determined. We also

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determined the thermal stability via incubation at multiple temperatures (30 to

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90 °C, at 10 °C intervals) for 10 min, and testing the residual activity of the

158

enzyme. The residual activity was assayed at 40 °C in Na3PO4 buffer (pH=8.0,

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50 mmol·L-1), using casein as the substrate. The activity of the non-heated

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protein was used as a control (100%).16,21

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Antiviral assay

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Plant material and TMV inoculum: Nicotiana benthamiana and N.

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tabacum cv. SunsamNN plants were cultured in a greenhouse of the Institute of

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Tobacco Research CAAS in Qingdao, China. Gooding & Helbert22 method was

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slightly modified and adopted for TMV solution preparation. N. tabacum cv.

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NC89 was inoculated by TMV, the infected upper leaves were ground in PBS

167

buffer (10 mmol·L-1, pH=7.4), followed by filtered with double layer gauze and

168

centrifuged (1000 ×g, 20 min), the percolate were treated with polyethylene

169

glycol twice. After centrifuged (10000 ×g, 30 min), the sediment were

170

resuspended with PBS buffer. The concentration of TMV was calculated

171

according to the following formula:

172

Virus concentration (mg/mL) = ( A260×Dilution Ratio)/ E0.1%1cm 260 nm

173

Note: E is the extinction coefficient of 0.1% (1 mg /1 mL) suspension, obtained

174

with the wavelength of 260 nm.

175

The titer value of TMV solution used in this manuscript was 1 μg·mL-1.

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Leaf-disk method: The ability of SAMP to inhibit TMV was assessed using

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the leaf-disk method with TMV 30B (a TMV strain with a GFP label). TMV 30B

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samples were inoculated on 4-week-old N. benthamiana plants by

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agroinfiltration, and the leaves were cut into 0.5 cm diameter disks. The leaf 8


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disks were put into 24-well plates containing liquid MS medium and SAMP was

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added at different concentrations (6.5, 12.5, 50 and 100 μg·mL-1). All

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treatments were kept at 25 °C for 5 days in the dark. Fluorescence intensity

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was detected with a FluorCam 7.0 (Photon Systems Instruments, Drasov,

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Czechia) to quantify the prevalence of TMV 30B.23

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Interference effect: 100 µL TMV 30B was agroinfiltrated into N.

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benthamiana leaves to form a 1 cm diameter circle. After 48 h, SAMP was

187

infiltrated into a circle surrounding the TMV 30B area. The intensity and spread

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of the fluorescence was observed under UV light. The control plants were

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treated with PBS buffer (10 mmol·L-1, pH=7.0).

190

Assessing inactivation, protective and curative effects: N. tabacum cv.

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SunsamNN was used to test the inactivation and protective effects of purified

192

SAMP, using the half-leaf method. The TMV inoculums was mixed with SAMP

193

at different concentrations (6.5, 12.5, 50, and 100 µg·mL-1), after 20 min, the

194

right side of the leaf was inoculated with TMV inoculum premixed with SAMP,

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and the left side of the leaf was inoculated with TMV mixed with PBS buffer (10

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mmol·L-1, pH=7.0).

197

For the protective effect test, the right side of the leaf was smeared with

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different concentrations (6.5, 12.5, 50 and 100 µg·mL-1) of 50 µL purified

199

SAMP, while the left side was treated with PBS buffer (10 mmol·L-1, pH=7.0).

200

After 1 h, leaves were treated with 20 µL TMV inoculums (1 µg·mL-1). For

201

curative effect assay, 20 μ L TMV solution (1 µg·mL-1) was inoculated on the

202

whole leaves of N. tabacum cv. SunsamNN, then washed with water. Six hours

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post inoculation, 50 μ L of SAMP solutions were smeared on the right hand

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sides of inoculated leaves, and PBS buffer (10 mmol·L-1, pH=7.0) on the left 9



205

hand sides. Ningnanmycin (NNM) and BSA were set as positive and negative

206

controls respectively.

