Silver nanoparticles in complex with bovine submaxillary mucin possess strong antibacterial

33 Pages·2017·4.53 MB·English

AEM Accepted Manuscript Posted Online 27 November 2017
Appl. Environ. Microbiol. doi:10.1128/AEM.02212-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
Silver nanoparticles in complex with bovine submaxillary
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mucin possess strong antibacterial activity and protect against
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seedling infection
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Running title: Antimicrobial activity of mucin-silver nanoparticles 4
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Daria Makarovsky,a* Ludmila Fadeev,b Bolaji Babajide Salam,a* Einat Zelinger,c Ofra 6 a
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Matan,a Jacob Inbar,d Edouard Jurkevitch,a Michael Gozin,b Saul Burdmana# 7
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Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of 9 :
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Agriculture, Food and Environment, The Hebrew University of Jerusalem, 10 m
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Rehovot, Israela; School of Chemistry, Faculty of Exact Sciences, Tel Aviv 11 m
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University, Tel Aviv, Israelb; Interdepartmental Core Facility, The Robert H. Smith 12 rg
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Faculty of Agriculture, Food and Environment, The Hebrew University of 13
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Jerusalem, Rehovot, Israelc; Department of Economics and Business 14 e
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Management, Faculty of Social Sciences and Humanities, Ariel University, Ariel, 15 e
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Israeld 16 ,
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*Daria Makarovsky is currently at the Goldschleger Eye Institute, Sheba Medical 18 g
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Center, Tel Hashomer, Israel. Bolaji Babajide Salam is currently at the 19 st
Department of Postharvest Science of Fresh Produce, The Volcani Center, 20
Agricultural Research Organization, Bet Dagan, Israel. 21
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Address correspondence to Saul Burdman, [email protected]. 23
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ABSTRACT A simple method for synthesis of nanoparticles (NPs) of silver (Ag) in a 24
matrix of bovine submaxillary mucin (BSM) was previously reported by some of the 25
authors of this study. Based on mucin characteristics such as long-lasting stability, 26
water solubility, and surfactant and adhesiveness characteristics, we hypothesized 27
that this compound, named BSM-Ag NPs, may possess favorable properties as a 28
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potent antimicrobial agent. The goal of this study was to assess whether BSM-Ag NPs 29 w
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possess antibacterial activity focusing on important plant pathogenic bacterial 30 a
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strains representing both Gram-negative (Acidovorax and Xanthomonas) and Gram- 31
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positive (Clavibacter) genera. Growth inhibition and bactericidal assays as well as 32
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electron microscopy observations demonstrate that BSM-Ag NPs, at relatively low 33 :
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concentrations of silver, exert strong antimicrobial effects. Moreover, we show that 34 m
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treatment of melon seeds with BSM-Ag NPs effectively prevents seed-to-seedling 35 m
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transmission of Acidovorax citrulli, one of the most threatening pathogens of 36 g
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cucurbit production worldwide. Overall, our findings demonstrate strong 37
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antimicrobial activity of BSM-Ag NPs and their potential application for reducing 38 e
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spread and establishment of devastating bacterial plant diseases in agriculture. 39 e
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IMPORTANCE Bacterial plant diseases challenge agricultural production and the 40
means available to manage them are limited. Importantly, many plant pathogenic 41
bacteria have the ability to colonize seeds, and seed-to-seedling transmission is a 42
critical route by which bacterial plant diseases spread to new regions and countries. 43
The significance of our study resides on the following aspects: i) the simplicity of the 44
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method of BSM-Ag nanoparticles’ (NPs) synthesis; ii) the advantageous chemical 45 w
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properties of the BSM-Ag NPs; iii) their strong antibacterial activity at relatively low 46 a
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concentrations of silver; and iv) the fact that, in contrast to most studies on effects 47
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of metal NPs on plant pathogens, the proof-of-concept of the novel compound is 48
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supported by in planta assays. Application of this technology is not limited to 49 :
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agriculture. BSM-Ag NPs could be potentially exploited as a potent antimicrobial 50 m
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agent in a wide range of industrial areas including medicine, veterinary, cosmetics, 51 m
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textile and household. 52 g
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KEYWORDS silver, metal nanoparticles, mucin, bacterial plant diseases, Acidovorax 54 m
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INTRODUCTION 55
Plant pathogenic microorganisms are a major cause of crop yield losses and 56
agricultural intensification has been possible through the use of chemical pesticides 57
to cope with them. However, despite the intensive use of antimicrobial compounds, 58
estimations of world crop yield losses due to plant disease range from 15 to 20% (1- 59
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3). Plant pathogenic bacteria are among the biotic agents causing significant losses in 60 w
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crop production. Almost every important crop suffers from at least one or several 61 a
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important bacterial diseases, which are often among the most significant diseases of 62
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the given crop (3). Moreover, the strategies available to manage bacterial plant 63
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diseases are generally limited, including the lack of efficient chemical bactericides for 64 :
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disease control (4). 65 m
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Importantly, many plant pathogenic bacteria are seedborne, namely, they 66 m
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can survive in the seed and be transmitted via contaminated seeds to new fields, 67 g
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regions and countries (4, 5). In a globalized world, in which a huge amount of plant 68
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material (mainly seeds) is transferred from one country to the other, inadvertent 69 e
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distribution of contaminated commercial seeds is one of the main ways by which 70 e
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bacterial plant diseases are spread worldwide (4). Therefore, new strategies to 71 ,
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manage bacterial plant diseases are highly demanded in general, and in particular, to 72 8