207

Interactions between metalloproteinase and TMV

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To investigate interactions between SAMP and TMV intracellularly,

209

SAMP-RFP and TMV 30B were infiltrated into N. benthamiana leaf

210

simultaneously and observed using a Leica SP8 confocal microscope 72 h

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post transient expression. An argon laser at 488nm and 552 nm was used to

212

excite GFP and RFP, respectively. The wavelengths of light emitted by GFP

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and RFP were 495-535 nm and 580-630 nm, respectively.24

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Microscale thermophoresis (MST) assay was performed using Nano

215

Temper Monolith NT.115 (NanoTemper Technologies, Germany), to confirm

216

the interaction of SAMP with TMV CP. In this assay, TMV CP-YFP and SAMP

217

were diluted and mixed as per protocol instruction.25 BAS was performed as

218

negative control.

219

The hydrolytic activity of the metalloproteinase to TMV CP was measured

220

as follows: the TMV CP solution and the SAMP solution were mixed at different

221

ratios and incubated at 37 °C. A portion of the reaction solution was withdrawn

222

at different intervals, and the proteins present were assessed using Western

223

blots.26

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To observe TMV particles changes after treated with SAMP, transmission

225

electron microscopy (TEM) and dynamic light scattering (DLS)27 were used to

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observe the TMV particles morphology and size. 60 μL TMV particles were

227

mixed with SAMP (50 μg·mL-1), and incubated at room temperature for 4 hours,

228

BAS (50 μg·mL-1) was set as negative control. The TEM images were obtained

229

using H-7650 transmission electron microscope (Hitachi, Japan), and particle 10


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size was detected with Zetasizer Nano S90 (Malvern, United Kingdom).

231

Statistical analysis

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All statistical analyses involved performing Duncan’s multiple range tests

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implemented by SPSS v18.0 (IBM SPSS, Chicago, USA).

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Results

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Isolation and identification of S. marcescens-S3 with anti-TMV effect

236

Samples of S. marcescens-S3 with excellent anti-TMV activity were

237

isolated from the rhizospheres of tobacco plants grown in Shanxi, China. S.

238

marcescens-S3 strain bacteria produce round, red, opaque colonies with wet,

239

convex, smooth surfaces, and scanning electron microscopy showed that

240

these colonies were composed of rod-shaped cells approximately 0.5-1.1 μm

241

long and 0.3-0.5 μm in diameter (Fig. 1a and b). The Biolog MicroPlates

242

results were shown in Table 1 and identified S3 as S. marcescens with a

243

probability of 61.7% (Table 1). The 16S rRNA gene sequence of S.

244

marcescens-S3 was about 1500 bp and submitted to NCBI (accession:

245

MK411566), permitted S3 to be classified as members of Serratia, where they

246

represented a lineage closely related to S. marcescens (Fig. 1c).

247

S. marcescens-S3 cultured in LB medium till OD600 reached 1.0 for

248

centrifugation (6000 ×g, 10 min), 1 mL supernatant was collected and mixed

249

with TMV inoculums (1 μg·mL-1) in the same volume. Mixture was tested for

250

inactivation effect through “half-leaf” method, and the results indicated that the

251

supernatant of S3 culture exhibited almost 100% inactivation activity against

252

TMV. However, after the supernatant treated at 95 °C for 10 min, the effect

253

decreased to 42% (Fig. 1d), indicating that the protein from S3 culture plays a

254

important role in the anti-TMV activity. 11



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Isolation and identification of the target protein

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The results of anion-exchange chromatography showed proteins with

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anti-TMV function detected when the pH reached 5.0 (Table 2) and collected

258

by the elution buffer with 0.2 mol·L-1 NaCl (Fig. 2 a). Functional sections were

259

collected and prepared for size-exclusion chromatography; the anti-TMV

260

results of sections eluted through Sephacryl S100 are displayed in Fig. 2b.

261

Finally, functional sections were also examined using SDS-PAGE, where only

262

one obvious band of approximately 50 kDa was found (Fig. 2c).

263

To obtain the amino acid sequence of the protein, we recovered protein

264

samples from the gel for MS/MS analysis (MS/MS Workflow and Protocol was

265

shown in supplementary file). Protein annotation using NCBI prot indicated the

266

best candidate, secreted alkaline metalloprotease (SAMP; Protein View No.

267

WP_015377659.1) isolated from a strain of S. marcescens, with 7 reliable

268

peptide

269

DTFVYFAAEESTAAAPDWIR (Fig. 2d) and the other six sequences were

270

exhibited in the supplementary file (supplement Fig. S1). After sequence

271

alignment with the alkaline metalloprotease genes obtained from the NCBI

272

database, we obtained the SAMP gene of S. marcescens-S3 via PCR. The

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amino acid sequence was aligned with three serralysin homologs that have

274

determined structures. One of these homologs is from S. marcescens (PDB

275

entry 1sat), while the other two were isolated from Serratia sp. Fs14 (PDB

276

entry 5d7w) and Serratia sp. E-15 (PDB entry 1srp).8,28-31 According to the

277

sequences, SAMP shares 99.79% identity with its thermostable homolog from

278

Serratia sp. Fs14 (Fig 3a). A predicted overall structure model of SAMP was

279

obtained from SWISS-MODEL (Fig 3b), suggested that SAMP possess

sequences

matched.

One

of

these

12


peptide

sequences

is

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proteolytic domain contacting with the N-terminal, one Zn2+-binding pocket,

281

and 7 Ca2+ ligand interaction sites.32

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Purification and properties of SAMP

283

SDS-PAGE image analysis showed that E.coli DE3 transformants with

284

recombinant expression vector pET-28a (+)-SAMP produced a large amount

285

of recombinant SAMP. The molecular weight of this protein is approximately

286

50 kDa, which was consistent with the theoretical molecular mass of SAMP

287

(Fig. 4a). For purification, the optimal concentration of imidazole was found to

288

be 200 mmol·L-1 (Fig. 4b), and this fraction was collected, renatured,

289

dispensed, and preserved at -80 °C for further testing.

290

We found that SAMP showed maximal enzymatic activity when

291

temperature reached to 40-50 °C in a pH range 8–9 (Fig. 4c and d), while its

292

activity decreased significantly above 50 °C. These results agreed with

293

previous reports of a homologous metalloproteinase.8,16,21 Compared with

294

SAMP purified via chromatography, the enzyme activity of recombinant SAMP

295

could recover to 12.5% after renaturation (Table 3).

296

Interestingly, SAMP exhibited a better stability in a pH range from 9-12 in

297

stability tests, while the recombinant SAMP showed a better thermal stability

298

when temperature exceeded 50 °C. When temperature reached 80 °C, the

299

SAMP purified from S3 retained about 43.19% activity, while recombinant

300

SAMP showed a slight higher residuary activity 45.09% (Fig. 4 e and f). The

301

difference may be related to the thermostable mechanism “self-digestion

302

hypothesis”, which described Serralysin-like protease B (SPB, approximately

303

50 kDa) from S. marcescens FS14 could digest itself above 50 °C (optimum

304

temperature about 60°C), the denaturation of SPB caused by higher 13



305

temperature (>60°C) may slow down or cease the self-digestion activity. When

306

temperature decreased, the unfold protease in supernatant could refold and

307

recover protease activity.33,8 In our tests, the renaturation ratio of recombinant

308

SAMP was only 12.5%, implied that there were plenty of unfolded SAMP in

309

protease solution, we speculated that the denaturated protein were recovered

310

protease activity after temperature decreased.

311

SAMP antiviral activity against TMV

312

Inhibition activity determined by the leaf-disk method: After 5 days,

313

leaf-disks treated with SAMP showed concentration-dependent decreases in

314

green fluorescence intensity (Fig. 5a). These results demonstrate that SAMP

315

may inhibit the growth of TMV in planta.

316

Interference in TMV growth: Under the UV light, no significant differences

317

were observed 5 d post agroinfiltration. However, samples containing

318

phosphate buffer solution (PBS, 10 mmol·L-1, pH 7.0) showed increased

319

fluorescence 7 d after agroinfiltration and exhibited bright green light in the

320

petiole and the stem near the root, while, plants treated with SAMP showed

321

bright green fluorescence only in the region that was agroinfiltrated. Moreover,

322

control plants showed plenty of local regions of fluorescence on apical new

323

leaves 9 d post agroinfiltrated, by contrast, few fluorescence observed on the

324

SAMP treated plants (Fig. 5b). These results suggest that SAMP inhibits the

325

growth and movement of TMV 30B from the primary infection site to new

326

organs, and may thereby suppress TMV infection in plants.

327

Inactivation, protective and curative effects against TMV: The purified

328

SAMP were diluted to concentrations of 100, 50, 12.5 and 6.5 µg·mL-1. Then

329

we used the “half-leaf” method to detect whether SAMP could inactivate TMV. 14


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We found a lack of observable lesions in N. tabacum cv. SunsamNN leaves

331

inoculated with the purified recombined proteins at concentrations above 50

332

µg·mL-1. Moreover, SAMP exhibited an efficient inactivation effect (i.e. >75%)

333

even when the SAMP concentration was as low as 6.5 µg·mL-1 (Fig. 5c). The

334

protective and curative effects of SAMP were shown in Table 4. Compared

335

with NNM, SAMP was superior in protective effect, but inferior in curative effect.