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prevent or reduce seed transmission of bacterial pathogens. 73 g
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The aim of this study was to assess the antimicrobial activity of silver (Ag) 74 st
nanoparticles (NPs) in complex with bovine submaxillary mucin (BSM), focusing on 75
plant pathogenic bacteria. Silver has been extensively used to control microbial 76
infections since ancient times (6, 7). Silver-based medical products have been shown 77
to be effective in reducing and preventing bacterial infections (8). Silver ions are 78
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highly reactive exerting a broad range of antimicrobial activities. They are able to 79
bind to and damage proteins and DNA leading to disruption of disulfide bonds in 80
proteins, structural changes in the cytoplasmic membrane and in the cell wall, 81
altered membrane permeability, cell distortion and inhibition of replication and 82
respiratory activity, leading eventually to cell death (9, 10). In recent years, the 83
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development of nanotechnologies has brought about a growing interest in the 84 w
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industrial and medical fields in generation of bioactive biomaterials that combine the 85 a
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relevant antibacterial properties of metals with the peculiar performance of the 86
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biomaterial. A variety of nanosilver compounds have been developed and tested for 87
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their antimicrobial properties (9, 11). Some nanosilver compounds were also 88 :
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generated and evaluated for their potential application in agriculture. However, this 89 m
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has been rather limited and very few studies were conducted to assess antibacterial 90 m
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or antifungal activities of the compounds in planta (12-16). 91 g
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Mucins are large, extracellular glycoproteins found as the main components 92
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of mucus in almost all animals (17, 18). With molecular weights ranging from 0.5 to 93 e
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20 MDa, mucins are highly glycosylated consisting of approximately 80% 94 e
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carbohydrates. Among their chemical properties, mucins may act as moisture 95 ,
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holders, adhesins and solubilizers as well as reducing and surfactant agents (18). 96 8

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Some of us have shown that BSM, a representative natural mucin, is capable of 97 g
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binding and solubilizing various types of polycyclic aromatic hydrocarbons in 98 st
aqueous solutions, leading to an increase of their antimicrobial activity (19). Further, 99
a simple method was developed to generate Ag NPs inside a BSM matrix (20). 100
Synthesis of the BSM-Ag NP compound was carried out in an aqueous solution 101
without the need of an external reducing agent, exploiting the natural solubilizing, 102
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reducing and stabilizing properties of mucin (20). The generated complex, named 103
BSM-Ag NPs, is highly soluble in water and biodegradable, thus having the potential 104
to be active at very low concentrations, constituting a potential for powerful and 105
environment friendly tool for crop protection. 106
In the present study, growth inhibition and bactericidal assays as well as 107
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electron microscopy observations demonstrate strong antibacterial effects of BSM- 108 w
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Ag NPs. Moreover, seed transmission assays reveal that BSM-Ag NPs effectively 109 a
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prevents seed-to-seedling transmission of Acidovorax citrulli, one of the most 110
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threatening pathogens of cucurbit production worldwide (21). Overall, our findings 111
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support the potential of BSM-Ag NPs as an efficient crop protection agent. 112 :
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RESULTS 114 m
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BSM-Ag NPs inhibit bacterial growth. BSM-Ag NPs tested in this report were 115 g
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produced as described earlier (20; see Materials and Methods). The size of the Ag 116
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NPs was found to be in the range of 5-20 nm, with an average diameter of about 10 117 e
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nm (20). The concentrated complexes carried Ag NPs at concentrations ranging 118 e
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between ~400 and 1000 mg l-1. We first assessed the antimicrobial activity of BSM- 119 ,
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Ag NPs by determining the effects of various Ag concentrations on growth of three 120 8