336

When the concentration of SAMP reached 100 µg·mL-1, the protective effect

337

was 74.28% which significantly greater than NNM (60.05%), the curative effect

338

was 35.36% just equivalent to NNM of 50 µg·mL-1 (39.15%), but still significant

339

higher than negative control (BSA).

340

Interactions between metalloproteinase and TMV

341

To investigate the anti-TMV mechanism of SAMP, we observed the

342

interactions between TMV 30B and SAMP under a confocal microscope.

343

Results showed that TMV 30B and SAMP-RFP were almost completely

344

localized in epidermal cells 72 h after infiltration into 4-week-old N.

345

benthamiana leaves, which suggest that SAMP may interact with TMV 30B in

346

plant cells (Fig. 6a).

347

MST analysis revealed that TMV CP-YFP (target protein) could interact

348

with SAMP (the ligand), these results are shown in Fig. 6b. TMV CP-YFP used

349

in this test was purified with His60 Ni2+ column (supplement Fig. S2 a).

350

SAMP is a type of hydrolytic enzyme, know as metalloproteinase. When

351

SAMP was added to TMV CP at concentrations of 1 and 6.5 μg·mL-1, a

352

significant portion of TMV CP were digested and more than three peptide

353

bands were found below the original band. However, when the concentration

354

of the added SAMP reached 12.5, 25, 50, and 100 μg·mL-1, a majority of TMV 15



Page 17 of 40

355

CP were digested and no obvious bands were displayed after incubation at 40

356

°C for 4 hours. When TMV CP was incubated with SAMP at a concentration of

357

6.5 μg·mL-1 for a different duration (0, 1, 5, 10, 20, 30 and 60 min), the

358

hydrolytic effect was approximately equal. These results concluded the

359

hydrolysis of TMV CP by SAMP was predominantly dose-dependent. All TMV

360

CP used in this experiment were recombinant (Supplement Fig. S2 c).

361

The TEM and DLS results demonstrated that SAMP was capable of

362

hydrolyzing TMV particles. After adding SAMP, TMV particles were destroyed,

363

i.e. showing particle cleavages and size reduction (Fig. 6d). The mean size of

364

TMV particles before treatment measured 252 nm, which was approximate to

365

the size of native TMV (300 nm),34 the difference may be caused by

366

mechanical damage during TMV particles extraction. After treated with SAMP,

367

the mean size of TMV decreased to 136 nm, while the BSA treatment detected

368

247 nm.

369

Discussion

370

Previous research suggests that extracellular proteins play a role in the

371

antiviral activities of beneficial microorganisms. Such extracellular proteins

372

include endoribonuclease L-PSP purified from Rhodopseudomonas palustris,1

373

an active protein of 39.4 kDa from Pseudomonas fluorescens CZ,2 and

374

activator proteins isolated from Alternaria tenuissima.35 Based on protein

375

chromatography

376

metalloprotease (SAMP) with a molecular weight approximately 50 kDa was

377

purified from S. marcestens-S3, and this protein showed strong antiviral

378

effects against TMV (Fig. 1-2).

379

and

bioactive-guided

assays,

secretory

alkaline

The metalloproteinase secreted from S. marcescens is a member of the 16


Page 18 of 40


380

serralysin proteinase family, of which protein crystal structures have been

381

published.19,31 Compared to homologous proteinases, the amino acid

382

sequence of SAMP is most similar to serralysin from Serratia sp. Fs14; only

383

the third amino acid was different between these proteins (Fig. 3a). The

384

predicted crystal structure of SAMP showed a proteolytic domain, Zn2+-binding

385

pocket, and Ca2+ ligand interaction sites. Prediction of its dimensional structure

386

suggested that SAMP was a serralysin proteinase from Serratia. Since the

387

1980s metalloproteinases from Serratia have been used as anti-inflammatory

388

agents, and recent studies have identified some metalloproteinases as

389

potential cardiovascular disease treatments due to their fibrinolytic activity.21,36

390

Many metalloproteinases have also been used in industry for similar

391

reasons.31

392

metalloproteinase was useful in agriculture, including in plant virus disease

393

management. In this study, we found that SAMP had strong proteolytic

394

properties at pH 8-9 and 40-50 °C (Fig. 4, Table 3). Moreover, SAMP showed

395

strong inactivation effects against TMV in tobacco. We found that 100 µg·mL-1

396

SAMP treatments eliminated TMV infection completely (Fig. 5). Compared with

397

NNM, SAMP exhibited outstanding protective effects but for the curative

398

effects. Perhaps SAMP (50kDa) with the large molecular weight was not easily

399

absorbed and transmitted in vivo by leaves.

At

present,

no

studies

have

yet

assessed

whether

400

Many anti-TMV proteins are active elicitors or activators against TMV which

401

can promote plant growth, induce phytoalexin (antibiotic) accumulation and

402

other plant defense reactions.35,36 However, few proteins that directly effect on

403

TMV—e.g. by destroying or eliminating virus particles—have been reported.

404

To date, only a few anti-TMV enzymes have been identified, including the 17



Page 19 of 40

405

endoribonuclease1 described above, ribonuclease (RNase) from Bacillus

406

cereus ZH144 and RIPs (ribosome-inactivating proteins) from Phytolacca

407

dioica L,37 although the anti-TMV mechanism for these have not yet been

408

reported. In this study, SAMP failed to induce plant resistance (data not

409

shown), however we found that SAMP could hydrolyze TMV directly. MST is

410

viable for analysis of protein interactions,38 Li et al. demonstrated the in vitro

411

inhibitory

412

technology.39,40 In addition, confocal microscopy is a widely used visual

413

technology capable of observing interactions in plant cells.23 In this study,

414

results of MST and confocal microscopy revealed that SAMP can interact with

415

TMV CP both in vitro and in vivo as a proteinase. Further tests demonstrated

416

that SAMP could hydrolyze the TMV CP into small peptide fragments (Fig. 6c)

417

and therefore destroyed TMV particles (Fig. 6d).

effect

of

NNM

on

TMV

CP

polymerization

using

MST

418

This study is the first report of the inhibitory effect of S. marcestens

419

metalloprotease on TMV and its anti-TMV mechanism. Unlike “activator

420

proteins”, SAMP can directly damage the virus particles themselves in vitro

421

and in vivo—thereby suppressing virus invasion and transmission. This study

422

may therefore improve our understanding of how this proteinase can be

423

useful for plant virus disease management and sustainable agriculture in the

424

future.

425

Abbreviations

426

SAMP, secrete alkaline metalloprotease; TMV, Tobacco mosaic virus; TMV

427

CP, TMV coat protein; TEM, transmission electron microscopy; DLS, dynamic

428

light scattering; MST, microscale thermophoresis; TMNV CP-YFP, TMV coat

429

protein with a yellow fluorescent protein label; SAMP-RFP, secrete alkaline 18


Page 20 of 40

430


metalloprotease with a red fluorescent protein label; NNM, Ningnanmycin.

431

Acknowledgements

432

We are grateful to Prof. L. X., College of Food Science and Engineering,

433

Shandong Agricultural University, for the providing of recombinant plasmid

434

TR-His-TMV-CP19. And thank to other laboratory members for the plant

435

material growing and TMV cultivating.

436

Supporting Information

437

The peptides of SAMP identified through MS/MS; primers used in

438

recombinant plasmids construction; and purification recombinant proteins

439

used in tests were submitted in supplementary file.

440 441

The Supporting Information is available free of charge on the ACS Publications website.

442

Funding

443

This research was supported by a grant from State Tobacco Monopoly

444

Bureau (110201601024(LS-04)), Development of Rapid Detection Method for

445

Tobacco

446

(201743010020088) and Jiangxi Institute of Tobacco Leaf Science

447

(201701002).

Virus

(SCYC201804),

Hunan

Tobacco

19


Science

Institute


448

References

449

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450

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YER057c/YjgF/UK114 protein family with antiviral properties, from the

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thermostable metalloprotease from the newly isolated Serratia sp. RSPB11. Int.

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tobacco mosaic virus in large quantities. Phytopathology. 1967, 57,

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1285-1290.

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Suppress Endoplasmic Reticulum-Mediated Immunity. Mol. Plant. 2018, 11,

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Molecular interaction studies using microscale thermophoresis. Assay Drug

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from full-length and GFP-tagged cDNA clones of Pepper mottle virus and

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stable systemic expression of GFP in tobacco and pepper. Virus Res. 2011,

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functionality. Nucleic Acids Res. 2017, 45, D313-D319.