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strains representing important seedborne plant pathogenic bacteria: the gram- 121 g
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negative Acidovorax citrulli M6 [bacterial fruit blotch (BFB) of cucurbits (21)] and 122 st
Xanthomonas euvesicatoria 85-10 (bacterial spot disease of pepper and tomato 123
(22)], and the gram-positive Clavibacter michiganensis subsp. michiganensis NCPPB 124
382 (Cmm 382; causal agent of bacterial canker and wilt of tomato (23)]. 125
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BSM-Ag NPs exerted strong growth inhibition effects on all tested strains at 126
very low Ag concentrations. Representative growth curve experiments in nutrient 127
broth (NB) are shown in Fig. 1A for A. citrulli M6 and in Fig. S1 (see supplemental 128
material) for Cmm 382 and X. euvesicatoria 85-10. A delay in the exponential growth 129
phase of all strains was achieved at Ag concentrations as low as 0.13 mg l-1. Much 130
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stronger inhibition effects were observed in the range of 0.67 to 2.68 mg Ag l-1. At 131 w
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these concentrations bacterial growth was delayed by 24 to 40 h relative to controls 132 a
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exposed to BSM alone. At Ag concentrations of 6.7 mg l-1 and higher, no growth 133
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could be detected for A. citrulli M6 and Cmm 382 after 168 h (one week) of 134
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incubation (Fig. 1A and Fig. S1A). In the case of X. euvesicatoria 85-10, substantially 135 :
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delayed growth occurred at 6.7 mg Ag l-1, but no growth occurred at 13.4 mg Ag l-1 136 m
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(Fig. S1B). In these experiments, we were not able to isolate bacteria following 137 m
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dilution plating of samples in which no growth was observed, indicating that under 138 g
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tested conditions, BSM-Ag NPs at relatively low Ag concentrations of 6.7 and 13.4 139
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mg l-1 have bactericidal effects on A. citrulli/Cmm and X. euvesicatoria, respectively. 140 e
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Confirmation of bactericidal activity of BSM-Ag NPs. We further verified the 141 e
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bactericidal activity of the BSM-Ag-NPs against A. citrulli. We selected this pathogen 142 ,
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for further experiments because BFB disease caused by this bacterium is considered 143 8

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as one of the most serious threats to the cucurbit industry worldwide, mainly to 144 g
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melon and watermelon (21). Moreover, seed transmission has been responsible for 145 st
the extraordinarily fast global spread of BFB, thus making of this disease a great 146
concern to the seed production sector (21). 147
Bacterial suspensions of A. citrulli M6 (107 CFU ml-1) in 100 mM phosphate 148
buffer (pH 7.0) were exposed to different concentrations of BSM-Ag NPs for 24 h at 149
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25C. Then the suspensions were serially diluted and dilution samples were plated 150
onto nutrient agar (NA) plates to determine live bacterial concentrations at the end 151
of the experiment. Results of these experiments confirmed the strong bactericidal 152
activity of BSM-Ag NPs. Under these conditions, exposure of A. citrulli cells to BSM- 153
Ag NPs at 0.4 mg Ag l-1 strongly reduced bacterial numbers by two orders of 154
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magnitude, and no bacteria could be retrieved after exposure to BSM-Ag NPs at 2.2 155 w
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mg Ag l-1 (Fig. 1B). 156 a
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BSM-Ag NPs damage bacterial cells. Scanning electron microscopy (SEM) 157
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was used to observe morphological effects of BSM-Ag NPs on A. citrulli M6 cells. 158
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While cells treated with BSM alone (without Ag NPs) possessed a typical rod-like 159 :
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shape and a well-defined cell wall (Figs. 2A and C), clear morphological alterations 160 m
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were observed in cells exposed to BSM-Ag NPs containing 10 mg Ag l-1: the latter 161 m
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cells had a disorganized and irregular shape, looked damaged and surface vesicles 162 g
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were detected on the surface of some of these cells (Fig. 2B and D). Backscattering 163 N
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analysis of non-coated samples with in-lens detector revealed that BSM-Ag NP- 164 e
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treated cells (Fig. 2D) but not BSM-treated cells (Fig. 2C) were covered with a high 165 e
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atomic dense material (yellow spots in Fig. 2D), supporting association of Ag NPs 166 ,
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with the bacterial surface. Transmission electron microscopy (TEM) supported the 167 8