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Y. Crystal structure of Serratia protease, a zinc-dependent proteinase from

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Serratia sp. E-15, containing a beta-sheet coil motif at 2.0 A resolution. J.

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Biochem. 1996, 119, 844-851.

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(31) Baumann, U. Crystal Structure of the 50 kDa Metallo Protease from

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Serratia marcescens. J. Mol. Biol. 1994, 242, 244-251.

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(32) Rose, A. S.; Bradley, A. R.; Valasatava, Y.; Duarte, J. M.; Prlic, A., Rose,

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P. W. NGL viewer: web-based molecular graphics for large complexes.

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Bioinformatics. 2018, 34, 3755-3758.

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(33) Wu, D.; Li, P.; Zhou, J.; Gao, M.; Lou, X.; Ran, T.; Wu, S.; Wang, W.; Xu,

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property from S. marcescens FS14. Int. J. Biol. Macromol. 2016, 93, 98-106.

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(34) Czapar, A. E.; Zheng, Y. R.; Riddell, I. A.; Shukla, S.; Awuah,S. G.;

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Lippard, S.J.; Steinmetz, N. F. Tobacco Mosaic Virus Delivery of

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Phenanthriplatin for Cancer therapy. ACS Nano. 2016, 10, 4119-4126.

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glycoprotein from Botrytis cinerea, elicits defence response and improves

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disease resistance in host plants. Biochem. Bioph. Res. Co. 2015, 457,

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627-634.

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(37) Iglesias, R.; Citores, L.; Ragucci, S.; Russo, R.; Di, M. A.; Ferreras, J. M.

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Biological and antipathogenic activities of ribosome-inactivating proteins from

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Phytolaccadioica L. Biochimica et Biophysica Acta. 2016, 1860, 1256-1264.

579

(38) Seidel, S. A.; Dijkman, P. M.; Lea, W. A.; van den Bogaart, G.;

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Jerabek-Willemsen, M.; Lazic, A.; Joseph, J. S.; Srinivasan, P.; Baaske, P.;

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Simeonov, A.; Katritch, I.; Melo, F. A.;Ladbury, J. E.; Schreiber, G.; Watts, A.;

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Braun, D.; Duhr, S. Microscale thermophoresis quantifies biomolecular

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interactions under previously challenging conditions. Methods. 2013, 59,

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301-315.

585

(39) Li, X.; GH, Wang, Q.; Chen, Z.; Ding, Y.; Yu, L.; Hu, D.; Song, B.

586

Ningnanmycin inhibits tobacco mosaic virus virulence by binding. Oncotarget.

587

2017, 47, 82446-82458

588

(40) Han, Y.; Luo, Y.; Qin, S.; Xi, L.; Wan, B.; Du, L. Induction of systemic

589

resistance against tobacco mosaic virus by Ningnanmycin in tobacco. Pestic.

590

Biochem.and Physiol. 2014, 111, 14-18.

25



591

Fig. 1 Identification of Serratia marcescens strain S3 and anti-TMV

592

activation of different fraction of S3 culture. (a) Scanning electron

593

microscopy images. (b) Colonies of S. marcescens S3, grown for 18 h. (c) A

594

phylogenetic tree based on the 16S rRNA gene sequence divergence,

595

indicating the relationships between S3 and other relatives. The numbers

596

represent the confidence levels from 1000-replicate bootstrap sampling. Bar,

597

1% sequence divergence. (d) in activation effect against TMV of S3 culture

598

supernatant, supernatant with heating treatment (95 °C), sediment, and crude

599

protein, n = 4; **, P < 0.01.

600

Fig. 2 Purification and MS/MS analysis of secretory alkalin

601

metalloproteinase (SAMP). (a-b) Anti-TMV effect of different eluent. (a)

602

Gradient elution with NaCl (0, 0.1, 0.2, 0.3, 0.4, 0.7, 1.0 mol·L-1) at 25

603

mmol·L-1 Tris-HCl (Ph=5.0) on a Q Sephacryl Fast Flow column. (b) Elution

604

fraction with 25 mmol·L-1 phosphate buffer solution performed on a Sephacryl

605

S100 HR column. (c) SDS-PAGE analysis of the purified protein revealed a

606

single band at a size 50 kDa with Coomassie brilliant blue R-250 staining.