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aforementioned alterations caused to the bacterial cell wall by BSM-Ag NPs (Fig. 2F) 168 g
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as compared with cells exposed to BSM alone (Fig. 2E). Moreover, Ag NPs were also 169 t
detected in TEM observations (Figs. 2F and G). 170
BSM-Ag NPs prevent seed-to-seedling transmission of A. citrulli. We asked 171
whether BSM-Ag NPs have the potential to reduce seed-to-seedling transmission of 172
A. citrulli in melon. To answer this question, we carried out seed transmission assays 173
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following two forms of applications of the compound: 1) treatment with BSM-Ag NPs 174
of seeds that were previously inoculated with A. citrulli M6 (“treatment”), or 2) 175
pretreatment of the seeds with BSM-Ag NPs followed by bacterial inoculation 176
(“pretreatment”). BSM-Ag NPs were tested using three concentrations of Ag: 5, 10 177
and 22 mg l-1. As controls, seeds were non-inoculated, inoculated only, or inoculated 178
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and treated/pretreated with BSM alone (without Ag NPs). Additional controls 179 w
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included treatment and pretreatment with a known bacterial disinfectant, sodium 180 a
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hypochlorite (NaClO), at a concentration of 0.6%. 181
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Three independent experiments with similar results revealed that BSM-Ag 182
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NPs, given either as seed treatment or as pretreatment, protected the emerging 183 :
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seedlings in all tested concentrations of Ag (Figs. 3, 4, and S2 and S3 in supplemental 184 m
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material). No significant differences in disease severity were observed among seed 185 m
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treatments or pretreatments with BSM-Ag NPs at Ag concentrations of 10 and 22 mg 186 g
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l-1 and these treatments did not significantly differ from non-inoculated (healthy) 187
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controls and from treatment with 0.6% NaClO (Fig. 3). Treatment and pretreatment 188 e
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of seeds with BSM-Ag NPs at an Ag concentration of 5 mg l-1 were slightly but 189 e
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significantly (p<0.05) less effective than treatment/pretreatment with the highest 190 ,
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concentrations of Ag. However, these treatments significantly (p<0.05) reduced 191 8

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disease severity as compared with inoculated controls (Fig. 3). 192 g
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In melon-A. citrulli seed transmission assays, disease severity negatively 193 st
correlates with plant growth parameters like seedling weight (24). In agreement with 194
the disease severity scores, all seedlings emerging from BSM-Ag NP-pretreated or 195
treated seeds had significantly (p<0.05) higher foliage weights in comparison with 196
inoculated, non-treated/non-pretreated controls (Fig. S2). In agreement with in vitro 197
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experiments, seed pretreatment and treatment with BSM alone (without Ag NPs), 198
did not protect the seedlings (Figs. 3, S2 and S3). In contrast to the effective 199
protection exerted by the different pretreatments with BSM-Ag NPs, pretreatment 200
with 0.6% NaClO did not protect the seedlings at all (Figs. 3, S2 and S3). Importantly, 201
seedlings emerging from BSM-Ag NPs treated/pretreated seeds did not show visible 202
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Young seedlings of melon are highly sensitive to A. citrulli, with seed 204 a
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inoculation at relatively high bacterial concentrations generally leading to seedling 205
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blight and death (24, 25). In seed transmission experiments we recorded the number 206
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of dead seedlings per treatment every day (Fig. 4). While most (above 90%) of the 207 :
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control (inoculated, non-treated/pretreated) seedlings died at 8 days after sowing, 208 m
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no seedling emerging from treated or pretreated seed with BSM-Ag NPs died in 209 m
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these experiments, except for a relative low percentage (less than 20%) of seedlings 210 g
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resulting from seeds treated with the lowest Ag concentration (5 mg l-1) (Fig. 4). As 211
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similar as inoculated controls, most seedlings emerging from seeds treated or 212 e
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pretreated with BSM without Ag and from seeds pretreated with NaClO, died in 213 e
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these experiments, although the progress of seedling death was slightly delayed 214 ,
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relative to untreated plants (Fig. 4). Representative pictures of selected treatments 215 8

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of seed transmission assays are shown in Fig. S3 (see supplemental material). 216 g
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Verification of antibacterial activity of BSM-Ag NPs in seeds. To further 217 st
assess the antibacterial activity of BSM-Ag NP treatment or pretreatment on seeds, 218
A. citrulli M6-inoculated and treated or pretreated melon seeds were put to 219
germinate in NA plates at room temperature. Representative pictures from some of 220
the treatments are shown in Fig. 5. As expected, no A. citrulli colonies developed 221
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