607

Lane 1: purified protein obtained via chromatography method; Lane M,

608

protein molecular mass marker. (d) Peptide of SAMP identified through

609

MS/MS.

610

Fig. 3 Mutiple sequence alignment and prediction overall structure of

611

SAMP. (a) Amino-acid sequence aligment of SAMP and three homologous

612

proteins isolated from Serratia spp.. The serralysins from Serratia sp. FS14

613

(5d7w), S. marcescens (1sat) and Serratia sp. E-15 (1srp). Identical residues

614

are shown on a gray background and differences are highlighted in colours.

615

(b) Cartoon view of SAMP structure, predicted via SWISS-MODEL. 26


Page 27 of 40

Page 28 of 40


616

Fig. 4 Recombinant, purification and properties of secretory alkalin

617

metalloproteinase (SAMP). (a) Expression of recombinant SAMP, line 1

618

were proteins expression of pet 28(a+), line 2 expressed by pet 28(a+)-SAMP.

619

The bold band in line 2 near to 50 kDa marker was recombinant SAMP. (b)

620

Purification of SAMP with Ni2+ superflow resin. From line 1 to 6 were proteins

621

eluted with different concentration of imidazole (50, 100, 150, 200, 250, 300

622

mmol·L-1). (c – d) Effect of temperature (c) and pH (d) on enzyme activity of

623

purified SAMP via chromatography and recombinant SAMP. (e – f) Enzyme

624

stability of the SAMP treated with different temperature (e) and pH (f).

625

Fig. 5 Anti-TMV effect of SAMP. (a) Inhibition effect of SAMP at different

626

concentrations (100, 50, 12.5, 6.5ug·mL-1), CK was treated with SM liquid

627

medium, and H means health leaf disks. (b) Anti-TMV 30B effect in N.

628

benthamiana plants. TMV 30B was infiltrated in red circles while SAMP was

629

in blue circles. As control treatment the blue circle regions were infiltrated with

630

phosphate buffer solution (PBS, 10 mmol·L-1, pH 7.0). (c) Inactivation effect of

631

different concentration SAMP against TMV.

632

Fig. 6 Interaction between TMV and SAMP. (a) TMV 30B and

633

SAMP-RFP almost completely colocalized in N. benthamiana leaf epidermal

634

cells through confocal microscope. (b) TMV coat protein was proved to

635

interact with SAMP performed with MST method, and there was no interaction

636

between TMV CP and BAS. (c) Hydrolysis effect of SAMP. The first WB was

637

TMV coat protein mixed with SAMP in different concentration (1-7

638

respectively were 0, 1, 6.5, 12.5, 25, 50 and 100 μg·mL-1), the second

639

displayed hydrolyzed results incubated for different interval with 6.5 μg·mL-1

640

SAMP (1-7 stands for 0, 1, 5, 10, 20, 30 and 60 min). (d) The hydrolysis of 27



641

SAMP to TMV particles visualized using TEM and DLS. The BSA treatments

642

were set as negative control.

28


Page 29 of 40

Page 30 of 40


Table 1 Identification of S3 through Biolog plates method with a GEN III plate Rank

PROB

SIM

DIST

Organism Type

1

0.617

0.617

5.580

GN-Ent

Serratia marcescens

2

0.184

0.184

6.054

GN-Ent

Serratia liquefaciens

3

0.062

0.062

6.999

GN-Ent

Cedecea lapagei

4

0.029

0.029

7.661

GN-Ent

Serratia odorifera

29


Species


Table 2 Inactivation effect of proteins bind onto the anion-exchange chromatography in Tris-HCl buffer (0.02 mol·L-1) with different pH. PH

Inactivation effect (%)

4

7.50±6.10 B

5

86.30±2.80 A

6

81.38±3.25 A

7

72.66±12.77 A

8

83.59±7.06 A

9

96.82±3.69 A

Note: Protein were eluted with 1 mol·L-1 NaCl and desalted with phosphate buffer solution (pH = 7.2, 0.02 mol·L-1) before applied in inactivation tests. In this test desalting buffer was set as mock controls All data are analyzed with Duncan’s multiple range test using the SPASS v18.0 data processing system, and the inactivation effect were shown as the means ± SD from four biological replicates, different capital letters means significant difference (P

Purification and Characterization of a Secretory Alkaline

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